Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a widely used molecular biology technology used to amplify specific DNA fragments in vitro. It can be regarded as special DNA replication outside organisms. Using PCR technology, millions of copies of a specific DNA sequence can be obtained in a short time. PCR technology has been widely used in molecular cloning and genetic diagnosis of genetic diseases. With the development of molecular biology technology and the needs of scientific research practice, some new PCR technologies have emerged, such as Reverse Transcription PCR (RT-PCR), Nested PCR, Multiplex PCR, Real-time PCR, etc.

Principle

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 also be denatured and melted at high temperatures, and can renature into double strands when the temperature is lowered. Therefore, by controlling the denaturation and renaturation of DNA through temperature changes, the in vitro replication of specific genes can be completed by adding designed primers, DNA polymerase, and dNTPs.

PCR technology actually relies on the enzymatic synthesis reaction of DNA polymerase in the presence of template DNA, primers and four kinds of deoxynucleotides. The specificity of PCR technology depends on the specificity of the binding of primers to template DNA. The reaction is mainly divided into three steps. ① Denaturation: Under high temperature conditions, the double strands of DNA dissociate to form two single strands of DNA. ② Annealing: When the temperature suddenly drops, the primer and its complementary template locally form a hybrid chain. ③ Extension: In the presence of DNA polymerase, dNTPs and Mg2+, the polymerase catalyzes the DNA chain extension reaction starting from the primer. The above three steps are a cycle, that is, three stages of high-temperature denaturation, low-temperature annealing, and medium-temperature extension. The product of each cycle can be used as a template for the next cycle. After dozens of cycles, the specific DNA fragment between the two primers is copied in large quantities, and the number can reach 106 to 107 copies.

Reagent Preparation

1. Soluble protein extraction buffer (62.5 mmol/L Tris-HCl pH 6.8, 10% glycerol, 2% β-mercaptoethanol, 1 mmol/L PMSF): Use a 5 mL micropipette to add 1.56 mL of 1 mol/L Tris-HCl (pH6.8) into a clean 50 mL beaker. Add 2.5 mL glycerol and 4.4 mg PMSF, then add approximately 15 mL double-distilled water, mix well, and adjust the volume to 24.5 mL. Place in a 4°C refrigerator until use (add 1/50 times the volume of β-mercaptoethanol before use).
2. Loading buffer (62.5 mmol/L Tris-HCl pH 6.8, 10% glycerol, 2% β-mercaptoethanol, 2% SDS): Use a 5 mL pipette to add 1.56 mL of 1 mmol/L Tris-HCl (pH 6.8) into a 50 mL beaker. Add 2.5 mL of glycerol and 5 mL of 10% SDS, then add 10 mL of double-distilled water and mix evenly. Adjust the volume to 24.5 mL and place it in a 4°C refrigerator for later use (add 1/50 times the volume of β-mercaptoethanol before use).

Procedures

A. PCR Primer Design

There are two primers in the PCR reaction, namely the 5' primer and the 3' primer. When designing primers, a single DNA strand is used as the benchmark (often based on the information strand). The 5'-end primer is identical to a short DNA sequence located at the 5' end of the fragment to be amplified, and the 3'-end primer is complementary to a short DNA sequence located at the 3' end of the fragment to be amplified.

Basic principles of primer design:

a. Primer length: 15 to 30 bp, commonly used is 20 to 25 bp.

b. Primer bases: The G+C content is preferably 40% to 60%. Too little G+C will result in poor amplification, and too much G+C will easily cause non-specific bands. ATGC is best distributed randomly to avoid clusters of more than 5 purine or pyrimidine nucleotides.

c. There should be no complementary sequences inside the primers.

d. There should be no complementary sequences between the two primers, especially complementary overlap at the 3' end.

e. The sequence homology between the primer and the non-specific amplification region should not exceed 70%. The 8 consecutive bases at the 3' end of the primer must not have a completely complementary sequence outside the region to be amplified, otherwise it will easily lead to non-specific amplification.

f. The bases at the 3' end of the primer, especially the last and penultimate bases, should be strictly matched. The best choices are G and C.

g. The 5' end of the primer can be modified. Such as adding restriction enzyme sites, introducing mutation sites, using biotin, fluorescent substances, and adding other short sequences, including start codons, stop codons, etc.

B. PCR Reaction System

The PCR reaction system generally consists of dNTPs, primers, template DNA, Taq DNA polymerase and PCR Buffer. Generally, the total volume is 10~50 µL, depending on the purpose of the experiment. The amount of reaction system added generally follows the following principles:

a. The final concentration of dNTP is generally 20~250 µmol/L.

b. The final concentration of primers is generally 0.1~0.5 µmol/L.

c. The amount of template DNA added is generally 1 to 20 ng/µL.

d. The amount of Taq DNA polymerase added is generally 0.05~0.1 U/µL.

e. 10×PCR Buffer is added according to the dilution factor.

After adding the above five reagents as required, add ddH₂O to the final volume. Mix well and centrifuge briefly to wait for reaction.

C. PCR Reaction Procedure

At present, PCR reaction procedures generally consist of the following steps:

a. Pre-denaturation: Complete denaturation of the template DNA and complete activation of the PCR enzyme are crucial to the success of PCR. It is recommended that the heating time refer to the reagent instructions, which is generally 94 °C for 3 to 5 minutes.

b. Denaturation: Generally, 94°C and 30 s in the cycle are enough to completely denature various target DNA sequences. If possible, the time of this step can be shortened. If the denaturation time is too long, the enzyme activity will be damaged, and if the denaturation time is too short, the target sequence will not be completely denatured, which can easily lead to amplification failure.

c. Annealing: The annealing temperature needs to be determined from many aspects. Generally, it is based on the Tm [Tm = 4(G + C) + 2(A + T)] value of the primer as a reference. It is about 5 °C lower than the Tm of the primer, between 50 and 65 °C. The annealing time is generally 30 s.

d. Extension: Primer extension is generally performed at 72 °C (the optimal temperature for Taq enzyme). The extension time depends on the length of the amplified fragment, and is generally set to 1 min/kbp.

e. Final extension: After the last cycle, the reaction is maintained at 72 °C for 10 to 30 minutes to complete the primer extension and anneal the single-stranded product into a double-stranded product.

The main cycles of PCR amplification are three steps: denaturation, annealing and extension. The number of cycles is generally 25 to 35 times, depending on the purpose of the experiment.

Note:

• The reaction system and reaction procedures described in this experiment are the basic principles of PCR reaction. The specific experimental system and procedures need to be set according to the experimental materials and experimental purposes.
• The number of PCR reaction cycles should not be higher than 35 cycles. After the number of cycles exceeds 30, the activity of DNA polymerase gradually reaches saturation, and the amount of product no longer increases with the increase of cycle number, resulting in the so-called "plateau period".

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