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With the fragmentation of DNA molecules using restriction enzymes and their recognition by gel electrophoresis, the next step is to multiply (clone) the obtained fragment and subject them to recombinant DNA technology.
The technique of DNA strand multiplication is called PCR (polymerase chain reaction).
In the 1980s, the PCR technique was used to make thousands of copies of a single piece of DNA. This technique is used in test tubes containing DNA and some other necessary compounds, such as primers (Primer DNAs) and the DNA polymerase enzyme (enzyme that makes DNA replication).
The primers they are DNA strands, with about 20 bases (A, T, C, G) complementary, that is, they bind by complementarity to the beginning of the DNA sequence to be multiplied. When a DNA molecule is to be multiplied, the double strand must be separated, thus forming two different but complementary strands. Each tape will serve as a template for duplication, so we need two different types of primers (see figure)
PCR technique, step by step
A minimal DNA sample is obtained from a human cell.
- The DNA sample, the replication enzyme (DNA polymerase), the DNA nucleotides, and the DNA sequence complementary primers are placed in a test tube.
- The test tube is placed in a PCR machine (a machine that raises and lowers the temperature according to a program). The following heating and cooling steps take place inside the machine controlled by the program.
- The tube is heated to 94 ° C to denature (separate the double strand) the DNA.
4. Each single strand of denatured DNA serves as a template for the synthesis of new complementary strands. For this it is cooled to 54ºC where the primers ring to the beginning of the two simple tapes, serving as primers for the polymerase enzyme.
5. The tube is reheated to 72 ° C (ideal DNA polymerase operating temperature) for duplication of the strip. DNA polymerase begins, after the end of the primer, to place the free nucleotides on the DNA strand by binding them together, thus forming a new double strand.