The application of thermostable DNA polymerases is at the heart of PCR technology. Thermostable DNA polymerases are generally classified into two classes, unreading activity and reading activity , depending on whether they have 3'→5' exonuclease activity . DNA polymerase without transcription activity ( represented by Taq enzyme) has high amplification efficiency, but due to lack of calibration activity, base mismatch is prone to occur, so there are many point mutations in the product. Taq DNA polymerase also has terminal transferase activity, which can add non-specific bases at the 3' end of the PCR product . Since the ratio of single A- bases is the highest, the PCR product can directly bind to the T- base with the 3' end . Vector ligation (ie, TA cloning) facilitates cloning, amplification, and sequencing of PCR products. However, Taq DNA polymerase can catalyze the mismatch primer extension or primer dimer formation during the first temperature rise of the PCR reaction, thus leading to non-specific amplification and affecting the synthesis amount of the target fragment. The hot-start Taq DNA polymerase designed for this phenomenon inhibits the polymerase activity at low temperatures, and the polymerase activity is restored by high temperature denaturation, catalyzing the specific binding of primers, thereby increasing the specificity of the target fragment. And production. Another type of DNA polymerase with proofreading activity ( represented by Pfu ) can selectively remove mis-incorporated dNTPs to maintain correct extension of the DNA strand; however, its amplification efficiency is usually lower than that of Taq DNA polymerase, especially The ability to extend long strand DNA strands is poor. Based on the characteristics of these two types of enzyme, an appropriate amount of Taq DNA polymerase with proof reading either with a mixed DNA polymerase activity, in a suitable reaction buffer system, amplification performance can be obtained with fidelity between the above A hybrid enzyme between two classes of enzymes for high-fidelity amplification of long fragments as well as complex templates. PCR experiments are influenced by many factors. High-quality commercial heat-resistant DNA polymerases and their accompanying buffers usually meet the needs of most DNA fragment amplification. The appropriate DNA polymerase should be selected based on the nature of the template (genomic, cDNA , plasmid, etc.) and the size of the fragment of interest, GC content, presence or absence of secondary structure (see GenStar® PCR Product Selection Guide). For fragments that are difficult to amplify, the reaction conditions should be optimized for different templates and primers to obtain the best amplification efficiency (see “ Optimization of conventional PCR reactions †for details ). A. Template dosage: Take 50 μl reaction system as an example B. Primer design principles: C. Primer dosage: Interventional Accessories,Introducer Sheath,Introducer Sheath Kit,arterial sheath introducer Anesthesia Medical Co., Ltd. , https://www.trustfulmedical.com
• Human genomic DNA : 0.1~1.0 μg
• E. coli genomic DNA : 10~100 ng
• λ DNA : 0.5~5 ng
• Plasmid DNA : 0.1~10 ng
• Primer length should meet the specific needs, generally between 18 and 25 bases; when expanding long fragments, it is preferably between 24 and 30 bases;
• ( G+C ) % content should be controlled as much as possible at 40~60% , and the ( G+C ) % content of the two primers should be as close as possible;
• Try to avoid the same base appearing more than three times in a row, and avoid using A or T at the 3' end ;
• Avoid the internal pairing of the primers to form a secondary structure;
• Paired bases should be avoided between the forward and reverse primers, especially the three bases at the 3' end, otherwise Primer dimers are easily formed ;
• The Tm values ​​of the two primers should be as close as possible, preferably no more than 5oC ;
• Calculation method of primer Tm value:
Below 20 nt : Tm = 2x ( A + T ) + 4x ( G+C )
20 nt or more: Tm = 81.5+0.41x ( G+C% ) -600/nt ( nt : number of bases of the primer)
• 0.1~1.0 μM , usually starting at 0.2 μM , adjusting the dosage according to the system;
• When using degenerate primers and random primers, increase the total amount of primers to compensate for the loss of yield; however, as the amount of primers increases, the specificity will decrease;
• When the template is larger or more complex (such as human genomic DNA ), the amount of primers needs to be reduced to increase the specificity;
• When the template is smaller and smaller (such as a plasmid template), increasing the amount of primer can increase the yield.
PCR technology overview
Polymerase chain reaction (Polymerase Chain Reaction, PCR) technology is the first major gene amplification technology innovation, is an important milestone in the history of molecular biology. Using PCR amplification technology, a very small amount of target DNA fragments can be specifically amplified by a million times, greatly improving the ability to analyze and detect DNA molecules. PCR technology has the advantages of high sensitivity, strong specificity, fast and simple, and has great application value and broad development prospects in many fields such as medicine, genetics, forensic science, microbiology, food inspection, and health inspection.