The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity relies on oligonucleotide primers that are complementary to both ends of the target sequence. Semi-conservative replication of DNA is an important pathway for biological evolution and generation. Double-stranded DNA can be denatured and unwinded into single strands under the action of various enzymes. With the participation of DNA polymerase, it can be copied into two identical molecular copies according to the principle of complementary base pairing.
In experiments, it was found that DNA can be denatured and unzipped at high temperatures, and can be renatured into double strands when the temperature is lowered. Therefore, by controlling the denaturation and renaturation of DNA through temperature changes, adding designed primers, DNA polymerase, and dNTPs, the in vitro replication of specific genes can be completed.
Cycling parameters
1. Pre-denaturation
Complete denaturation of template DNA and complete activation of PCR enzyme are crucial to the success of PCR. It is recommended to refer to the heating time. According to the reagent instructions, the general activation time for unmodified Taq enzyme is two minutes.
2. Denaturation step
Generally, 95°C and 30 seconds in the cycle are enough to completely denature various target DNA sequences. This step time can be shortened if possible. If the denaturation time is too long, the enzyme activity will be damaged. If the denaturation time is too short, the target sequence will not be completely denatured, which can easily lead to amplification failure.
3. Primer annealing
The annealing temperature needs to be determined from many aspects. Generally, the Tm value of the primer is used as a reference, and the annealing temperature is appropriately lowered according to the length of the amplification. Then make an estimate based on this experiment. Annealing temperature has a great impact on the specificity of PCR.