At that time, the impact on PCR was general, but there was no problem in operation. But think about it, like other experiments, I have little knowledge, and so does PCR. So I think it is necessary to re-learn various technologies and principles related to PCR.
Get down to business
PCR (polymerase chain reaction) Polymerase chain reaction, also known as in vitro DNA amplification technology, was initiated by Kary Mullis of Cetus Company in the United States in 1985, which can amplify a small amount of DNA fragments by more than one million times. Kary Mullis herself won the 1993 Nobel Prize in Chemistry.
I have shared the songs of PCR before, so I will review them here first, and then systematically sort out the relevant knowledge of PCR.
First, the principle of PCR
Under the catalysis of DNA polymerase, mother DNA was used as template and specific primers were used as extension starting point. The process of replicating the daughter DNA complementary to the mother template DNA in vitro through denaturation, annealing, extension and other steps.
Polymerase chain reaction is used to amplify known short DNA fragments, which can be a single gene or just a part of a gene. Unlike living organisms, PCR can only replicate very short DNA fragments, usually no more than 10kbp. DNA is a double-stranded molecule, so its size is measured by the structural unit (nucleotide) of complementary DNA double-stranded, and the unit is base pair (bp).
Second, PCR reaction components
template
Too many:
Nonspecific bands increased.
Too little:
The yield of PCR decreased.
primer
The known sequences at both ends of the preamplified nucleic acid fragment determine the specificity.
High:
Amplification and mismatch of nonspecific products increased the formation of primer dimer between primers, and the yield decreased.
Low:
The output has decreased.
polymerase
High heat resistance
High:
Amplification of nonspecific products of primers.
Low:
The number of products has decreased.
dNTP
dATP、dGTP、dCTP、dTTP
Too high:
Accelerating the reaction speed will also increase the wrong incorporation rate of bases and the cost of laboratory testing.
Too low:
The decrease of reaction speed can improve the accuracy of the experiment.
buffer
magnesian ion
Taq enzyme is Mg2+-dependent, which significantly affects the specificity of the reaction and the yield of amplified fragments.
Excess:
Will increase non-specific amplification and affect yield.
Too low:
The enzyme activity decreased significantly.
10~50 mm Tris- Cl (pH8.4)
Maintain the alkaline environment of Taq enzyme.
25~50 mm KCl
Promote primer annealing, and > > 50 mM will inhibit Taq enzyme activity.
100 μ g/ml bovine serum albumin (BSA)
It has a certain protective effect on enzymes, and if the quality is not good, it will be counterproductive. Acetylated BSA is recommended. Gelatin, Tween -20 and dithiothreitol (DTT) have similar effects.
Third, the basic steps of PCR reaction
The general polymerase chain reaction consists of 20 to 35 cycles, and each cycle includes the following three steps:
Transmutation:
Annealing or joining:
Extension (extension):
4. Optimization of 4.PCR reaction conditions
1, denaturation temperature and time:
Ensuring the melting of template DNA is the key to ensure the success of PCR amplification.
When heated at 90 ~ 95℃ for 30 ~ 60 seconds, even the most complex DNA molecules will become single-stranded.
Too high temperature or high temperature for too long will destroy Taq enzyme activity and dNTP molecules.
2. Renaturation temperature and time:
The specificity of PCR amplification depends on the combination of primers and templates during renaturation.
The higher the renaturation temperature, the higher the product specificity. The lower the renaturation temperature, the lower the product specificity.
It needs to be set according to the Tm value of the primer.
3. Extension temperature and time:
Generally, the optimum temperature of Taq enzyme is between 70℃ and 75℃. When the primer is less than 16 nucleotides, too high extension temperature is not conducive to the combination of primer and template, and the temperature can be slowly increased to 70 ~ 75℃.
The extension reaction time may depend on the length of the fragment to be amplified, and less than 1 KB and 1 min is enough. If it is greater than 1kb, the extension time should be extended. Taq enzyme can increase the time according to 1kb/min.
It should be noted here that if the extension time is too long, nonspecific amplification may occur. Therefore, it is necessary to set a suitable extension time.
4. Cycle times:
After selecting other parameters, the number of PCR cycles mainly depends on the concentration of template DNA.
Theoretically 20? After 25 cycles, the accumulation of PCR products can reach the maximum. In practice, the yield of each reaction can't reach 100%, so no matter what the template concentration is, 20~30 cycles is a reasonable number of cycles. The more cycles, the more nonspecific amplification.
Verb (abbreviation of verb) PCR extension technology
There are many technologies extended from PCR, and here are just a few commonly used in daily experiments.
1. touchdown PCR
Login PCR is mainly used to optimize the conditions of PCR. In many cases, the design of primers brings difficulties to PCR, such as insufficient specificity and easy mismatch. If the annealing temperature is too high, the PCR efficiency will be too low, but if the annealing temperature is too low, the non-specific amplification will be too high.
Therefore, the initial annealing temperature of the previous cycle is set to be several degrees higher than the highest melting temperature (Tm) of the primer. In the first few cycles, the temperature gradually drops to the set final Tm. After the template with high specificity matching was obtained at higher temperature, it was amplified efficiently at lower temperature.
2. Reverse transcription polymerase chain reaction
The principle of reverse transcription-polymerase chain reaction (RT-PCR) is to extract total mRNA from tissues or cells, and reverse transcribe it into cDNA with reverse transcriptase Oligo(dT) or random primers. Then PCR amplification is carried out using cDNA as a template to obtain the target gene or detect gene expression.
3. Real-time PCR/ quantitative PCR
Real-time fluorescence quantitative PCR technology refers to the method of adding fluorescent groups to the PCR reaction system, monitoring the whole PCR process in real time by using fluorescence signal accumulation, and finally quantitatively analyzing the unknown template by standard curve.
4. nested PCR
First, several cycles were carried out with low specificity primers to increase the number of templates, and then amplification was carried out with high specificity primers.
5.SOE PCR
Overlapping extension PCR can be divided into two types: overlapping extension PCR site-directed mutation and overlapping extension PCR sequence deletion mutation, that is, overlapping extension PCR gene splicing (SOE PCR).
5. 1 overlapping extension PCR site-directed mutation (because the principle is simple, it is directly shown in the figure).
Pfu enzyme must be used in this step, not Taq enzyme, because Taq enzyme is easy to add an A at the end of PCR product, which will cause frame shift mutation of the product.
5.2 Sequence deletion mutation by overlapping extension PCR
6. High GC content PCR
It has a high GC content (>: 65%), and it is difficult to amplify the DNA template due to the strong hydrogen bond between G and C bases. GC-rich sequences also include secondary structures. Therefore, GC-rich sequences will cause DNA polymerase to "block" along the template and interfere with DNA synthesis.
In order to amplify the fragment with high GC content, it is necessary to dissociate the double-stranded template, so that the primer can combine with the template and DNA polymerase can read the sequence. In order to overcome the strong GC interaction, the most common method is to use PCR additives such as DMSO or auxiliary solvents to help DNA denaturation. However, these reagents usually reduce the Tm of primers, so the annealing temperature needs to be adjusted accordingly.
DNA polymerase with high synthesis ability is beneficial to complete PCR with high GC content because of its stronger binding ability with template. Ultra-high thermal stability DNA polymerase is also beneficial to PCR with high GC content, because higher denaturation temperature (for example, 98℃ instead of 95℃) may promote double-stranded dissociation and PCR amplification.
7.AS-PCR
PCR (AS-PCR) is the base mismatch between the indicator and the template, which can effectively inhibit the PCR reaction, thus achieving the purpose of template identification (allele identification).
Because primer extension starts from the 3' end in PCR, the base at the 3' end is very important for primer extension. If this base is complementary to the template, the primer can be extended continuously, and PCR can be carried out normally to obtain an amplification band with a specific length, otherwise it cannot be extended. Therefore, as long as mutant bases different from normal alleles are arranged at the end of primer 3', when primers containing mutant sequences are used for PCR, if specific bands are obtained, it indicates that the tested genes contain the mutation. There is no specific amplification band, indicating that there is no such mutation.
Note: Because only the base mismatch at the 3' end of the primer is used here, it is necessary to find a suitable Tm to achieve the detection purpose.
refer to
Mullis, Kary B. et al. "Methods for amplifying, detecting and/or cloning nucleic acid sequences" U.S. Pat. No.4,683, 195.