PCR Technology Summary

1、 Setting of temperature and time

Three temperature points of denaturation annealing extension are set based on PCR principle in three steps. In the standard reaction, the three temperature point method was used. Double stranded DNA was denatured at 90~95 ℃, then rapidly cooled to 40~60 ℃. The primer was annealed and bound to the target sequence, and then rapidly heated to 70~75 ℃. Under the action of Taq DNA polymerase, the primer chain was extended along the template.

For shorter target genes (when the length is 100~300bp), the two temperature point method can be used. In addition to the denaturation temperature, the annealing and extension temperature can be combined. Generally, 94 ℃ is used for denaturation, and 65 ℃ is used for annealing and extension (Taq DNase still has high catalytic activity at this temperature).

1. Denaturation temperature and time: low denaturation temperature and incomplete chain breaking are the main reasons for PCR failure. Generally, 93 ℃~94 ℃ for 1min is enough to denature the template DNA. If it is lower than 93 ℃, the time needs to be extended, but the temperature cannot be too high, because the high-temperature environment has an impact on the enzyme activity. If the target gene template or PCR product cannot be completely denatured in this step, the PCR will fail.

2. Annealing (renaturation) temperature and time: Annealing temperature is an important factor affecting the specificity of PCR. After denaturation, the temperature was rapidly cooled to 40 ℃~60 ℃, and the primer and template could be combined. Because template DNA is much more complex than primers, the chance of collision between primers and templates is much higher than that between complementary chains of templates.

The annealing temperature and time depend on the length of primer, base composition and concentration, as well as the length of target sequence. For primers with 20 nucleotides and about 50% G+C content, 55 ℃ is the ideal starting point for selecting the optimal annealing temperature. The refolding temperature of primers can be selected by the following formula:

Tm value (chain breaking temperature)=4 (G+C)+2 (A+T)

Refolding temperature=Tm value - (5~10 ℃)

Within the allowable range of Tm value, selecting a higher refolding temperature can greatly reduce the non-specific binding between primer and template, and improve the specificity of PCR reaction. The refolding time is generally 30~60sec, which is enough to make the primer and template combine completely.

3. Extension temperature and time: biological activity of Taq DNA polymerase:

70 ～ 80 ℃ 150 nucleotides/S/enzyme molecule

70 ℃ 60 nucleotides/S/enzyme molecules

55 ℃ 24 Nucleotide/S/enzyme molecule

When the temperature is higher than 90 ℃, DNA synthesis can hardly be carried out.

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 primer and template. The extension reaction time of PCR can be determined according to the length of the fragment to be amplified. Generally, for DNA fragments within 1Kb, the extension time of 1min is sufficient.

3~4kb target sequence takes 3~4min; Amplification of 10Kb needs to be extended to 15min The presence of nonspecific amplification bands will be caused by the long extension of the insertion space. For the amplification of low concentration template, the extension time is slightly longer.

2、 Number of cycles

The number of cycles determines the extent of PCR amplification. The number of PCR cycles mainly depends on the concentration of template DNA. Generally, the number of cycles is 30~40. The more cycles, the more non-specific products.

Five elements of PCR reaction: there are mainly five substances participating in the PCR reaction, namely primer, enzyme, dNTP, template and Mg2+

1） , Primer

Primer is the key to PCR specific reaction. The specificity of PCR products depends on the complementary degree of primer and template DNA. In theory, as long as any segment of template DNA sequence is known, complementary oligonucleotide chains can be designed according to it as primers, and template DNA can be amplified in large quantities in vitro using PCR.

1. The primer design shall follow the following principles:

1) Primer length: 15-30 bp, usually about 20 bp.

2) Primer amplification span: 200-500bp is appropriate, and the fragment can be expanded to 10kb under specific conditions.

3) Primer base: The content of G+C should be 40-60%. Too little G+C has poor amplification effect, and too much G+C is easy to produce non-specific bands. ATGC should be distributed randomly to avoid string arrangement of more than 5 purine or pyrimidine nucleotides.

4) Avoid the secondary structure inside the primer, and avoid the complementation between two primers, especially the complementation of the 3 'end, otherwise the primer dimer will be formed, producing non-specific amplification bands.

5) The base at the 3 'end of primer, especially the last and penultimate base, should be paired strictly to avoid PCR failure due to mismatching of terminal base.

6) The primer has or can be added with an appropriate restriction site, and the target sequence to be amplified should have an appropriate restriction site, which is very useful for restriction analysis or molecular cloning.

7) Primer specificity: The primer should have no obvious homology with other sequences in the nucleic acid sequence database.

2. Primer quantity: the concentration of each primer is 0.1~1 umol or 10~100 pmol. It is better to produce the required result with the lowest primer quantity. High primer concentration will cause mismatch and non-specific amplification, and increase the chance of forming dimer between primers.

2） , enzyme and its concentration

At present, there are two kinds of Taq DNA polymerase available, one is a natural enzyme purified from hydrothermal bacteria, and the other is a genetically engineered enzyme synthesized by Escherichia coli. A typical PCR reaction requires about 2.5U of enzyme (when the total reaction volume is 100ul). If the concentration is too high, it can cause non-specific amplification. If the concentration is too low, the amount of synthetic products will be reduced.

3） DNTP mass and concentration

The quality and concentration of dNTP are closely related to the PCR amplification efficiency. The dNTP powder is granular, and will lose biological activity if it is not stored properly. DNTP solution is acidic, so it should be prepared into a high concentration when used, adjusted to pH of 7.0~7.5 with 1M NaOH or 1M Tris. HCL buffer solution, packed in small quantities, and frozen at - 20 ℃. Repeated freezing and thawing will degrade dNTP.

In the PCR reaction, dNTP should be 50~200umol/L. In particular, it should be noted that the concentration of the four dNTPs should be equal (equimolar preparation). If any concentration is different from the others (higher or lower), it will cause mismatch. If the concentration is too low, the yield of PCR products will be reduced. DNTP can combine with Mg2+to reduce the concentration of free Mg2+.

4） Template (target gene) nucleic acid

The amount and purification degree of template nucleic acid is one of the key links in the success or failure of PCR. Traditional DNA purification methods usually use SDS and proteinase K to digest and process samples. The main functions of SDS are: to dissolve the lipids and proteins on the cell membrane, thus dissolving the membrane proteins to destroy the cell membrane, and to dissociate the nuclear proteins in the cell. SDS can also combine with proteins to precipitate;

Protease K can hydrolyze and digest proteins, especially histones bound to DNA, extract proteins and other cell components with organic solvent phenol and chloroform, and precipitate nucleic acids with ethanol or isopropanol. The extracted nucleic acid can be used as a template for PCR reaction.

In general, the clinical test samples can be dissolved by a fast and simple method, the pathogen can be lysed, the protein of chromosome can be digested and removed to free the target gene, and directly used for PCR amplification. Generally, guanidine isothiocyanate or proteinase K method is used for RNA template extraction to prevent RNA degradation by RNase

5） Mg2+concentration

Mg2+has a significant effect on the specificity and yield of PCR amplification. In general PCR reaction, when the concentration of dNTP is 200umol/L, the appropriate concentration of Mg2+is 1.5~2.0mmol/L. If the concentration of Mg2+is too high, the specificity of the reaction will be reduced, and non-specific amplification will occur. If the concentration is too low, the activity of Taq DNA polymerase will be reduced, and the reaction products will be reduced.