Semi-conservative replication of DNA is an important way of biological evolution and passage. Double-stranded DNA can be denatured and unrolled into single strand under the action of many enzymes, and copied into the same two-molecule copy according to the principle of base complementary pairing with the participation of DNA polymerase.
The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence.
PCR consists of three basic reaction steps: denaturation-annealing-extension:
(1) denaturation of template DNA: After the template DNA is heated to about 93℃ for a certain period of time, the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is dissociated, so as to combine with primers and prepare for the next round of reaction;
(2) Annealing (renaturation) of template DNA and primer: after the template DNA is denatured into single strand by heating, the temperature is reduced to about 55℃, and the primer and the complementary sequence of the template DNA single strand are paired and combined;
③ Primer extension: DNA template-primer conjugate, under the action of DNA polymerase (such as TaqDNA polymerase) at 72℃, uses dNTP as reaction raw material and target sequence as template, and synthesizes a new semi-conservative replication chain complementary to the template DNA chain according to the principles of base complementary pairing and semi-conservative replication.
More semi-conservative replication chains can be obtained by repeating the three processes of cyclic denaturation-annealing-extension, and this new chain can be used as a template for the next cycle. It takes 2 ~ 4 minutes to complete a cycle, and the target gene to be amplified can be amplified by a million times in 2 ~ 3 hours.
Extended data:
The specific determinants of highly specific PCR reaction are:
① Correct combination of specificity of primer and template DNA;
② the principle of base pairing;
③ The authenticity of ③Taq DNA polymerase synthesis reaction;
④ The specificity and conservation of the target gene.
The correct combination of primers and templates is the key. The combination of primer and template and the extension of primer chain follow the principle of base pairing. Because of the fidelity of polymerase synthesis reaction and the high temperature resistance of Taq DNA polymerase, the combination (renaturation) of template and primer in the reaction can be carried out at higher temperature, the specificity of combination is greatly increased, and the amplified target gene fragment can maintain high accuracy. By selecting highly specific and conservative target gene regions, its specificity will be higher.
The amount of PCR products with high sensitivity increases exponentially, and the initial template to be tested can be amplified from picogram level (pg= 10- 12g) to microgram level (ug= 10-6g). A target cell can be detected from 6.5438+0 million cells; In virus detection, the sensitivity of PCR can reach 3 RFU (plaque forming unit); In bacteriology, the lowest detection rate is 3 bacteria.
The extension temperature of PCR reaction is generally 70 ~ 75℃, and the common temperature is 72℃. Too high extension temperature is not conducive to the combination of primers and templates. The time of PCR extension reaction depends on the length of the fragment to be amplified. Generally, for DNA fragments within 1Kb, the extension time of 1min is enough.
The target sequence of 3 ~ 4 KB takes 3 ~ 4min；; The amplification of 10Kb needs to be extended to 15min. Excessive extension will lead to the appearance of non-specific amplification bands. For the amplification of low concentration template, the extension time is slightly longer.
The number of cycles determines the degree of PCR amplification. The number of PCR cycles mainly depends on the concentration of template DNA. Generally, the number of cycles is between 30 and 40, and the more cycles, the more nonspecific products.
References:
Baidu encyclopedia -PCR amplification