PCR (polymerase chain reaction) polymerase chain reaction, also known as in vitro DNA amplification technology, was pioneered by Kary Mullis of Cetus in the United States in 1985, which can amplify a small amount of DNA fragments more than one million times. Kary Mullis himself won the 1993 Nobel Prize in Chemistry.
Advantages: high sensitivity, strong specificity, high yield, good repeatability, fast and simple, etc., widely used in microbiology, archaeology, forensic medicine, sports and other fields, and has been popularized in many ordinary laboratories, greatly simplifying the tradition Molecular cloning technology, which makes it easier to analyze and identify the target gene.
Principle of PCR
It is used to amplify a DNA fragment located between two known sequences, similar to the replication process of natural DNA.
The DNA molecule to be amplified is used as a template, and a pair of oligonucleotide fragments complementary to the 5’end and 3’end of the template are used as primers. Under the action of DNA polymerase, it follows the template according to the half-reserved replication mechanism. Chain extension until the completion of new DNA synthesis, repeat this process, the target DNA fragment can be amplified.
Basic PCR reaction steps
The specificity of amplification depends on the specific binding between the primer and the template DNA. The basic reaction steps are divided into three steps:
1. Denaturation: Heating breaks the hydrogen bond between the template DNA double strands to form two single strands. 94°C 30〃
2. Annealing: After a sudden drop in temperature, the template DNA and the primer are complementary to each other according to the base pairing principle. There is also a combination between the two template strands, but due to the high concentration of the primer and the simple structure, the main The binding occurs between the template and the primer. 55°C 30
3. Extension: Adjust the reaction temperature to the optimum temperature of the enzyme. In the presence of DNA polymerase, 4 types of dNTPs and magnesium ions, start from the 3’end of the primer and bind to mononucleotide to form A new DNA strand complementary to the template strand. 72°C 1〃
The above 3 steps are a cycle. After each cycle, the amount of DNA in the sample should double, and the newly formed strand can become a template for a new cycle. After 25 to 40 cycles, DNA can be amplified by 106 to 109 Times.
The specificity of PCR amplification is determined by a pair of oligonucleotide primers.
In the early stage of the reaction, the original DNA is responsible for the template. As the number of cycles increases, the number of primer-mediated extension fragments increases sharply and becomes the main template. The final amplified product is between the 5’ends of the two primers. DNA fragments.
The composition and function of PCR
- Buffer: 10-50 mM Tris-Cl (pH 8.4) to maintain the alkaline environment of Taq enzyme action. 25-50 mM KCl promotes primer annealing, and >50 mM will inhibit Taq enzyme activity. 100μg/ml bovine serum albumin (BSA) has a certain degree of protection to enzymes. If the quality is not good, it will have the opposite effect. It is recommended to use acetylated BSA. Gelatin, Tween-20, and dithiothreitol (DTT) also have similar effects.
- MgCl2: L5~2e0 mMTaq enzyme is Mg2+ dependent, which significantly affects the specificity of the reaction and the yield of amplified fragments. Excessive amount can increase non-specific amplification and affect the yield. If it is too low, the enzyme activity will decrease significantly.
- dNTPs: dATP, dGTP, dCTP, dTTP—substrate 0.02~0.2 mM dNTPs can bind to Mg2+. Pay attention to the relationship between Mg2+ concentration and dNTPs concentration. Mg2+ concentration is 0.2~2.5 mM higher than dNTPs concentration. Too high: speed up the reaction speed, but also increase the base error incorporation rate and laboratory cost. Too low: The reaction speed decreases, which can improve the accuracy of the experiment.
- Primer-P: Pre-amplify the known sequence at both ends of the nucleic acid fragment to determine the specificity. 0.2~1 μM is too high: non-specific product amplification and mismatches increase the formation of primer dimers between primers and reduce yield. Low: lower output.
- Taq DNA polymerase: high heat resistance 0.5~5 U/100μL 1U/25~50μL high: primer non-specific product amplification. Low: The amount of product is reduced.
- Template DNA: Minimum of 102~105 bp DNA fragments. The actual amount far exceeds this amount. The amount needs to be explored in the experiment. 1~5μL. Too high: non-specific products increase Too low: yield decreases
- Water: Deionized water to make up the entire reaction volume.
Optimization of PCR reaction conditions
1. Denaturation temperature and time: Ensuring the template DNA melting is completely the key to ensuring the success of the entire PCR amplification. Heating at 90~95°C for 30~60s, no matter how complex DNA molecules are changed into single strands. According to the complexity of the template DNA, the denaturation temperature and time can be adjusted. Under normal circumstances, choosing 90°C 30〃 can completely denature various complex DNA molecules.
Too high temperature or long duration of high temperature can cause damage to Taq enzyme activity and dNTP molecules.
2. Refolding temperature and time: PCR amplification specificity depends on the combination of primer and template during the renaturation process. The selection of the renaturation temperature can be determined according to the length of the primer and the G+C content. When the length is between 15 and 25 bp, the renaturation temperature Tm=4(G+C)+2(A+T) can be calculated, generally Located at 40~600°C, 30~60s.
The higher the renaturation temperature, the higher the product specificity. The lower the refolding temperature, the lower the product specificity. In general, choosing 55°C 30” is enough to make the primer and template completely bind.
3. Extension temperature and time: Generally, the optimum temperature for Taq enzyme is between 70 and 75°C. When the primer is less than 16 nucleotides, the excessively high extension temperature is not conducive to the combination of the primer and the template, and the temperature can be slowly raised to 70~75°C.
The extension reaction time can be determined according to the length of the fragment to be amplified, <1Kb, 1 minute is sufficient; >1Kb requires longer extension time, and 10Kb fragment extension time can reach 15 minutes. If the extension time is too long, non-specific amplification may occur, and 72°C 1 is commonly used.
4. Cycle number: After other parameters are selected, the number of PCR cycles mainly depends on the concentration of template DNA.
In theory, after 20-25 cycles, the accumulation of PCR products can reach the maximum value. In actual operation, since the yield of each reaction step cannot reach 100%, no matter what the template concentration is, 20-30 times is more reasonable. The number of cycles. The more cycles, the more non-specific amplification.