One of the methods of DNA replication is using the PCR method. Due to its many uses and advantages, this method has expanded rapidly in molecular laboratories, and this method is now commonly used in these laboratories.
Polymerase Chain Reaction (PCR)
In 1983, Mollis worked on a polymerase chain reaction to amplify certain DNA sequences. In terms of practical principles, PCR has a great similarity to DNA replication and is actually taken from it.The DNA replication process is exponential in this way, and thus a large amount of DNA can be obtained. Primers complement parts of two DNA strings and determine the area to be multiplied.
- Initialization step: This step is only necessary for polymerase DNA that requires heat activation in the hot start PCR method. At this stage, the reaction temperature of the reaction solution is 94 to 96 degrees for a certain period.
- Denaturation step: At this stage, due to the breakdown of hydrogen bonds between nucleotides, two strands of DNA patterns are separated and single strands of DNA are obtained. This stage is the first part of the thermal cycles and involves heating the reaction solution for a specified period at 94 to 96 degrees.
- Annealing step: At this stage, the reaction temperature is reduced to 50 to 65 ° C, and primers, which are similar to the two sides of the DNA segment to be reproduce, are designed to complement their complementary DNA sequences in the two opened DNA strands. To be These two open-ended pieces provide DNA polymerase activity.
- Extension / elongation step: At this stage, the DNA polymerase enzyme, using the four open source DNA builders in the solution, forms a new DNA sequence in the order of 5 to 3 versus the strands. The length of this step should also be proportional to the type of DNA polymerase and the length of the DNA strand.
- Final elongation: This step is performed after a final PCR cycle at a temperature of 70 to 74 degrees, to ensure that all single strands of DNA are replicated.
- Final hold: At this stage, the final solution can be stored at 4 to 15 degrees for a short time.
Temperature cycle and target gene amplification in the PCR test
- Electrophoresis in Agarose Gel: Finally, PCR products can be electrophoresed on an agarose gel with a molecular weight marker (DNA-ladder containing specific DNA fragments), and the PCR results are examined.
RNA forms mRNA and rRNA in large numbers in animal, plant, and fungal eukaryotic cells as well as in bacteria. In the RT-PCR technique, because RNA is used as a replication template, the RNA phase should be converted to a cDNA. The extracted RNA is converted to the cDNA using an enzyme called an inverse copy vector. Then the cDNA produced by the PCR technique is duplicated.
Real time PCR
The PCR method is a technique with high sensitivity and specificity for the detection of nucleic acids, which is well established and used, but it is not easy to determine the specific nucleic acids present in the sample and the changes that may occur during preparation, storage, Or performing a reaction, it is difficult to determine precisely the amount of specific nucleic acids.