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Why do many DNA solutions contain additional compounds?

Why do many DNA solutions contain additional compounds?



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DNA solubility data in only water is scarce.

A previous question asked for a quantification of DNA solubility in water. It seemed like it would be easily answerable, however isn't quite that simple since no data seems to exist for DNA solubility in exclusively water.

Even the small amount of data about water was in the context of washing away organic compounds from the solution. This got me thinking. DNA is in an aqueous solution in biological situations, and is usually handled in aqueous solutions in labs. So why is solubility data so scarce for pure water? What is important about organic compounds for DNA solutions?

Why are other additives important for laboratory DNA solutions?

So my question is why is DNA dissolved in exclusively water seemingly "uncommon" in laboratories? Is there something about pure water that is bad for DNA storage? Is it that functionally DNA never needs to be in water because it is inaccessible to proteins? Or have I misinterpreted the lack of solubility data; water is common-place and no data exists because there is no need?

I imagine PCR is where most of the data on DNA solutions exists. What about the polar organic solvents/compounds makes them important for DNA solutions?


DNA in pure water.

The only time that nucleic acids would encounter pure water would be in a laboratory setting--for example after an oligonucleotide is synthesized in vitro, the protecting groups are removed from the reactive atoms in the finished sequence and the final product is cleaved from the supporting matrix. At that point you can lyophilize (freeze dry) the ammonium hydroxide solution, and resuspend the single-stranded oligo in pure water.

Additional compounds enhance heavy oligomer solubility.

However, as noted in the comments, nucleic acid solubility, particularly high molecular weight DNA, like genomic DNA, is enhanced in solutions with dilute monovalent cations. 10 mM TrisHCl, pH 8.0, 1 mM EDTA suffices for almost every application. The EDTA inhibits any errant DNAses, and also slightly inhibits microbial growth.

Compounds act as pH buffers.

Tris is not the best biological buffer but has high solubility and is relatively cheap and stable. If the solution becomes too basic the DNA strands will melt, and if the solution becomes too acidic the purines will start to deaminate.

DNA never experiences pure water in biology.

From the moment that DNA is synthesized in a cell until that cell dies and its DNA is ultimately degraded, it does not encounter a pure water environment.


While physiological experiments could be conducted without organic solvents, chemical syntheses of DNA or analogs, chemical modification of DNA, and purification after chemical reaction could be performed in the presence of organic solvents. Such experiments are not directly relevant to physiology, but we use chemically synthesized DNA and DNA analogs. In addition, chemical properties of DNA in the presence of organic solvents may tell how DNA behave in physiological conditions. People doing PCR and/or molecular biology may not be interested in the solubility of DNA, because under the condition they are using, the concentrations are far less than the saturated condition.


Why do many DNA solutions contain additional compounds? - Biology

From the time of discovery of nucleic acids by Fredrick Miescher in 1870, they were long regarded as something of a curiosity until the structures of the monomer units, the nucleotides, was established in 1909 and that of RNA was proposed by Levene and Tipson in 1935. Nucleic acids are basically of two types- DNA and RNA, which are polymers of nucleotide chains. Each nucleotide comprises of three parts, firstly - a sugar moiety which is a pentose (five-membered-ring) joined to a second part-phosphate groups. The anomeric carbon of each sugar is bonded to the nitrogen atom of a third part - heterocyclic compound in a ß- glycosidic linkage. The linkage is named so, as the substituents at C-1 and C-4 are on the same side of the furanose ring.

As the heterocyclic compounds which formed a part of the nucleic acids contained amine groups and in general amines act as bases, these heterocyclic compounds were also arbitrarily referred to as bases. Moreover, since the hetero atom in the ring is Nitrogen- these compounds were named as Nitrogen Bases. Nitrogen bases are planar, aromatic, heterocyclic molecules which for the most part are derivatives of purine or pyrimidine. There are only four bases in DNA—two are substituted purines (adenine and guanine), and two are substituted pyrimidines (cytosine and thymine). RNA also contains only four bases. Three (adenine, guanine, and cytosine) are the same as those in DNA, but the fourth base in RNA is uracil instead of thymine. Thymine and uracil differ only by a methyl group—thymine is 5-methyluracil.

Why do Nucleic acids contain only Ribose or Deoxy-Ribose sugars ?
In RNA the five-membered-ring sugar is D-ribose. In DNA it is 2’-deoxy-D-ribose (D-ribose without an OH group in the 2-position). The choice of five membered ring is by default as these rings are the most stable amongst aromatic compounds except in the case of benzene, a six membered ring which is stabilized by resonance. Amongst the pentoses, Ribose is selected during evolution, as the exclusive sugar component of nucleic acids. The selection is explained by using molecular models and by eliminating most of the other common sugars by looking at their chemical structure and envisioning how they would fit in a nucleic acid model. Comparisons of sugar pucker conformations and configurations of pentoses indicate that ribose was not randomly selected but the only choice, since ß-D-ribose fits best into the structure of physiological forms of nucleic acids. In other nucleotides containing arabinose, xylose, or lyxose, the C2'-OH and/or the C3'-OH are above the furanose ring, causing steric interference with the bulky base and the C5'-OH group.

Why Nucleic acids are negatively charged?
Phosphoric acid links the sugars in both RNA and DNA. The acid has three dissociable OH groups with pKa values of 1.9, 6.7, and 12.4. Each of the –OH groups of the phosphoric acid is equally capable of reacting with an alcohol (acid + alcohol -> ester) to yield a phosphomonoester, a phosphodiester, or a phosphotriester. In nucleic acids the phosphate group forms only a phosphodiester. Although, the phosphoric acid is capable of forming three ester bonds, it is forming only two bonds, leaving the third –OH group free, which upon dissociation imparts negative charge. In case of RNA, the additional 2’-OH group also adds to the negative charge. Therefore RNA has more charge and is therefore less stable and exhibits greater mobility upon electrophoresis than DNA.


Heating of phosphoric acid molecules results in formation of pyrophosphoric acid, which is a phosphoanhydride. formed by loss of a water molecule. The name pyrophosphate is derived from the the Greek word - pyr, which means “fire.” Thus, pyrophosphoric acid is prepared by “fire”—that is, by heating. In the above said manner, Triphosphoric acid and higher polyphosphoric acids can also be formed, due to the ability of phosphoric acid to form an anhydride.

Nucleotides can exist as monophosphates, diphosphates, and triphosphates and are named by adding monophosphate or diphosphate or triphosphate to the name of the nucleoside. These phosphoanhydride bonds are High energy bonds which means that when these bonds are broken , they release lot of energy, which can be simultaneously used for driving other chemical reactions in the cell.

The purines and pyrimidines are bonded to the anomeric carbon of the furanose ring—purines at N-9 and pyrimidines at N-1—in a -glycosidic linkage. A compound containing a base bonded to D-ribose or to 2’-deoxy-D-ribose is called a Nucleoside. In a nucleoside the ring positions of the sugar are indicated by primed numbers to distinguish them from the ring positions of the base. This is why the sugar component of DNA is referred to as 2’-deoxy-D-ribose. For example, adenine is the base, whereas adenosine is the nucleoside. Similarly, cytosine is the base, whereas cytidine is the nucleoside, and so forth. Uracil is found only in RNA and is therefore is attached to D-ribose but not to 2-deoxy-D-ribose. Similarly, because thymine is found only in DNA, it is attached to 2-deoxy-D-ribose but not to D-ribose.

A nucleotide is a nucleoside with either the 5’ or the 3’ -OH group bonded in an ester linkage to phosphoric acid. The nucleotides of RNA—where the sugar is D-ribose—are more precisely called ribonucleotides, whereas the nucleotides of DNA—where the sugar is 2-deoxy-D-ribose—are called deoxyribonucleotides.

Biological functions of Nucleotides:
Nucleotides apart from acting as integral units of DNA and RNA, tperform various cellular activities such as donors of chemical energy, secondary messengers in cell signalling, cofactors for enzymes, as vasidialtors, carriers of subunits required in cell wall synthesis and lipid biosynthesis.

ATP- Currency of the cell:
All living systems need energy to ensure their survival and reproduction and they derive energy by converting nutrients into a chemically useful form of energy. The most important form of chemical energy is adenosine -triphosphate (ATP). The importance of ATP to biological reactions is shown by its turnover rate in humans—each day, a person uses an amount of ATP equivalent to his or her body weight. ATP is known as the universal carrier of chemical energy as it is commonly used in all living systems from prokaryotes to eukaryotes. ATP has the ability to convert thermodynamically unfavorable reactions to favorable ones as “the energy of hydrolysis of ATP converts endergonic reactions into exergonic reactions.”

The secret behind the stability of ATP:
As it can be noticed from the structure of ATP, that it is highly negatively charged as any nucleotide triphosphate has four units of negative charge and must be highly reactive and thereby making it less stable. Considering the above fact, although ATP reacts readily in enzyme-catalyzed reactions, but surprisingly, its reaction rate is quite slow in the absence of an enzyme. When we compare with carboxylic acid anhydrides which hydrolyze in a matter of minutes, ATP takes several weeks to hydrolyze. The low rate of ATP hydrolysis is a favourable consequence for the cell as it can be retained in the cell for a longer period of time and be available when needed for an enzyme-catalyzed reaction. It is very interesting to know that the reason behind slow reactivity of ATP in the absence of enzymes is its negative charge which repels the approach of nucleophiles. When ATP is bound at an active site of an enzyme, it complexes with magnesium which decreases the overall negative charge on ATP. This is why ATP-requiring enzymes also require metal ions The other two negative charges can be stabilized by positively charged groups such as arginine or lysine residues at the active site. In this form, ATP is readily approached by nucleophiles, so ATP reacts rapidly in an enzyme-catalyzed reaction, but only very slowly in the absence of the enzyme.

  • ATP is not the only biologically important nucleotide. Guanosine –triphosphate (GTP) is also used in place of ATP in some phosphoryl transfer reactions. GTP drives the peptide bond formation during translation. GTP and GDP are also involved in G-protein coupled Receptor (GPCR) mediated signaling.
  • UDP serves as the carrier of Glucose moiety in the form of UDP-Glucose, required for cell wall synthesis in bacteria.
  • CDP-Choline acts as the carrier of Choline group for the synthesis of membrane phospholipid- Phosphotidyl Choline.
  • Dinucleotides such as FAD, FMN are used as oxidizing agents and NADH, NADPH are used as reducing agents.
  • Another important nucleotide is adenosine – monophosphate, commonly known as cyclic AMP. Cyclic AMP is called a “second messenger” because it serves as a link between several hormones (the first messengers) and certain enzymes that regulate cellular function. Secretion of certain hormones, such as adrenaline, activates adenylate cyclase, the enzyme responsible for the synthesis of cyclic AMP from ATP. Cyclic AMP then activates an enzyme, generally by phosphorylating it.
  • Adenosine acts as Sleep inducer and is commercially available as “Adenocarp”

The reducing agent is NADPH and every NADPH produced in the cell can generate three ATPs. Based on the this fact, the usage of NADPH to reduce dihydrofolate comes at the expense of ATP. Although the process of synthesis of thymine is energetically expensive still the cell prefers to recruit Thymine in DNA rather than Uracil.
The presence of uracil in DNA is mutagenic and presence of thymine prevents potentially lethal mutations. The chemical phenomenon behind this is chemical instability of Cytosine which can tautomerize to form an imine, which subsequently gets hydrolyzed to uracil. The overall reaction is called a deamination since it removes an amino group.
If a cytosine in DNA is deaminated to a uracil, uracil will specify incorporation of an adenine into the daughter strand during replication instead of the guanine that would have been specified by cytosine. This would have adverse effects as it leads to changes in the nucleotide sequence and can cause GC basepairs to AT base pair mutations. Inorder to avoid such complications presence of a U in DNA is recognized as a “mistake” by celllular enzymes before an incorrect base can be inserted into the daughter strand. Uracil-N-glycolsylase and dUTPase are the two enzymes which ensure the elimination of U from DNA. dUTPase cleaves UTP molecules present in the nucleus and prevents them for getting into the active site of DNA polymersase. In case of any U being incorporated into the DNA, Uracil-N-glycolsylase cleaves the glycosydic bond and removes the nucleotide frorn DNA. This generates a apyrimidinic site which is recognized as a lesion and is further repaired by repair enzymes.
If U’s were normally found in DNA, the enzymes could not distinguish between a normal U and a U formed by deamination of cytosine. Having T’s in place of U’s in DNA allows the U’s that are found in DNA to be recognized as mistakes. Unlike DNA, which replicates itself, any mistake in RNA does not survive for long because RNA is constantly being degraded and then resynthesized from the DNA template. Therefore, it is not worth spending the extra energy to incorporate T’s into RNA.

Properties of nucleic acids:
Effect of temperature
Duplex nucleic acid structures get disrupted or denatured at high temperature into single stranded molecules and the process is termed as ‘melting’, the process of melting of double helices can be monitored easily because base stacking and base-pairing interactions significantly reduce UV absorption. As the temperature of a solution containing a polynucleotide duplex is raised, the absorption of UV light will greatly increase around the temperature at which the two strands separate into single-stranded polynucleotides. This is due to the fact that single stranded molecules exhibit higher UV absorption than the double stranded molecules at a wave length of 260 nm. The transition in UV absorption typically occurs over a 48?C to 88?C range. When a plot is drawn with the Absorbance on X-axis and Temperature on Y-axis, it is termed as Melting curve. The midpoint of the transition or melting curve at which half of the polynucleotide species is duplex and half is single-stranded is identified as the Melting temperature (Tm). In general, DNA duplexes are relatively less stable to thermal denaturation than RNA duplexes or RNA-DNA duplexes.

Hyperchromic shift: Increase in the absorption at 260nm upon denaturation of double stranded DNA resulting in formation of single stranded DNA is termed as Hyperchromic shift.

Hypochromic shift: Decrease in the absorption at 260nm upon renaturation of single stranded DNA molecules to yield double stranded DNA is termed as Hypochromic shift. Renaturation bt slow cooling is called Annealing.

The Tm for a given duplex is otherwise affected by a number of factors. The major determinants include sequence length, G-C content and salt concentration. Longer duplexes or those having a higher proportion of G-C pairs are more stable and therefore will have a higher Tm. Monovalent cations, such as sodium (Na+) and potassium (K+), stabilize double helices by partially neutralizing the negative charge along the phosphodiester backbone. Consequently, higher salt concentration will also increase the Tm of a given polynucleotide duplex. In addition the presence of mismatched bases in a duplex will generally have a destabilizing effect and decrease Tm.

  1. Absorbance At 260 nm in decreasing order.
    Absorbance of free nucleotides is more their polymeric forms and single stranded molecules absorb more than double stranded forms. Similarly absorption of RNA is more than DNA due to the presence of 2’-OH group. Purine absorb more than pyrimidines due to the presence of double ring structure. Amongst the purines, Adenine absorbs more than Guanine due to the presence of more free electrons. Consequently two DNA molecules equal in length but differing in GC content absorb differently with the DNA having more AT content absorbing more than the DNA with more GC content.
    A260: Nucleotides > ss DNA > ds DNA
    A260: RNA > DNA
    A260: Purines > Pyrimidines
    A260: Adenine > Guanine > Thymine = Cytosine
  2. Osmotic pressure increases upon denaturation. Organic solvents and detergents interferes hydrophobic interactions and causes the DNA denaturation.
  3. Viscosity decreases upon denaturation. DNA solutions are highly viscous and high viscosity is due to the long length and rigidity of the molecules. Upon denaturation, viscosity decreases as double stranded DNA is more rigid and hence viscous when compared to single stranded DNA.
  4. Optical Rotation becomes more negative upon denaturation as the plane polarized light shifts slightly towards left (more negative).
  5. Density of nucleic acids increases upon denaturation as double stranded DNA is less dense when compared to single stranded DNA as it occupies more mass per given volume.

Effect of pH on nucleic acids:
Nucleic acids are stable between a pH range of 5-9. Beyond this range, they undergo denaturation spontaneously due to the tautomeric shift of Nitrogen bases altering the H- bonding between the nucleotides of the opposite strands.

Effect of Acid on nucleic acids:
Both DNA and RNA undergo acid hydrolysis. The glycosidic bonds of purine nucleosides are susceptible to acid hydrolysis by protonation at N7 of guanosine or adenosine. Under mild acidic conditions DNA becomes Apurinic DNA. Depurination of DNA is more favourable than that of RNA, although the formation of an abasic site in the polynucleotide results in either case. A complete loss of sequence information represented by an abasic site in DNA can likewise cause a permanent mutation.

Effect of Alkali on nucleic acids:
RNA undergoes alkali hydrolysis due to the presence of 2’-OH group but DNA doesn’t undergo alkali hydrolysis.
Although both RNA and DNA can be degraded through cleavage of the phosphodiester backbone, their chemical stability is drastically different. RNA is distinctively subjected to a transesterification reaction in which the C2’ oxygen serves as a nucleophile for attack on the adjacent phosphorus. This particular reaction proceeds through a pentacoordinate phosphate intermediate, in which the bond formed between the C2’ oxygen and the phosphorus centre which is in line with the bond to be broken between the phosphorus centre and the C5’ oxygen. As a aresult, the reaction products terminate with a C5’ hydroxyl and C2’, C3’-cyclic phosphate. This cyclizing mechanism of RNA transesterification occurs easily because the nucleophile is inherently positioned in proximity to the phosphorus centre in the phosphodiester backbone. Moreover, the reaction is promoted either by deprotonation of the C2’ hydroxyl or protonation of the C5’ oxygen. Therefore, RNAs are easily degraded in alkaline or acidic solutions. A similar mechanism applicable to both RNA and DNA polymers, operates for cleavage of phosphodiester , in which a second molecule serves as the attacking nucleophile. The reaction is similarly promoted by deprotonation of the attacking group and protonation of the leaving group. However, the products terminate with a C5’ phosphate and C3’ hydroxyl. Water can serve as a nucleophile in the spontaneous hydrolytic degradation of RNA or DNA polymers, although the reaction is approximately 100 000 times less likely to occur than RNA transesterification. Based on the above principle and the fact that each sugar lacks a C2’ hydroxyl group, the phosphate and sugar backbone of DNA exhibits a relatively huge chemical stability. In fact, DNA is the most stable of the biological polymers enabling it to maintain the integrity of genetic information- a prerequisite to act as genetic material.

Effect of bisulfite and nitrous acid:
Bisulfite and nitrous acid promote deamination or removal of an amino group from cytosine, resulting in conversion of cytosine to uracil. In DNA, cytosine deamination will ultimately lead to conversion of a G-C base pair to an A-T base pair if left uncorrected.

Effect of Ionizing radiations and Oxidizing agents:
Strong oxidizing agents such as hydroxyl radicals generated by ionizing radiation or incomplete metabolic reduction of oxygen producing superoxide ions, hydrogen peroxide or hydroxyl radicals react extremely with DNA. Hydroxyl radicals can nonspecifically damage DNA by breaking the phosphodiester backbone. Strand scission occurs by a number of mechanisms, each of which produces polynucleotide fragments with different 5’and 3’ terminal products. In addition, hydroxyl radicals can directly attack and alter the chemical composition of the bases in nucleic acids generating compounds like 8-hydroxyguanine and thymine glycol. These modifications result in formation of lesions in the sequence of DNA making them susceptible to mutations, which may ultimately lead to cancer. Therefore, ionizing radiation and other compounds that cause DNA damage are typically carcinogenic or cancer causing.


Mechanism of Action

DTT is involved in disulfide exchange reactions. DTT is used at 1-10 mM for protein SS reduction and is capable of crossing biological membranes.

Reducing properties

Dithiothreitol is a strong reducing agent with a redox potential of -0.33 V at pH 7. The reduction of a disulfide bond is followed by two sequential thiol-disulfide exchange reactions (see Fig. 2 below). The reduction generally continues past the mixed-disulfide species due to the second thiol in DTT having an increased tendency to close the ring. This causes the formation of an oxidized DTT and leaves behind a reduced disulfide bond. DTT’s reducing power is limited to pH values greater than 7, since only the negatively charged thiolate form is reactive. The thiol groups have pKa values of 9.2 and 10.1.

Fig. 2: Reduction of a typical disulfide bond by DTT via two sequential thiol-disulfide exchange reactions


LABORATORY PROCEDURES

DNA extraction

Key steps leading to successful DNA extractions are grinding the tissue sufficiently and identifying the best extraction protocol(s) for the purpose at hand. The availability of lab equipment and infrastructure is also a consideration: suggestions for a minimum set of lab equipment and basic molecular biology protocols are given in Appendix 1.

Efficient grinding of the plant tissues is the first step toward high yields of DNA. For efficient and simultaneous homogenization of multiple tissue samples, we used a modified version of a grinder based on a reciprocating saw (Alexander et al., 2007 Appendix 1) as an inexpensive alternative to commercially available bead beaters. Pestles and mortars with the addition of molecular-biology-grade silver sand to aid grinding by hand can be used as an alternative. If a minimal sample size is needed and the tissues are soft, they can be ground in microfuge tubes with micropestles (e.g., Geneaid catalog no. MP050 Geneaid Biotech Ltd., New Taipei City, Taiwan).

The DNA extraction protocol needs to be considered carefully. Plants, especially tropical plants, synthesize a wide range of compounds, such as polysaccharides and polyphenols (Coley and Barone, 1996 ), that can be co-purified with DNA and may reduce yield and/or inhibit subsequent PCR reactions. In some cases, the extraction protocol will need to be tailored to meet the specific challenges of the tissue, and it may be difficult to find a single method that works well for all samples.

Most widely used DNA extraction methods can be placed into one of two groups: those that use DNA-binding columns to purify DNA, and those that use chemical methods to partition DNA from cellular contents in solution. DNA-binding columns are reliable and produce consistent results, require less technical expertise to use effectively, and generate little or no hazardous waste. The major disadvantage is that they can be expensive, although cheaper versions are becoming available, and consideration is needed of the savings in time and labor achieved with kits.

If a partition-based method is chosen, we recommend searching the literature for successes using that particular method to extract DNA from closely related taxa, or from taxa with similar extraction challenges (e.g., excess polysaccharides). There are numerous simple DNA extraction methods that have been used successfully on a variety of samples including cashew and corn (Sika et al., 2015 ), potato (Hosaka, 2004 ), Rosaceae (Antanaviciute et al., 2015 ), and rice (Sajib et al., 2017 ) that could be tested and may be successful. Otherwise, a CTAB method modified by adding agents to remove specific secondary metabolites is a good starting point see Allen et al. ( 2006 ) and Neubig et al. ( 2014 ). Many of these methods require toxic chemicals such as phenol and chloroform, which must be handled in a fume hood and be disposed of safely in accordance with local regulations using established protocols. Safety Data Sheets (SDS) that accompany all purchased chemicals and are available online (e.g., at www.sigmaaldrich.com) are a good source of safety information.

To find the best extraction protocol for our needs, we assessed two relatively inexpensive and reasonably simple CTAB-based methods, modified to be carried out in microfuge tubes. Both of these protocols have been used successfully by the LIPI Molecular Systematics Laboratory for taxon-specific projects. Initially, we extracted DNA from the tissues of 75 specimens using the extraction method of Tel-Zur et al. ( 1999 ), modified by Wendel (Appendix 1). After PCR, 63 specimens did not yield enough PCR product for sequencing both rbcL and matK (discussed in detail below). Therefore, we extracted DNA from these and a further 331 specimens, using the extraction method of Porebski et al. ( 1997 ), which generated a smaller volume of hazardous chemical waste but included one extra overnight step compared to the Wendel extraction method (Appendix 1). In total, we extracted DNA from the tissues of 406 specimens. For an additional comparison, we used the column-based DNeasy Plant Mini Kit (QIAGEN, Venlo, The Netherlands Appendix 1) to extract DNAs from the tissues of a subset of 48 specimens that were previously subject to CTAB extractions. The molecular biology workflow we used is shown in Figure 1, DNA extraction methods are detailed in Appendix 1, and DNA extraction data are shown in Appendix S1.

Two general approaches are widely used to determine the quantity and quality of DNA extracts. Gel electrophoresis of DNA samples and a ladder for quantification allow estimation of DNA concentrations and determination of whether the sample is degraded or contains mostly fragments of high molecular weight. Spectrophotometry allows quantity to be estimated as well as the identification of some common contaminants such as proteins and phenol. We used gel electrophoresis because we did not have access to an appropriate spectrophotometer. We attempted PCR for all samples regardless of the evidence of DNA degradation or low yield that we obtained from the gel, although lower PCR success is expected from attempts to amplify loci from DNAs that are highly degraded.

PCR primers and amplification

Published, taxon-specific primers for the group of interest are a good starting point for clade-focused studies. If such primers are not available or, as is the case in our study, a wide range of taxa are being studied, universal primers designed to work across phylogenetically diverse taxa are a good option (e.g., those recommended by the CBOL Plant Working Group 2009 ). The criteria for CBOL-recommended primers are based on universality (successful amplification across multiple taxa), sequence quality and coverage (amplification of regions that return high-quality sequence data), and discrimination (enable the most species to be distinguished). Relevant taxon-specific primer sequences can still be useful for troubleshooting if the project is broad in scope but poor PCR results are associated with particular taxa. If these approaches are not successful, primers can be designed based on publicly available sequence data. Ideally, sequence alignments should be generated from multiple taxa related to the target taxa so that suitable, conserved regions can be identified as primer sites. Primers can then be designed to amplify the region of interest using software such as PrimerDesign (Brodin et al., 2013 ) or Primaclade (Gadberry et al., 2005 ). Lorenz ( 2012 ) offers general guidelines for PCR primer design.

PCR can be challenging and, in order to achieve reproducible amplification, it is critical to use DNAs of high quality whenever possible, and to always use well-designed primers and properly prepared and stored reagents. Storing DNA is challenging (Anchordoquy and Molina, 2007 ) and is discussed in detail, along with details on using frost-free freezers for storing DNA and other reagents, in Appendix 1. Water quality is often a problem, and if reliable Milli-Q (MilliporeSigma, Burlington, Massachusetts, USA) or equivalent water is not available, it is recommended to purchase molecular-biology-grade water from a reliable reagent company.

We selected PCR primers for the plant DNA barcodes rbcL and matK based on recommendations from the CBOL Plant Working Group ( 2009 ). Appendix 1 details primer sequences and PCR conditions. We performed two 12.5-μL PCR reactions for every DNA sample extracted using a CTAB-based protocol (Fig. 1). Two small-volume reactions were used instead of one large-volume reaction to give two independent attempts at amplification while conserving expensive PCR reagents. PCR products were examined using gel electrophoresis as described above. If no PCR product was generated after two attempts, no further PCRs were performed. However, if some product was present, additional PCRs were performed until there was enough DNA for sequencing. There are trade-offs associated with performing additional PCRs to obtain enough product vs. attempting to optimize the PCR protocol for template and primer combinations that produce marginal yields. Optimization may not be practical when a project, as in this case, samples individuals from across a region or a community. When sampling closely related taxa, however, optimization could ultimately save time and resources. Suggestions for optimization and troubleshooting can be found in Appendix 1.

To obtain rbcL barcodes, we performed up to four PCR reactions on 75 DNA samples extracted using the Wendel protocol and up to six PCR reactions on 386 DNA samples extracted using the Porebski protocol (55 samples represent extractions from specimens previously extracted with the Wendel protocol Fig. 1). In total, we attempted to generate rbcL barcodes from 406 specimens (Appendix S1). We used gel electrophoresis to determine PCR yield we assigned yields to qualitative categories in order to determine which DNA samples should be targets of additional PCR reactions to accumulate sufficient DNA for sequencing. The categories we used were “no product” when there was no visible product band “some product” when a faint band of the expected size was visible and “adequate product” when a bright band of the expected size was visible. These categories were based on empirical results from sequencing faint vs. bright bands. We used the same categories as described above to categorize pooled DNA from multiple PCR reactions in order to send samples for sequencing (Fig. 1). Regardless of the DNA extraction method used, we most commonly needed to carry out three or four 12.5-μL PCR reactions to obtain enough PCR product for sequencing. Of the 75 samples extracted with the Wendel protocol, 19 were sequenced, and of the 386 samples extracted with the Porebski protocol, 76 were sequenced. A summary of these data is shown in Figure 2. A further two specimens were sequenced by pooling the PCR products from both Wendel and Porebski extractions, giving a total of 97 barcodes generated from 406 specimens (24%).

To obtain matK barcodes, we performed up to six PCR reactions on 73 samples extracted using the Wendel protocol and up to seven PCR reactions on 386 samples extracted using the Porebski protocol (56 samples represent extractions from specimens previously extracted with the Wendel protocol Fig. 1). In total, we attempted to generate matK barcodes from 405 specimens (Appendix S1). As described above, PCR products for each extraction method were divided into three categories (no product, some product, and adequate product) based on yield estimated by gel electrophoresis. The PCR results are summarized in Figure 2. We most commonly needed to perform two (Wendel) or four (Porebski) 12.5-μL PCR reactions per sample to obtain enough product for sequencing. Of the 73 samples extracted with the Wendel protocol, 18 were sequenced, and of the 386 samples extracted with the Porebski protocol, 116 were sequenced. A further 10 specimens were sequenced from pooled products from both Wendel and Porebski extractions, giving a total of 144 barcodes from 405 specimens (35%). Overall, the Wendel and Porebski DNA extraction methods performed similarly (Fig. 2).

The DNAs extracted using the QIAGEN DNeasy Plant Mini Kit protocol (Appendix 1) were each subject to a single PCR reaction (Fig. 1). A single PCR reaction from the corresponding CTAB-extracted DNA was carried out at the same time. As before, PCR products were divided into three categories (no product, some product, adequate product) based on yield estimated by gel electrophoresis. The success of these single PCR reactions for matK and rbcL are shown in Figure 3, and complete details are given in Appendix S1. In terms of DNAs that could be used to generate PCR product, the QIAGEN-extracted DNA performed similarly to the CTAB-extracted DNA. Using the DNAs extracted using the QIAGEN kit, we generated an additional 18 rbcL sequences to give a total of 115/406 specimens (28%) and 10 matK sequences to give a total of 154/405 specimens (38%). GenBank accessions are given in Appendix S1.

Although PCR failure rates appear high, the specimens that had at least some PCR product (Fig. 2, Appendix S1) could likely be sequenced after PCR optimization to increase yield. A total of 51 specimens had some PCR product for rbcL (combined from all three DNA extraction methods). PCR optimization and successful sequencing of these would increase the overall success rate to 41%. Similarly, there were 75 specimens for matK, which if successfully sequenced, would increase the overall success rate to 57%.

As discussed above, plant taxonomic groups differ by the presence of compounds that hinder DNA extraction and amplification, and universal primers may not work for all families. Therefore, we expected our overall success to vary among plant families. We found significant association of taxonomic family with overall success of generating DNA barcodes for both matK and rbcL (respectively, χ 2 = 57.6, df = 11, P = 2.57 × 10 −8 χ 2 = 25.5, df = 11, P = 0.00768 Table 1, Appendix S1). It is important to note the almost total failure of samples from Clusiaceae and Phyllanthaceae for both markers, and the differences in success between matK and rbcL for Annonaceae and Myristicaceae.

Familya a Families differed significantly in success rate (matK: χ 2 = 57.6, df = 11, P = 2.57 × 10 -8 rbcL: χ 2 = 25.5, df = 11, P = 0.00768). See Appendix S1 for full lists of success by family.
matK rbcL
Barcode generated Barcode not generated Barcode generated Barcode not generated
Annonaceae 18 9 1 26
Apocynaceae 7 7 3 11
Clusiaceae 0 11 1 10
Dipterocarpaceae 12 8 8 12
Lauraceae 6 5 1 10
Meliaceae 7 8 6 9
Moraceae 7 14 7 14
Myristicaceae 12 2 0 14
Phyllanthaceae 1 30 7 24
Primulaceae 4 9 5 8
Rubiaceae 7 28 9 26
Other 73 120 67 127
TOTAL 154 251 115 291
  • a Families differed significantly in success rate (matK: χ 2 = 57.6, df = 11, P = 2.57 × 10 -8 rbcL: χ 2 = 25.5, df = 11, P = 0.00768). See Appendix S1 for full lists of success by family.

Overall, our data suggest that multiple extraction methods can be used successfully, indicating that other factors, such as kit costs, access to appropriate chemicals and infrastructure, and previous successful experience with similar samples, should be considered when choosing a method.

Reducing contamination

Contamination can be a major problem in any molecular biology laboratory. Previously amplified PCR products are of particular concern because they may amplify much more readily than the original target locus, which may be located in a long fragment of genomic DNA. The lab should be laid out in a way that minimizes the risk of contamination. Ideally, there should be separate rooms with separate equipment and micropipettes for DNA extraction vs. PCR and all post-PCR processes. If this is not possible, separate areas of the lab with separate micropipettes should be used for DNA extraction and PCR. Filter tips effectively reduce the amount of cross-contamination by aerosols during pipetting and should be used if at all possible. The additional cost of filter tips is offset by reducing the generation of unusable data. Pipettes should be cleaned regularly, and fresh gloves should be worn at all times and changed frequently. It is very easy for fluids, or aerosols from fluids, to adhere to skin or gloves, and to be transferred to the next processing step. Care should be taken when handling specimens so as not to spread leaf fragments around the work area, or to cross-contaminate samples. Forceps for sample manipulations can be sterilized by flaming or cleaned in alcohol. Negative controls (complete reaction mixes without DNA template) should be included in every set of PCR reactions to allow contamination to be detected quickly before costly sequencing is performed. The keeping of detailed records in log books on all PCR experiments is indispensable to the task of finding the source of contamination. If access to automated processing of samples is available, this presents further possibilities for reduction in contamination as well as for increasing reproducibility.

DNA sequencing

The cost of DNA sequencing continues to decrease, and more sequencing services and platforms are becoming available. High-throughput sequencing of barcodes (e.g., Liu et al., 2017 ) and metabarcoding (Deiner et al., 2017 ) are good options for barcoding projects that target a very high number of samples and/or ecological networks. Even whole genome shotgun sequencing at low coverage to “skim” the organellar and high-copy nuclear loci from the sequencing reads is becoming cost-effective (Twyford and Ness, 2017 ). For projects that target a small number of barcodes from specimens numbering in the hundreds to a few thousand, Sanger sequencing remains a reasonable option. The main decision is whether to outsource the sequencing of PCR-generated barcodes, or to complete it within the institution. We recommend outsourcing to a high-quality, affordable sequencing service as it is often cheaper than importing reagents, performing repeat reactions and troubleshooting, and maintaining instruments. Sequencing services are also in a much better position than are individual laboratories to keep up with the rapid pace of technological change in DNA sequencing approaches. Various companies offer single-pass sequencing from as little as US$3 per sample. There are usually even greater discounts for submitting larger numbers of samples in plate format, and free shipping is available for submitting larger, but still modest, numbers of samples. Additional services such as PCR product purification are also offered by many companies, which may be more cost-effective than importing reagents. Unlike specimens or genomic DNA, PCR products for DNA sequencing can usually be sent out of the country of origin because the samples are only a small fragment of the genome, which cannot be used for other purposes, and the sequencing reaction uses up the entire sample. We used the Sanger sequencing service at Macrogen Korea, where the requirements for sample submission were 25 μL of product at 100 ng/μL, plus 2 μL of the sequencing primer at 10 pmol/μL. Macrogen also offers a reasonably priced primer synthesis option. Shipping is free for more than 20 reactions, and one free repeat reaction is provided for failed samples, making this a very cost-effective way to generate sequence data for a small-scale laboratory.

High-quality sequencing data can usually be obtained when appropriate quantity and quality standards are met, although certain sequence characteristics (e.g., high GC content and presence of simple sequence repeats) can interfere. Guidelines for quantity and quality typically are available from sequencing services, and an excellent resource for troubleshooting DNA sequence traces has been made available by the Nucleic Acid PCR Research Core Facility (NAPCore Facility, Philadelphia, Pennsylvania, USA https://napcore.research.chop.edu/problems.php).


Applications

Transmission electron microscopy DNA sequencing is not yet commercially available, but the long read lengths that this technology may one day provide will make it useful in a variety of contexts.

De novo genome assembly

When sequencing a genome, it must be broken down into pieces that are short enough to be sequenced in a single read. These reads must then be put back together like a jigsaw puzzle by aligning the regions that overlap between reads this process is called de novogenome assembly. The longer the read length that a sequencing platform provides, the longer the overlapping regions, and the easier it is to assemble the genome. From a computational perspective, microfluidic Sanger sequencing is still the most effective way to sequence and assemble genomes for which no reference genome sequence exists. The relatively long read lengths provide substantial overlap between individual sequencing reads, which allows for greater statistical confidence in the assembly. In addition, long Sanger reads are able to span most regions of repetitive DNA sequence which otherwise confound sequence assembly by causing false alignments. However, de novo genome assembly by Sanger sequencing is extremely expensive and time consuming. Second generation sequencing technologies, [19] while less expensive, are generally unfit for de novo genome assembly due to short read lengths. In general, third generation sequencing technologies, [11] including transmission electron microscopy DNA sequencing, aim to improve read length while maintaining low sequencing cost. Thus, as third generation sequencing technologies improve, rapid and inexpensive de novo genome assembly will become a reality.

Full haplotypes

A haplotype is a series of linked alleles that are inherited together on a single chromosome. DNA sequencing can be used to genotypeall of the single nucleotide polymorphisms (SNPs) that constitute a haplotype. However, short DNA sequencing reads often cannot be phased that is, heterozygous variants cannot be confidently assigned to the correct haplotype. In fact, haplotyping with short read DNA sequencing data requires very high coverage (average >50x coverage of each DNA base) to accurately identify SNPs, as well as additional sequence data from the parents so that Mendelian transmission can be used to estimate the haplotypes. [20] Sequencing technologies that generate long reads, including transmission electron microscopy DNA sequencing, can capture entire haploblocks in a single read. That is, haplotypes are not broken up among multiple reads, and the genetically linked alleles remain together in the sequencing data. Therefore, long reads make haplotyping easier and more accurate, which is beneficial to the field of population genetics.

Copy number variants

Genes are normally present in two copies in the diploid human genome genes that deviate from this standard copy number are referred to as copy number variants (CNVs). Copy number variation can be benign (these are usually common variants, called copy number polymorphisms) or pathogenic. [21] CNVs are detected by fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH). To detect the specific breakpoints at which a deletion occurs, or to detect genomic lesions introduced by a duplication or amplification event, CGH can be performed using a tiling array (array CGH), or the variant region can be sequenced. Long sequencing reads are especially useful for analyzing duplications or amplifications, as it is possible to analyze the orientation of the amplified segments if they are captured in a single sequencing read.

Cancer

Cancer genomics, or oncogenomics, is an emerging field in which high-throughput, second generation DNA sequencing technology is being applied to sequence entire cancer genomes. Analyzing this short read sequencing data encompasses all of the problems associated with de novo genome assembly using short read data. [22] Furthermore, cancer genomes are often aneuploid. [23] These aberrations, which are essentially large scale copy number variants, can be analyzed by second-generation sequencing technologies using read frequency to estimate the copy number. [22] Longer reads would, however, provide a more accurate picture of copy number, orientation of amplified regions, and SNPs present in cancer genomes.

Microbiome sequencing

The microbiome refers the total collection of microbes present in a microenvironment and their respective genomes. For example, an estimated 100 trillion microbial cells colonize the human body at any given time. [24] The human microbiome is of particular interest, as these commensal bacteria are important for human health and immunity. Most of the Earth's bacterial genomes have not yet been sequenced undertaking a microbiome sequencing project would require extensive de novo genome assembly, a prospect which is daunting with short read DNA sequencing technologies. [25] Longer reads would greatly facilitate the assembly of new microbial genomes.


Recent Trends in Animal Biotechnology

Animal biotechnology has been defined in various ways. One of the most recent quoted definitions of animal biotechnology is: “The application of scientific and engineering principles to the processing or production of materials by animals or aquatic species to provide goods and services”.

Animal Biotechnology has developed rapidly since early 1980s when the first transgenic mice and first in vitro produced bovine embryos were created.

An attempt has been made in the present article to describe the information sources and processes which are available in the field of animal biotechnology.

Examples of animal biotechnology include generation of transgenic animals or transgenic fish using gene knockout technology to generate animals in which a specific gene has been inactivated, production of nearly identical animals by somatic cell nuclear transfer, or production of infertile aquatic species.

To cater to the need of our fast growing population, searching cheap sources of proteins either from animals or from plants have been the most daunting task for us. Animal protein is an excellent source of dietary amino acids essential for human nutrition. In addition, we are able to harvest meat, milk and fibre from live animals and use them for different welfare programmes. The production of food from animals is expensive and less efficient than plants. In spite of these disadvantages, we continue to farm animals on commercial basis for food, fibres and by-products.

Our ancestors first domesticated animals many thousands of years ago and since then they have been our constant companions. From that time onwards, the animals themselves started to change. With the passage of time and continued isolation from their wild relatives, domestic animals started to display different characteristics. Some would have produced more or better milk than others, some would have grown faster or been more fecund, some would have been more vigorous.

Our ancestors might .have noticed these variations and mated animals with similar desirable traits, but they did so without understanding the basis of heredity. Restrictive breeding and selective mating gradually led to the many diverse breeds of livestock we have today, although most breeds of the dominant domestic cattle, Bos taurus, arose in the last couple of centuries. Herds of ancestral species, Bos primigenius, lived in Europe in the 18 th century.

Thus animal improvement is no longer a matter of interest but it is a matter of necessity. Biotechnology provides us powerful tools that engage us learning the basics of these tools for increase in animal production world wide. In this article I will discuss what these tools are and their applications.

A. Reproduction in Animals/Reproductive Technology:

Reproduction has some important roles in production system. It must occur at a rate that ensures replenishment of stock is greater than its use and improving genetic quality of stock. The aim of reproductive technologies is to increase the number of progeny while improving the genetic quality of stock in general and most intensively managed animals like dairy cattle and domestic pigs in particular. This has been done by various procedures like artificial insemination, embryo transfer, in vitro fertilization and embryo cloning.

A. 1. Artificial Insemination (AI):

Artificial insemination, the first animal biotechnology, takes advantages of the male’s excess gametes (sperm) production capacity. This has been the single-most important factor in increasing the productivity. There are many variations of artificial insemination to consider. There is animal-animal artificial insemination, fresh-extended (chilled) semen insemination and frozen semen insemination.

Before insemination the sperm ejaculated is collected, diluted and examined under microscope to determine the number and mortality of sperms. Techniques of AI include vaginal deposition, surgical implant, and trans-cervical insemination. Studies have been published with convincing evidence that every semen preparation in fresh, fresh-extended or frozen will produce larger litters if semen is deposited into the uterus, especially with trans-cervical technique. Fresh and fresh-extended semen produce good results with vaginal deposition.

For cattle, AI is done in standing animals without using anesthesia, using a procedure known as “rectal palpation”. Of the different types of semen preparation, it is again obvious that fresh animal-animal collection and insemination will give the best semen preparation. Fresh-extended or “chilled” semen has performed very well for many years, but not that well as does the fresh semen.

The worst results are with the frozen semen, no matter what process is used for the freezing. Females are inseminated after ovulation, which last only a few hours and occurs most commonly at night. In many breeds of farm animals it is very difficult to detect ovulation (oestrous).

The alternative is to induce females to ovulate in a synchronized manner. In practice, it is rather impossible to achieve total synchrony of ovulation but about 80% of females would respond to the inducer. With ruminants, ovulation in females can be induced by administration of hormone like progesterone or prostaglandin.

Single sex is being preferred by all livestock industries. In dairy and beef cattle, females are more accepted than males because females are not efficient in reproduction and having desirable production traits. Same thing happens in pig industry. The sex of the embryo is determined solely by sperm containing either X chromosome or Y chromosome.

Since half of the sperm in an ejaculate contain the X and the remaining half the Y, the sex ratio of progeny is near to 1 : 1. Separating sperm containing the X chromosome from that of the Y would allow us to manipulate the sex progeny by AI. Separation of the two populations has been done using a fluorescent activated cell sorter (FACS). The method is very expensive and very slow too. Monoclonal antibody that binds specifically to Y-bearing sperm cells could be used in future to separate Y- bearing and X-bearing sperm. The flow sorting of sperm cells to separate X from Y bearing cells has been successful in most cases tested and in cattle and swine has resulted in offspring of the desired sex.

A. 2. Embryo Transfer:

The propagation of genetically valuable animals is enhanced by transferring embryo. To do this one has to increase the number of mature eggs produced by a selected female. Following fertilization, those fertilized eggs are implanted into foster mother, one for each egg. Donor females are injected with prostaglandin F2a (PGF2a) to induce a synchronized oestrous before treatment is started. Ten days after oestrous period they are injected with FSH for a period of four days, followed by PGF2a to induce oestrous.

Superovulated donors are then mated by artificial insemination. Six to eight days after insemination, the fertilized eggs are recovered from cattle and six days from sheep and goat. They are immediately transferred into synchronized recipients. Alternatively, they can be frozen for indefinite storage. With good practice, pregnancy rates of 50-60% can be achieved in cattle from transferred embryo.

Pregnancy output rates can be increased by embryo splitting a method which produces identical twins. Recently this has become a routine production tool, requiring micromanipulation equipment and minimal training. Embryos were obtained from donor at the blastocyst stage (Fig. 21.1) and transferred into a standard cell culture medium containing hypertonic sucrose and bovine serum albumin.

The embryo is then transferred to a plastic Petridish containing standard culture medium, where it sinks to the bottom and sticks to it. It occurs because of the electrostatic interactions between the BSA-induced negative charge on the outer membrane and the positive charge of the plastic disc.

The embryo is then bisected using a micromanipulator fitted with a fine blade under the inverted microscope. During embryo splitting care must be taken to ensure that the inner cell mass is split into equal halves. After splitting, they are transferred into the oviduct of the recipient female following the same procedure as for normal transfer. Routine biopsy has been carried out to determine the sex of progeny, a production trait. Presence of Y chromosomal DNA in cells of an embryo biopsy indicates that the embryo is a male: if no Y chromosomal DNA is present it is female. This is being done using PCR to amplify Y-chromosome specific DNA.

A. 3 In Vitro Fertilization (IVF):

Embryo transfer is not used widely because of many factors including high costs, technical difficulty and limited availability. We can overcome these problems if oocytes, collected from a donor female are fertilized in vitro. It would ensure fertilization of more eggs using less amount of semen. Thus IVF is more efficient method than AI. During oestrous cycle a number of Graafian follicles start growing.

Eventually one of the follicle matures and ruptures, releasing the eggs for fertilization. Oocytes are recovered from these follicles from super ovulated donors by laparoscopic surgery. Eggs are then incubated to be matured and fertilized in vitro. This method demands high technical skill and, therefore, not always cost effective.

The success of IVF depends on collection of large number of oocytes and there maturation in vitro. This can be done by removing an ovary from a selected female and is induced to mature in vitro. Numbers of scientists across the globe have been carrying out research on oocyte maturation in vitro. However, it may be worthwhile to mention here that majority of Graafian follicles never attain maturity. The highest success rates with embryo transfer are reported when blastocysts are implanted into recipients.

This can be done if embryos are maintained in culture. Many laboratories have shown that 60% of IVF cattle embryos can be cultured to blastocyst stage. High rate of abortion is recorded from culture embryos during the first two months of pregnancy. This may be due to genetic defect in the oocyte and or fertilizing sperm and environmental mutagenesis of egg, sperm or embryo (mainly caused by oxygen free radicals).

B. Cloning:

Another application of animal biotechnology is the use of somatic nuclear transfer to produce multiple copies of animals that are merely identical copies of other animals. This process is called ‘cloning’. So cloning is the production of one or more identical plants or animals that are genetically identical to another plant or animal.

It would facilitate increased production of numbers of one particular embryo having desirable characteristics. Nature itself is the greatest cloning agent. In about one of every 75 conception, the fertilized embryo splits and produces monozygotic twins.

B. 1. DNA Cloning:

To do further manipulations and analysis of individual recombinant DNA molecules (e.g. DNA containing insulin gene), we must have many copies of the molecules, usually in a purified form. DNA cloning is a technique to produce large quantities of specific DNA segment. The DNA segment to be cloned is inserted to a vector, which is a vehicle for carrying inserted DNA into a suitable host cell, such as the bacterium E. coli.

The vector contains sequences that allow it to be replicated within the host cell and usually refer to as cloning vector. There are numerous cloning vectors in current use and choice among them depends on the size of the DNA fragment that needs to be cloned or the use to which the clone will be put.

Bacterial plasmids are small circular DNA molecules that are distinct from the main bacterial chromosome. They replicate their DNA independently of the bacterial chromosome. The plasmids that are routinely used as vectors are those that carry genes for drug resistance. The drug resistance genes are useful because drug- resistant phenotype can be used to select for those cells that contains the recombinant plasmid.

The process by which bacterial cells take up DNA from the medium is called transformation. This forms the basis for cloning plasmid in bacterial cells (Fig. 21.2). In most commonly used methods recombinant plasmids are added to a bacterial culture that has been pretreated with Calcium ions. Bacterial cells are then activated (by heat-shock) to take up DNA from the medium.

Normally a small number of cells are able to take up and retain one of the recombinant plasmid molecules. Once a bacterial cell has taken up a recombinant plasmid from the medium, the cell gives rise to a colony of cells containing the recombinant DNA molecule. These bacteria containing a recombinant plasmid are selected from the rest by growing the cells in the presence of the antibiotic specific to drug-resistance gene of plasmids.

B. 1b. Bacteriophage Vectors:

There are different classes of bacteriophage vectors, depending on whether chromosomal DNA inside the bacteriophage is single stranded or double stranded and the size of the donor DNA insert.

B. 1c. Bacteriophage (Lambda):

It is used as a cloning vector for double-stranded DNA inserts up to approximately 25 kb. Lambda phage heads harbor a linear DNA (genome) of about 50kb in length. The central part of the genome is not required for replication or packaging and so a central part can be cut out by using restriction enzyme and discarded.

The two remaining ‘arms’ at either end of the genome are ligated to restriction-digested donor DNA (Fig. 21.3). The recombinant molecules can be introduced into E. coli by transformation. Once in the bacteria, the donor DNA insert is amplified along with the lambda/phage DNA and packaged into a new generation of virus particles, which are released when the cell is lysed. The released particles infect new cells. This results into the occurrence of a clear spot (or plaque) in the bacterial plate at the site of infection. Each plaque harbors millions of large particles, each carrying a single copy of same donor DNA insert.

B. 1d. Vectors for Larger DNA Inserts:

The maximum size of the donor DNA that can be inserted into a standard plasmid or vector is about 30 kb in length. To meet these demand, several vectors have been engineered. The largest prokaryotic inserts use the BAC (bacterial artificial chromosome) vector system. It is based on the 7-kb F plasmid and has the ability to accept larger DNA inserts (up to about 300 kb).

For inserts larger than 300 kb, YAC (yeast artificial chromosome) — a eukaryotic vector system based on yeast chromosomes introduced into yeast cells by transformation, is being used to clone recombinant molecules as large as 1,000 kb in length.

B. 1e. Formation of a DNA Library:

DNA cloning is frequently used to produce DNA libraries, which are collections of cloned DNA fragments. There exists two basic types of DNA libraries: genomic libraries and cDNA libraries. Genomic libraries are produced from total DNA obtained from nuclei and contain all of the DNA sequences of the species.

Once a genomic library of a species is created, scientists can use the library to isolate specific DNA segments. cDNA libraries are derived from DNA copies of mRNA population. cDNA libraries are produced from mRNA present in a particular cell type and this corresponds to the genes that are active in that type of cell.

B. 1f. Identification of DNA Molecules of Interest:

Immediate after cloning, the next task is to find that particular clone which contains the desired gene. This has been done either by using a specific probes (for finding DNA or protein) or by probing a specific nucleic acid.

B. 1g. Use of Recombinant DNA Technology for Genetic Engineering:

The use of sophisticated recombinant DNA techniques to alter the genotype and phenotype of an organism is called genetic engineering. This is being done by introducing genes into eukaryotic cells, where they are transcribed and translated. There are number of strategies to achieve this. The most frequently used technique is the viral-mediated gene transfer, which is termed transduction.

Here the engineered DNA is incorporated into the genome of a non-replicating virus and allow the virus to infect the cell. It is the type of virus that would determine whether gene of interest can be expressed temporarily or integrated into the genome of the host cells. Retroviruses have been used in gene therapy to transfer a normal gene into the cells of a patient having a defective gene. Transfection is a process by which naked DNA can be introduced into cultured cells.

During the process the cells are treated with either calcium phosphate or DEAE-dextran, both of which form a complex with the added DNA that promotes its adherence to the cell. It is observed that only a few cells take up the DNA and incorporates it stably into the chromosomes to make a transgenic eukaryote (Fig. 21.4). Electroporation and lipofection—the two other methods have also been used to transfect cells.

In lipofection, foreign DNA binds to positively charged lipids (liposomes) which are capable of fusing with lipid bilayer of the cell membrane and supplying the DNA to the cytoplasm. In electroporation, foreign DNA finds their way through the modified plasma membrane, induced by electric current, into the nucleus and become integrated into the genome. Foreign DNA can also be introduced into a cell by microinjecting directly into the cell nucleus.

For a long time Xenopus oocytes have been used to study the expression of foreign genes. It contains all ingredients for mRNA synthesis. When foreign DNA is delivered into the nucleus, it initiates transcription and eventually m-RNA is transported to the cytoplasm, where they are translated into proteins that can be detected immunologically.

Another important target for injected DNA is the nucleus of a mouse embryo. Here the foreign DNA become integrated into the egg’s chromosome, which will pass on to all the cells of the embryo and finally to the adult. Those animals that have been genetically engineered are called transgenic animals.

The first transgenic animal was created by Ralph Brinster of the University of Pennsylvania and Richar Palmiter of the University of Washington in the year 1981. They succeeded in introducing a gene for rat growth hormone (GH) into the fertilized eggs of mice. The injected DNA was constructed in such a way that the rat GH gene (coding protein) is located downstream from the promoter region of the mouse metallothionein gene.

In a normal rat, the synthesis of metallothionein gene is accelerated following the treatments of metals like cadmium, zinc, or glucocorticoid hormones. In the transgenic mice, synthesis of the GH gene has seen to have enhanced following treatment with metals and glucocorticoids. Mice are the most important models for mammalian genetics. The technology developed in mice is applicable to human.

There are two strategies for transgenesis in mice: ectopic insertion and gene targeting. The procedure involved in ectopic insertion is simply to inject bacterially cloned DNA solutions into the nucleus of early-stage embryos, which has been discussed earlier. Gene targeting is a bit rare event and a multi-step process is needed involving the use of embryonic stem cells. Embryonic stem cells (ES) have the ability to form any and all parts of a mouse, that is why they are called totipotent cells.

The process of gene targeting can be recognized by one of its outputs, the substitution of a nonfunctional gene for the normal gene. This type of targeted inactivation is called a gene knockout. Here embryonic stem cells are isolated from a mouse strain (albino) and embryos are transfected with a DNA insert containing an inactive, mutant allele of the gene to be knocked out.

The ES cells are then injected into the recipient young mouse embryo (blastocoel) collected from an albino strain. In the next step, the embryo containing the ES cells grows to term in surrogate mother. The resulting progeny are chimeric, having tissue derived from both the donor (transplanted ES) and recipient strains. Chimeric mice are then mated with their sibling to produce homozygous mice with the knock-out in each copy of the gene (Fig. 21.5). These are knock-out mice that lack a functional copy of the gene.

The term ‘cloning’ usually refer to three different procedures. The three types of cloning are: embryo cloning, adult DNA cloning, and therapeutic cloning.

B. 2. Embryo Cloning:

It might be more accurately called ‘artificial twining’ because it stimulates the mechanism by which twins naturally develop. It involves removing one or more cells from an embryo and encouraging a cell to develop into a separate embryo with the same DNA as the original.

It has been successfully carried out for years on many species of animals including cattle, sheep, pigs, goats, horses, moles, cats, rats and mice. The technology of embryo cloning has been done in two ways: nuclear transfer and embryonic stem cell. Cloning of embryos has been used in mice since the late 1970s and animal breeding since 1980s.

B. 2a. Nuclear Transfer:

The technique involves culturing somatic cells from an appropriate tissue (preferably fibroblast) from the animals to be cloned. Nuclei from the cultured somatic cells are then micro-injected into an enucleated oocyte obtained from another individual in same or closely related species.

Through a process that is not yet understood, the nucleus from the somatic cells is reprogrammed to a pattern of gene expression suitable for directing normal development of the embryo. After further culture and development in vitro, the embryos are transferred to a recipient female, and, ultimately, would result in the birth of live offspring.

The success rate of producing animals by nuclear transfer is less than 10% and depends on many factors including the species involve, source of recipient ova, cell type of donor nuclei, treatment of donor cells prior to nuclei transfer and techniques used for nuclear transfer.

The first publicly announced human cloning was done by Robert J. Stillman and his team at the George Washington Medical Centre in Washington D.C. They took 17 genetically defective human embryos. These embryos were derived from ovum that had been fertilized by two sperms. This has resulted into an extra set of chromosomes which ruined the ovum’s fate. None could have developed into a fetus. These ovum were successfully split in 1994, each producing one or more clones.

B. 3. Adult DNA Cloning (Reproductive Cloning):

This technique is used to produce a duplicate of an existing animal. It has been used to clone a sheep and other mammals. Reproductive cloning was earlier thought to be impossible in all mammals until it was activated in 1996 by a scientist, Dr. Ian Wialmut of the Roslin Institute in Roslin, Scotland, U. K. “Dolly”, a seven-month old sheep, was displayed to the media on February 23 rd , 1997. She is the first large cloned animal using DNA from another adult.

A cell was taken from the mammary tissue of a mature six-year old sheep while its DNA was in dormant state. It was fused with sheep ovum which had its nucleus removed. The ‘fertilized’ cell was then stimulated with an electrical pulse. Out of 277 attempts at cell fusion, only 29 began to divide. These were all implanted in ewes, 13 became pregnant but only one lamb, Dolly, was borne. On March 4, 1997, President Clinton ordered a widespread ban on the Federal funding on human cloning in the USA. Research on human cloning continues in other countries.

Over the past 25 years tremendous advances have been made in the analysis of eukaryotic genomes. This progress began as molecular biologists learned to construct recombinant DNA molecules, which are molecules containing DNA sequences derived from more than one source.

It involves the isolation from the genome of a particular segment of DNA that codes for a particular polypeptide, isolation of enzymes which would cut DNA at precisely defined locations (restriction endonucleases) and those which would covalently join DNA fragments (ligases).

The Main Steps of DNA Cloning are given in (Fig. 21.6) and Involve the Following Procedures:

1. Isolation of the DNA to be cloned.

2. Insertion of the isolated DNA into another piece Of DNA called a vector, which is a vehicle for carrying foreign DNA into suitable host cell such as the bacterium E. coli. Although (Fig. 21.2). shows the use of a plasmid vector, other types of vector, can also be used such as the Bacteriophage lambda.

3. Transfer of the recombinant vectors in bacterial cells either by transformations by infection using viruses.

4. Selection of those cells which contain the desired recombinant vectors.

5. Growth of the bacteria to give as much cloned DNA as is needed.

B. 3a. Isolation of DNA to be Cloned:

The organism under study, which will be used to donate DNA for the analysis, is called the donor organism. Three sources of DNA segment can be considered: genomic DNA, complementary DNA and chemically synthesized DNA.

These DNA sequences exist in the chromosomes of the organism under study and thus are the most easy source of DNA.

Complementary DNA (cDNA):

Complementary DNA or cDNA, is essentially a double-stranded DNA version of an mRNA molecule. cDNA is made from mRNA with the use of a special enzyme called reverse transcriptase, originally isolated from retroviruses. With the use of an mRNA molecule as a template, reverse transcriptase synthesizes a single-stranded DNA molecule that can be used as a template for double- stranded DNA synthesis. Synthesis of cDNA is very important in the analysis of gene structure and gene expression (Fig. 21.6).

Chemically Synthesized DNA:

Techniques for the chemical synthesis of oligonucleotides have been developed to produce DNA sequence for the purpose of recombinant DNA.

B. 3b. Construction of Recombinant DNA:

Isolated DNA molecules are broken into small fragments by enzymatically digesting it with endonucleases (restriction enzymes) that cleave at specific sites. Restriction enzymes cut at specific DNA target sequences (involving 4 to 8 nucleotides) and this property is one of the key features that make them suitable for DNA manipulation.

It is purely, by chance, any DNA molecule, be it derived from viruses, bacteria, plant and animal, contains restriction enzyme target sites. Thus, in the presence of appropriate restriction enzyme, the DNA would be cut into a set of small fragments according to the location of the restriction sites.

The Restriction Enzyme EcoR1 (from E. coli) Recognizes the following 6-Nucleotide-Pair Sequence in the DNA of Any Organism :

This type of segment is called a DNA palindrome, which means that both strands have the same nucleotide sequence but in antiparallel orientation. EcoR1 recognizes and cuts only within the GAATTC palindrome sequence.

The Cuts are Between The G and The A Nucleotides on Each Strand of the Palindrome:

This staggered cuts leave a pair of identical five-base long single stranded ‘sticky ends’. The ends are called sticky because, being single stranded, they can be hybridized through base-pair hydrogen bonding to a complementary copy. The production of this sticky ends is another feature of many restriction enzymes that makes them suitable tools for recombinant DNA technology.

If two DNA molecules are cut with the same sticky end-producing restriction enzyme, the fragments of each will have the same complementary sticky ends, enabling them to hybridize with each other under the appropriate conditions in a test tube. (Fig. 21.7). illustrates the restriction enzyme making a single cut in the circular DNA molecule such as a plasmid (vector) the cut opens up the circle, and the resulting a liner molecule has two sticky ends.

If such a molecule is mixed with a different DNA molecule (donor DNA fragment containing insulin—gene cut with EcoR1, as shown in (Fig. 21.7), the two can then hybridize to each other through complementary sticky- ends to form a recombinant molecule. There are two reasons why the donor DNA containing insulin gene must be attached to plasmid DNA segments to form useful recombinant molecules.

First, a fragmented donor DNA segment does not have a necessary DNA sequences, on its own, to enable it to be replicated in a test tube or inside a host organism. The donor DNA must be physically attached to other DNA segments that can support replication in a test tube or inside a host cell. Second, an experiment may demand that multiple fragments be glued together to form a functional unit (e.g. a transcriptionally active gene).

Most commonly, both donor and plasmid DNA are digested together in the presence of DNA ligase. During the incubation, two types of DNAs become hydrogen bonded to one another by their sticky ends and then ligated to form circular DNA recombinants.

B. 4. Therapeutic Cloning:

This is a procedure whose initial stages are identical to adult DNA cloning. However, the stem cells are removed from the pre-embryo with the interest of producing tissue or a whole organ for transplant back into the person who supplied the DNA. The pre-embryo dies in the process. The goal of therapeutic cloning is to produce a health copy of a sick person’s tissue or organ for transplant.

The tissue or organ, would have the sick persons original DNA and, therefore, the patient need not have to take immunosuppressive drugs for the rest of life. Scientists are attempting to create transgenic pigs which have human genes. Their heart, liver or kidneys might be used as organ transplants in humans. This will save many lives because thousand of people die each year waiting for human organs.

Once achieved, transgenic animals could be cloned to produce as many organs as are needed. Researchers have produced transgenic animals typically in order to produce human hormones or proteins in its milk. These substances can be separated from the milk and be used for treating human ailments.

Gene therapy is a technique for correcting genes responsible for disease development. The promise of gene therapy was evident from the first trials initiated in 1989. The disease addressed was adenosine deaminase (ADA) deficiency, a rare autosomal recessive disorder in which the body’s lack of a housekeeping enzyme is harmful in the immune system.

There, the absence of ADA results in premature death of T lymphocytes, leading to an immunodeficiency state (SCID – severe combined immunodeficiency). At the National Institute of Health, USA investigators transferred a normal ADA gene into the lymphocytes of children born with the disorder.

In later trials, the gene was put into bone marrow cells. In both strategies, the manipulation was ex vivo. Patient was injected with her own blood cells transfected with an wild type ADA gene. Parallely she was given adenosin deaminase obtained from cattle blood. Although long-term clinical benefits have yet to be appreciated, these early efforts served to establish the potential of gene delivery as a therapeutic strategy (Fig. 21.8).

Researchers do Use One of Several Approaches for Correcting Defective Genes:

a. A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most commonly practised.

b. An abnormal gene could be swapped for a normal gene through homologous recombination.

c. The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.

d. The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

In most gene therapy studies, a “normal” gene is inserted into the genome to replace an “abnormal”, disease-causing gene. A vector must be used to deliver the therapeutic gene to the patient’s target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their gene to human cells in a pathogenic manner. Scientists have tried to advantage this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes.

Target cells such as the patient’s liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into
the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

There are Number of Gene Therapy Vectors. Some of the Vectors are:

A class of viruses that can create double-stranded DNA copies of their RNA genome. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus.

A class of viruses with double-stranded DNA genome that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus.

Adeno-Associated Viruses:

A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19.

Herpes Simplex Viruses:

A class of double-stranded DNA viruses that infect a particular cell type, neurons, Herpes simplex virus type is a common human pathogen that causes cold sores.

A gene is given a more or less artificial carrier in some cases, a lipid encapsulation, so as to facilitate passage across cell membrane in others, a tiny gold bead on which the gene is plated so it can be shot into the skin in still others, a protein to which the gene is attached.

Requirements of Gene Therapy:

The gene (for example ADA gene) is identified and cloned. It is then inserted into patients own cells (ex vivo), treating them in tissue culture and returning them to the patient. It must be inserted into DNA, transcribed and translated with sufficient efficiency so that worthwhile amount of the enzyme is produced.

Retroviruses are used as the gene vector. Retroviruses have several advantages for introducing genes into human cells – their envelop protein enables the virus to infect human cells. RNA copies of the human ADA gene can be incorporated into the retroviral genome using a packaging cell.

Packaging cells express (i) an RNA copy of the human ADA gene along with a packaging signal (P) needed for the assembly of fresh virus particles and inverted repeats (R) at each end to aid insertion of the DNA copies into the DNA of the target cells,

(ii) an RNA copy of the retroviral gag, pol and env genes but with no packaging signal so that then genes cannot be incorporated in fresh viral particles.

Treated with these Two Genomes, the Packaging Cell Produces a Crop of Retroviruses with:

(i) The envelope protein needed to infect the human target cell,

(ii) An RNA copy of the human ADA gene, complete with R sequences at each end

(iii) Reverse transcriptase needed to make a copy of the ADA gene that can be inserted into the DNA of the target cell,

(iv) None of the genes (gag, pol, env) that would enable the virus to replicate in its new host. Once the virus has infected the target cells, this RNA is reverse transcribed into DNA and inserted into the chromosomal DNA of the host.

Besides, there are several other options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amount of DNA.

Current Status of Gene Therapy Research:

The Food and DRUG Administration (FDA) has not yet approved any human gene therapy for sale. Current gene therapy is experimental and has not proven very successful in clinical trials. Little progress has been made since the first gene therapy clinical trial began in 1990.

In 1999, gene therapy suffered a major setback with the death of 18-year-old Jesse Gelsinger. Jesse was participating in a gene therapy trial for ornithine transcarboxylase deficiency (OTCD). He died from multiple organ failures 4 days after starting the treatment. His death is believed to have been triggered by a severe immune response to the adenovirus.

Another major blow came in January 2003, when the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. FDA took this action after it learned that a second child treated in a French gene therapy trial had developed a leukemia like condition. Both the child and another, who had developed a similar condition in August 2002, had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID), also known as “bubble baby syndrome”.

FDA’s Biological Response Modifiers Advisory Committee (BRMAC) met at the end of February 2003 to discuss possible measures that could allow a number of retroviral gene therapy trials for treatment of life-threatening diseases to proceed with appropriate safeguards. FDA has yet to make a decision based on the discussions and advice of the BRMAC meeting.

B. 4b. Problems with Gene Therapy:

Short-lived nature of gene therapy—before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.

Anytime any foreign body is introduced into human tissues, the immune system is designated to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Further, the enhanced immune response to invaders makes it difficult for gene therapy to be repeated in patients.

Problems with Viral Vectors:

Viruses, the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient such as toxicity, immune and inflammatory responses. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders such as heart disease, high blood pressure, Alzheimer’s disease, arthritis, and diabetes are caused by the combined effects of variations in many genes. Multi-genic or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.

C. Uncertainties with Animal Biotechnology:

As with new technologies, animal biotechnology faces a variety of uncertainties, safety issues and potential risks. For example, concerns have been raised regarding: the use of unnecessary genes in constructs used to generate transgenic animals, the use of vectors with the potential to be transferred or to otherwise contribute sequences to other organisms, the potential effects of genetically modified animals on the environment, the effects of biotechnology on the welfare of the animal, and potential human health and food safety concerns for meat or animal products derived from animal biotechnology. Before animal biotechnology will be used widely by animal agriculture production systems, additional research will be needed to determine if the benefits of animal biotechnology outweigh these potential risks.


PCR Technique: Step By Step

7. DNA Ladder "Unzips" Into Two Separate Strands

D NA polymerase and a mixture of all four nucleotides are added to a test tube containing the extracted DNA sample. When the double-stranded parental (template) DNA is heated to 95 degrees Celsius, the individual strands unwind and separate from each other. The objective is to replicate the section of each strand containing the target gene using the enzyme DNA polymerase. Each single parental strand of DNA has the remarkable property of rebuilding the missing complementary strand as nucleotides attach in the 5 prime (5') to 3 prime (3') direction. Each newly-formed complementary strand (one for each parental strand) is called a "daughter strand."

When the double-stranded, parental DNA molecule (DNA ladder) is heated to 95 o C, the two individual strands separate from each other. DNA polymerase facilitates the attachment of the complementary nucleotides to rebuild each strand, resulting in two double-stranded molecules. P = phosphate, D = deoxyribose, A = adenine, T = thymine, G = guanine, and C = cytosine. The extended phosphate "tail" represents the 5' position of each strand.

When the double-stranded, parental DNA molecule (DNA ladder) is heated to 95 o C, the two individual strands separate from each other. DNA polymerase facilitates the attachment of the complementary nucleotides to rebuild each strand, resulting in two double-stranded molecules. The pink section represents the actual target gene that will be replicated.

8. Primer Attaches To One End Of DNA Strand

I n order for DNA polymerase to find the start of a specific target gene in each section of DNA, a short segment of DNA called a primer must be attached (annealed) to each "mother" DNA strand upstream (toward 3' end) from each gene. The primer does not overlap the target gene, because it is complementary to the base sequence that appears just before the gene on the mother strand of DNA. The complementary "daughter" strand is produced in the 5' to 3' direction. Primers contain about 20 bases and they have been synthesized for many of the genes that are commonly amplified using the PCR technique. They may be purchased from biotechnology supply companies. The primer for a specific gene is added to the mixture of single-stranded DNA after it has cooled down to 52-54 o C (126-129 o F).

Short sections (oligonucleotides) called primers attach upstream from each gene (toward 3' end of parental "mother" strand). Now DNA polymerase can recognize the start of the gene and rebuild the complementary strand in the 5' to 3' direction.

A s the double-stranded, parental (template) DNA ladder unzips and nucleotides attach to each of the two single parental strands, something very interesting happens. One daughter strand, called the "leading strand," forms continuously as nucleotides attach in the 5' to 3' direction. But in the other daughter strand, called the "lagging strand," the nucleotides attach in discontinuous sections. These sections are called Okazaki fragments, named after the Japanese scientist Reiji Okazaki who discovered them. Since the lagging strand is complementary to the leading strand, its 3' end is opposite the leading strand's 5' end, and vice versa. The only way this strand can lengthen in the 5' to 3' direction as the parental DNA molecule unzips, is is for it to grow in sections or fragments. This remarkable discovery is shown in the following illustration.

When the parental (template) DNA strands replicate, the daughter strands are synthesized in two different ways. The leading strand is formed continuously as single nucleotides attach one-by-one in a 5' to 3' direction. The lagging strand is formed discontinuously as preformed sections of nucleotides (called Okazaki fragments) attach in a 5' to 3' direction.

9. Telomeres: A Major Molecular Fix To The End Replication Problem

T he structure and function of DNA are certainly two of the most significant discoveries that have revolutionized the science of biology. Even though DNA appears to be a perfect storage molecule for genetic information, it has a serious replication problem. Chromosomes of eukaryotic cells are composed of linear DNA. In order for cell division to take place, the DNA molecule must replicate. In other words, the single chromosome must become a doubled chromosome composed of two chromatids. The problem is that each time DNA replicates the new molecules get slightly shorter. After a number of consecutive divisions, this degradation could result is serious gene loss at the ends of the chromosomes. Both Alexey Olovnikov and James Watson independently described this phenomenon called "end replication problem" in the early 1970s. In fact, Olovnikov's "A Theory of Marginotomy" predicted that the loss of terminal sequences resulting from end replication problem would lead to senescence (Olovnikov, 1973). [James Watson and Francis Crick discovered the structure of DNA in 1953 and received the Nobel Prize in Medicine in 1962.]

T o cope with the devastating end problem replication problem, eukaryotic cells have evolved protective "caps" on the ends of chromosomes called telomeres. For their discovery of how chromosomes are protected by telomeres and the enzyme telomerase, Elizabeth Blackburn, Jack Szostak and Carol Greider were awarded the Nobel Prize in Medicine in 2009. With their ingenious genetic research and meticulous biochemical studies, they not only solved a fundamental problem in biology but also opened a new field of research and initiated the development of potential therapies against the aging process and cancer. It should be noted here that more than 60 years earlier, Barbara McClintock was studying telomeres in corn. In the early 1940s she turned her attention to the study of transposable elements (transposons) in corn, another remarkable genetic phenomenon with important medical implications in people. For her lifelong research on transposons, she received the Nobel Prize in Medicine in 1983.

T elomeres are repetitive strands of DNA (sequences of repetitive bases) at the terminal ends of linear chromosomes. They play an essential role in maintaining the integrity of the chromosome by protecting it from degradation and from end-to-end fusion with other chromosomes. Telomeres are essentially protective "end caps" of non-coding DNA at the extreme ends of chromosomes. Telomeres have been metaphorically compared with the tips of shoelaces that keep the laces from unraveling. Each time a cell divides, the telomeres lose a small amount of DNA. Eventually, when all of the telomere DNA is gone, the cell can no longer divide and dies. End replication problem is not an issue in prokaryotic cells because they have circular DNA molecules without ends.

T he number of times a population of normal cells can divide is called the Hayflick limit, named after its discoverer Leonard Hayflick. In 1961, Hayflick demonstrated that normal human fetal cells in a culture divide between 40 and 60 times. It is now clear that cell division occurs until the telomeres reach a critical length. An estimated length for human telomeres ranges from 8,000 base pairs at birth to 3,000 as people age, and as low as 1,500 in elderly people. Starting with 8,000 base pairs, a loss of 100 to 200 with each division would completely erode away the telomeres in 40 to 80 divisions.

1. When DNA Unzips, DNA Polymerase Must Attach To 3' End of Mother Strand.
2. It Must Add Nucleotides (Synthesize Daughter Strand) In The 5' to 3' Direction.
3. An RNA Primer Must Attach First To Give DNA Polymerase A Place To Start.

C hromosome duplication starts with the unzipping of the double stranded DNA into two strands (mother strand #1 & mother strand #2). These complementary mother strands serve as templates to build two DNA molecules. An RNA primer attaches to the 3' end of mother strand #1, thus giving DNA polymerase a place to start. The primer attaches just before the initial attachment of DNA polymerase. DNA polymerase moves in the 3' to 5' direction along mother strand #1, adding nucleotides to form a continuous complementary daughter strand of DNA the entire length of the mother strand #1 template. This complementary strand is called the "leading strand" and it is synthesized in the 5' to 3' direction. [5' and 3' refer to specific carbon atoms of deoxyribose sugar in DNA building blocks called nucleotides.] When 5' and 3' directions are mentioned, it is important to specify whether you are referring to the mother strand or the complementary daughter strand.

A s the original mother DNA unzips, DNA polymerase cannot attach to the top of mother strand #2 at the 5' position (top right in following diagram). Even if it could attach to the top of mother strand #2 at the 5' position, it could not move down the mother strand and synthesize a daughter strand in the 3' to 5' direction. Therefore, DNA polymerase attaches farther down on mother strand #2 and produces a series of DNA sections in the 5' to 3' direction. These sections are named "Okazaki fragments" after the Japanese scientist Reiji Okazaki who discovered them. The sections collectively form a daughter strand called the "lagging strand" to the top of mother strand #2. This is nicely explained by R. Ohki, T. Tsurimoto and F. Ishikawa ( Molecular and Cellular Biology Vol. 21, 2001). Short RNA primers must attach ahead of each DNA section in order to form a starting point for DNA polymerase.

T here is a problem at the 3' end of mother strand #2. When the last RNA primer reaches this end, there is no more DNA template for it to keep ahead of DNA polymerase. The last primer attaches to the 3' end, but DNA polymerase cannot add the last section of the lagging strand, leaving a gap where the primer was attached. Therefore, the 5' end of each newly synthesized lagging strand is cut short. About 100 base pairs are shaved off with each round of replication, thus shortening the telomere. In the following diagram, mother strand #2 has a gap at the 5' end of the newly formed lagging strand.

The following animated gifs show replication of DNA in six consecutive divisions without any shortening, compared with the end replication problem on lagging strand and the gradual shortening of DNA.

T elomeres can be restored by the enzyme telomerase. This enzyme lengthens telomeres in germ cells (cells that produce eggs and sperm), thus restoring telomeres to their maximum length in the zygote. It is also present in other cells that must continually divide, including bone marrow stem cells that produce large numbers of generations of red blood cells necessary to sustain life, the epithelium of skin, and cells lining the intestine. Telomerase is generally not active in normal somatic cells. This enzyme adds noncoding DNA sequence repeats TTAGGG in vertebrates to the 3' end of DNA strands in the telomere region of eukaryotic chromosomes. The presence of active telomerase in cancer cells may be useful in the diagnosis and treatment of some cancers with telomerase inhibitors.

T he following paragraph comes from Science and Technology (9 November 2007): Sharks have telomerase in all of their cells. Their telomeres don't shorten and sharks do not have a genetically programmed life span like humans. In fact, sharks keep growing throughout their life. The limit to their life span is the fact that they must keep moving in order to circulate air through their gills for the uptake of oxygen. Sharks are exceptionally genetically stable, having changed very little in hundreds of millions of years. In addition, sharks rarely get cancer.

T elomeres and telomerase also occur in plant cells. Plant telomere biology is summarized by T.D. McKnight and D.E. Shippen in The Plant Cell Vol. 16: 794-803 (2004). In most flowering plants, telomeres consists of the DNA base repeats TTTAGGG. Like the somatic cells of animals, there is little or no active telomerase in vegetative tissue, although it is reactivated during flowering, probably to ensure that gametes and embryos inherit telomeres restored to their maximum length. Like cancer cells in in animals, telomerase is fully functional in cells of plant tissue cultures, as might be expected for cells with an unlimited capacity for proliferation. The monocot order Asparagales that contains about 27,000 species (roughly10 percent of all angiosperms) has 6-base repeats of TTAGGG, the same sequence found in mammalian telomeres. This order includes many familiar plant families, such as orchids, iris, amaryllis, agave, onion and asparagus. Since plants and mammals evolved into multicellular organisms along completely separate pathways, this appears to be yet another example of parallel evolution (homoplasy).

T elomeres do not prevent the shortening of DNA, they just postpone the erosion process. The telomere shortening mechanism normally limits cells to a fixed number of divisions. Eventually, when all of the telomere is gone, the cell can no longer divide, thus terminating the cell cycle. Most cancer's are the result of "immortal" cells which have evaded programmed cellular death due to erosion of telomeres. Chromosomes of malignant cells usually do not lose their telomeres, thus resulting in uncontrolled cell division. Animal studies suggest that telomere length may be related to the aging process on the cellular level and the life span of animals. There are even studies suggesting that regular exercise and stress reduction may help to minimize telomere erosion. In fact, a study published in the May 3, 2005 issue of the American Heart Association journal Circulation found that weight gain and increased insulin resistance were correlated with greater telomere shortening over time.

I t is interesting to speculate on the origin of telomeres. If another version of DNA polymerase existed that attached to the 5' end of mother strand #2 and added nucleotides in the 3' to 5' direction, then theoretically a continuous strand could be synthesized to the end of the mother strand template without the end replication problem. This theoretical version has never been found and therefore telomeres are essential to prevent the gradual shortening of DNA and erosion of genes. Why is there only one form of DNA polymerase that synthesizes daughter strands in the 5' to 3' direction? This is like asking why living systems only have L-form (left handed) amino acids and D-form (right handed) sugars. Did the evolution of telomeres solve a replication problem inherent in the original DNA, or were telomeres present in the original DNA of the first eukaryotic cells?

Evolutionary Significance of End Problem Replication & Telomeres

W hen I first wrote this section about end replication problem, I concluded that it was a defect in DNA that literally shortened the life of a cell by limiting the number of divisions. Telomeres serve as mitotic time clocks that prolong life by a certain number of consecutive erosions. However, there is another side to this story where limiting the life span of organisms could actually be beneficial. In a rapidly changing environment, survival of a species depends on genetic variability through DNA mutations and the ability to pass these genes on to future generations. A species with exceedingly long generation times may not be able to compete because adaptive mutations can't keep up with environmental changes however, longer generation spans could also slow population growth as long as fecundity (number of offspring per female) remains constant. To an individual, immortality may seem good however, this may not be good for the species. This logic is mentioned in Star Trek 2: "The Wrath of Khan" when Spock said: "The good of the many outweighs the good of the few, or the one." Actually, this logic is mentioned two thousand years earlier in John 11:49-50. Of course, one caveat to the benefit of end replication loss is the shark, which apparently has active telomerase in all of its cells and telomeres lengths that don't decline significantly with age. Sharks (class Chondrichthyes) are a very successful group and they have been around for more than 200 million years. In fact, some species have age estimations of 100 years or more. Undoubtedly, environmental changes in the ocean have not been as rampant as on land.

10. Single DNA Strands Replicate Into Doubled Strands

T he DNA mixture is heated to 72 o C (162 o F) and DNA polymerase recognizes the primer annealed to each strand and proceeds to synthesize the complementary strand all the way down the gene. Nucleotides attach along the gene from all the adenines, thymines, cytosines and guanines that are already in the mixture. Now the mixture contains two identical copies of the gene (two complete DNA ladders). DNA polymerase from the bacterium Thermus aquaticus (called TAQ polymerase) is used for the reaction because it is immune to the high temperatures. Unlike most protein enzymes that are destroyed at temperatures above 40 o C (104 o F), DNA polymerase from Thermus aquaticus can survive the 72 o C of the reaction. In fact, this bacterium normally lives in hot springs and can survive temperatures approaching the boiling point of water.

Bacteria Of Boiling Hot Springs In Yellowstone National Park

B oiling hot springs in Yellowstone National Park are colored by colonies of thermophilic cyanobacteria, eubacteria and archaebacteria. Orange-colored cyanobacteria generally occur in water that has cooled below 73 o C (163 o F). The green chlorophylls in these photosynthetic bacteria are masked by orange carotenoid pigments. Like the bright red halobacteria of salt lakes, carotenoids protect the delicate cells from intense solar radiation, especially during the summer months. Warmer, whitish areas of the ponds contain stringy masses of nonphotosynthetic eubacteria. Thermus aquaticus survives in temperatures too high for photosynthetic bacteria, up to 80 o C (176 o F). Thermus aquaticus is heterotrophic and survives on minute amounts of organic matter in the water. TAQ polymerase used in the amplification of DNA using the polymerase chain reaction (PCR) was originally isolated from a colony of T. aquaticus collected in a hot spring at Yellowstone National Park.

A boiling hot springs in Yellowstone National Park. The orange-red coloration is caused by dense colonies of photosynthetic cyanobacteria.

A rchaebacteria thrive in boiling water at Yellowstone National Park, at temperatures of 92 o C (198 o F). These bacteria also thrive near steam vents at the bottom of the ocean at temperatures exceeding 115 o C (239 o F). Scientists from throughout the world are studying the amazing bacteria flora at Yellowstone National Park. This is one of the best places on earth to study these organisms in their natural protected habitats. In other parts of the world, similar hot springs have been destoyed for the production of geothermal energy.

Boiling hot springs in Yellowstone National Park. The orange-red coloration is caused by thriving colonies of photosynthetic cyanobacteria. Stringy masses of nonphotosynthetic eubacteria occur in the whitish areas of warmer water.

A cid hot springs in Yellowstone National Park with a pH of below 4.0 support the eukaryotic alga Cyanidium caldarum . This remarkable photosynthetic alga can even survive in a pH of zero! Some acidophilic hot springs bacteria utilize the oxidation of sulfur and iron for the synthesis of ATP. Alkaline hot springs support colonies of bacteria that utilize hydrogen sulfide for their energy source.

L ife as we know it may have first arisen more than three billion years ago in a high temperature environment of boiling water. Thermophilic bacteria in hot springs of Yellowstone National Park may be relict populations of the first life on earth. In fact, these thermophilic bacteria may be the ancestors of all other life forms, including humans!

11. Two DNA Ladders "Unzip" Into Four Separate Strands

N ow the mixture is once again heated to 95 o C and the double-stranded DNA molecules containing the target genes separate into single strands. But now there are four single strands from two double-stranded genes. The mixture is once again cooled to 52-54 o C and the primers anneal to the strands at start positions before each gene. DNA polymerase once again catalyzes the rebuilding of each single strand into four complete double-stranded genes. PCR is called polymerase chain reaction because the reaction occurs repeatedly in cycles as duplicate copies of genes are produced exponentially. After only 40 cycles there would be 1.0995116 X 10 12 or more than one trillion copies of the original gene!

The two double-stranded DNA molecules (DNA ladders) separate into four strands. DNA polymerase will rebuild the complementary strand for each ladder, resulting in four double-stranded DNA molecules. Note: This illustration does not show the end replication problem where DNA shortens during repetitive divisions.

12. Using Lice DNA To Date The First Clothing Worn By People

O ne of the most novel uses for DNA sequencing is the determination of when humans first began wearing clothing. According to Mark Stoneking and his colleagues at the Max Planck Institiute for Evolutionary Anthropology in Leipzig, Germany, we started wearing clothing about 70,000 years ago. This date is based on genes of human sucking lice. It correlates with the approximate time when the body louse evolved from the human head louse and corresponds to the time when the body louse's habitat (clothing) became widespread. This is also the time when Homo sapiens sapiens began moving out of Africa into cooler regions of Europe.


Human head louse
S ucking lice belong to the wingless, parasitic insect order Anoplura. Human sucking lice include body lice, crab lice and head lice ( Pediculus humanus ). Anoplurans use a set of long hypodermic-like stylets to pierce the skin and withdraw blood. After ingesting blood their body becomes swollen and shows a dark clot of blood in their abdomen. There are two forms of human sucking lice, the head louse ( P. humanus capitis ) and the body louse ( P. humanus humanus ). The head louse infests the hair of the scalp and the body louse lives in clothing near the body surface. Human lice are also known as "cooties" and their eggs attached to hairs are called "nits." Human lice cause local itching, but the discomfort is minor compared with the misery of the bacterium they can transmit called Rickettsia prowazeki . This minute bacterium causes "Epidemic Typhus," a serious disease that has devastated populations in medieval Europe.

S toneking and his colleagues Ralf Kittler and Manfred Kayser compared mitochondrial DNA sequences from head and body lice. The greater the difference in sequences between the two forms of lice, the older their evolutionary split. Human lice from Africa are more genetically diverse than lice from other parts of the world, indicating that the species originated in Africa. Head lice are more diverse than body lice, showing that they are the older group. By comparing the mitochondrial DNA of body lice to chimpanzee lice, Stoneking's team was able to approximate the origin of body lice to around 70,000 years ago. This date correlates well with the growing evidence that modern humans evolved in Africa and migrated northward around 100,000 years ago.

S toneking is also studying human crab lice ( Pthirus pubus ) which typically inhabit pubic hair. Human pubic lice are more closely related to gorilla lice than to head lice. Since this sucking louse only inhabits hairy places on the body, it might shed some light on when humans lost their heavy body hair. Crab lice are typically transferred from person to person through sexual intercourse, although they may also be picked up from infested linen, clothing and other sources.


INTRODUCTION

Polycyclic aromatic molecules are known to intercalate into double-stranded DNA ( 1, 2 ). Apart from the theoretical treatment of such host–guest interactions, the consequences of DNA intercalation by exogeneous molecules have attracted considerable interest in medicinal chemistry, because such a complex formation leads to a significant modification of the DNA structure and may result in a hindered or suppressed function of the nucleic acid in physiological processes ( 3-10 ). Because such an influence on the biological system is a main requirement for DNA-targeting drugs, the intercalation of small molecules into DNA may be applied in therapeutic approaches in which the suppression of DNA replication and gene transcription is used to destroy tumor cells or infected tissue. As a result, many studies have been performed to gain more insight in different aspects of the association process of large and small molecules with DNA in order to obtain highly selective and efficient intercalators ( 11 ).

Cationic organic dyes are usually regarded as classical intercalators. Representative examples are acridine derivatives such as proflavine ( 1 ) or acridine orange ( 12 ), azine dyes such as methylene blue ( 13 ), phenanthridinium salts such as ethidium bromide ( 14 ) or cyanine dyes such as thiazole orange (aggregates of which may also bind to the minor groove) ( 15-17 ). The association of these compounds with DNA usually influences their absorption and emission properties, so that their interaction with DNA may be evaluated qualitatively and quantitatively by spectrophotometric and spectrofluorometric titrations ( 18 ). These simple and straightforward spectroscopic methods are especially advantageous because organic dyes absorb and emit at wavelengths that do not interfere with the absorption of the DNA bases (λmax= 260 nm). Thus, spectrophotometric and spectrofluorometric titrations usually indicate the association of a dye with DNA. Moreover, the resulting binding isotherms can be used to estimate the binding constants and the binding site size, i.e. the number of occupied binding sites. Moreover, the absorption of circularly or linearly polarized light can be used in circular dichroism (CD) and linear dichroism (LD) spectroscopy to gain further knowledge of the orientation of the dye molecule relative to the DNA and to deduce the binding mode ( 19 ). Steady-state fluorescence polarization measurements as well as fluorescence energy transfer from the DNA bases to the bound dye have been used as additional reliable criteria to elucidate the binding mode ( 20 ). In addition to the spectroscopic methods, hydrodynamic and thermodynamic criteria such as the viscosity, the sedimentation coefficient, or the DNA melting temperature may be used to evaluate the binding mode ( 20 ).

It is commonly established that a positive charge enhances the propensity of a molecule to bind to DNA due to attractive ionic interactions between the cation and the phosphate backbone ( 21 ). In most cationic dyes, this positive charge is established by an exocyclic ammonium functionality or by an endocyclic pyridinium moiety. These functionalities are usually quaternized by alkylation or protonation. In the latter case, however, this leads to a significant influence of the pH on the DNA-binding properties. In contrast, cationic dyes with an endocyclic quaternary bridgehead nitrogen atom are rather rare and few systematic studies exist for this class of compounds. In this article we will summarize the studies of the DNA-binding properties of quinolizinium (Scheme 1) derivatives ( 22 ) as representative examples for dyes with a quaternary bridgehead nitrogen atom, and we will demonstrate that annelated quinolizinium derivatives may serve as a useful platform for the design of intercalating dyes.


MATERIALS AND METHODS

Materials

Nuclease P1 (from Penicillium citrinum ) was purchased from Calbiochem, EMB Biosciences, Inc. (San Diego, CA). Calf thymus (ct)-DNA, snake venom phosphodiesterase and calf spleen phosphodiesterase were obtained from Sigma Chemical Co. (St Louis, MO). Alkaline phosphatase was purchased from Roche Diagnostics Corporation (Indianapolis, IN). 2′-Deoxyadenosine-5′-triphosphate-1,3,7,9- 15 N 4 -(4-amino- 15 N) (dATP- 15 N 5 ) and 2′-deoxyguanosine-5′-triphosphate-1,3,7,9- 15 N 4 -(2-amino- 15 N) (dGTP- 15 N 5 ) were from Medical Isotopes, Inc. (Pelham, NH). (5′ S )-cdA was obtained from Berry & Associates, Inc. (Ann Arbor, MI). Acetonitrile (HPLC grade) was from Burdick and Jackson (Muskegon, MI). Biomax5 ultra filtration membranes (molecular mass cutoff of 5 kDa) were purchased from Millipore (Bedford, MA). Water (HPLC-grade) for LC/MS analyses was from J.T. Baker (Phillipsburg, NJ). Water purified through a Milli-Q system (Millipore) was used for all other applications. 2′-Deoxyadenylyl(3′→5′)-(5′ S )-8,5′-cyclo-2′-deoxyadenosine 3′-monophosphate [d(AcA)-3′-p], 2′-deoxycytidylyl(3′→5′)-(5′ S )-8,5′-cyclo-2′-deoxyadenosine 3′-monophosphate [d(CcA)-3′-p], 2′-deoxyguanylyl(3′→5′)-(5′ S )-8,5′-cyclo-2′-deoxyadenosine 3′-monophosphate [d(GcA)-3′-p] and 2′-deoxythymidylyl(3′→5′)-(5′ S )-8,5′-cyclo-2′-deoxyadenosine 3′-monophosphate [d(TcA)-3′-p], and oligodeoxynucleotides containing (5′ S )-cdA and with no end phosphate groups were synthesized as described elsewhere ( 10 , 20 ).

Irradiation of DNA

An aqueous buffered solution of DNA (10 mM phosphate buffer, pH 7.4, 0.3 mg/ml) was saturated with N 2 O and irradiated with gamma rays in a 60 Co gamma-source at a dose of 2 Gy (dose rate 24 Gy/min). Subsequently, irradiated and unirradiated DNA solutions were dialyzed against water for 18 h. Water outside the dialysis tubes was changed three times during the course of dialysis. Aliquots of 50 μg of DNA were dried in a SpeedVac under vacuum.

Isolation of DNA from pig liver

Frozen pig liver was obtained from Pel-Freeze, Inc. (Rogers, AK). Two methods of DNA isolation were used. The first method (phenol–chloroform method) was essentially as described previously ( 21 ). Briefly, tissues were homogenized in 10 vols of homogenizing buffer (1% SDS + 1 mM EDTA), then 500 μg/ml Proteinase K was added and incubated for 30 min at 37°C. The homogenates were then extracted with phenol, phenol–Sevag (chloroform/isoamyl alcohol in the ratio 24:1), and then with Sevag. The DNA was precipitated with 1 vol. of cold ethanol and dissolved in 0.01× SSC (1.5 mM NaCl, 0.15 mM sodium citrate, pH 8.0). The DNA was treated with RNase T1 (50 U/ml) and RNases A (50 μg/ml) for 30 min at 37°C, then extracted with equal volumes of Sevag. The DNA was precipitated from the aqueous layer using cold ethanol and washed with 70% ethanol. The DNA was then dissolved in 0.01× SSC at ∼1 mg/ml. We also tested the effect of adding 1.25 mM desferoximine (Sigma) to the homogenizing buffer prior to homogenization.

The second method was a high-salt extraction method as described previously ( 22 , 23 ). Frozen tissue (1 g) was homogenized in 8 ml Lysis solution (0.5 M Tris, pH 8.0, 20 mM EDTA, pH 8.0, 10 mM NaCl, 1% SDS). Proteinase K (0.5 mg/ml) was added to the mixture followed by incubation overnight at 37°C. The next day, 4 ml of saturated NaCl solution was added to the mixture, which was vortex-mixed for 1 min, and then incubated at 56°C for 15 min. The mixture was centrifuged for 30 min with 16 000 g and the supernatant was collected. After another centrifugation, the DNA in the supernatant was precipitated using 1 vol. of cold ethanol, washed with 70% ethanol and dissolved in 0.01× SSC. The DNA was then treated with RNases, extracted with Sevag, precipitated and dissolved as described above.

Enzymic hydrolysis of dinucleotides, oligodeoxynucleotides and DNA

For enzymic hydrolysis, 2 pmol of (5′ S )-cdA- 15 N 5 as an internal standard was added to 100 pmol of dinucleotides and oligodeoxynucleotides. The amounts of (5′ S )-cdA- 15 N 5 added to 50 μg of unirradiated and irradiated ct-DNA samples were 0.2 and 1 pmol, respectively. After addition of the internal standard, dinucleotides, oligodeoxynucleotides and DNA samples were dried in a SpeedVac under vacuum, and then dissolved in 50 μl of 10 mM Tris–HCl solution (pH 7.5) supplemented with 2.5 μl of 1 M sodium acetate containing 45 mM ZnCl 2 (final pH 6.0). Aliquots of nuclease P1 (5 U), snake venom phosphodiesterase (0.004 U) and alkaline phosphatase (32 U) were added and the samples were incubated at 37°C for 6, 24 or 48 h. In some instances, spleen phosphodiesterase (0.004 U) was also used. The 3′-phosphate groups of dinucleotides were removed by hydrolysis with alkaline phosphatase for 6 h in the same manner to obtain dinucleoside monophosphates d(AcA), d(CcA), d(GcA) and d(TcA). After hydrolysis, the samples were filtered using ultrafiltration membranes with a molecular mass cutoff of 5 kDa by centrifugation at 6000 g for 30 min.

To determine whether the enzymic hydrolysis conditions used in the 32 P-postlabeling studies reported previously ( 20 ) would also release (5′ S )-cdA, 100 pmol of dinucleotides or oligodeoxydinucleotides were incubated in 10 μl of a solution containing 30 mM sodium succinate (pH 6), 10 mM CaCl 2 , 500 mU micrococcal nuclease and 10 mU spleen phosphodiesterase (Worthington Biochemicals, Vineland, NJ). Digestion continued for 3.5 h at 37°C. Then, 4 μl of a solution containing 225 mM sodium acetate (pH 5.0), 0.55 mM ZnCl 2 and 5.6 μg nuclease P1 was added and incubation continued for 45 min at 37°C. The reaction was neutralized by adding 2.6 μl of 0.75 M CHES [2-(cyclohexylamino)ethanesulfonic acid] buffer (pH 9.7), and the samples were frozen on dry ice. The samples were subsequently thawed and treated with alkaline phosphatase as described above.

Analysis by LC/MS

Analysis by LC/MS with the API-ES process using isotope-dilution technique and selected-ion monitoring (SIM) was performed as described previously ( 17 ). A Zorbax Eclipse XDB C18-reversed-phase column (15 cm × 2.1 mm i.d., 5 μm particle size) (Agilent Technologies, Rockville, MD) was used. A guard column packed with the same stationary phase (1 cm × 2.1 mm i.d.) was attached to the column head. The solvent A was a mixture of water and acetonitrile (98:2, v/v) and the solvent B was 100% acetonitrile. A gradient of 0.5% of the solvent B per minute was used. The flow rate was 0.2 ml/min. The column temperature was kept at 30°C. The isotopically labeled internal standard (5′ S )-8,5′-cyclo-2′-deoxyadenosine-1,3,7,9- 15 N 4 -(4-amino- 15 N) [(5′S)-cdA- 15 N 5 ] was prepared using dATP- 15 N 5 as described previously ( 18 ). Aliquots (20 μl) of filtered enzymic hydrolysates were injected onto the LC-column without any further treatment. When necessary, the effluents were passed through a UV-spectrophotometer for monitoring of the absorbance of (5′ S )-cdA and dinucleoside monophosphates, before they were introduced into the ion chamber of the mass spectrometer. The identification and quantification of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and 8-hydroxy-2′-deoxyadenosine (8-OH-dA) were performed as described elsewhere ( 24 , 25 ). The isotopically labeled internal standards 8-OH-dG- 15 N 5 and 8-OH-dA- 15 N 5 were isolated from gamma-irradiated solutions of dGTP- 15 N 5 and dATP- 15 N 5 , respectively, using semi-preparative LC and the same procedure as described previously for the isolation of (5′ S )-cdG- 15 N 5 and (5′ S )-cdA- 15 N 5 ( 18 ).


Affiliations

State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, PR, China

Xian Zhang, Ling Chen & Hong-Wen Gao

Key Laboratory of Yangtze River Water Environment of Ministry of Education, College of Environmental Science and Engineering Tongji University, Shanghai, PR, China, 200092

Environmental Engineering College, NanJing Forestry University, Nanjing, 210037, PR, China


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