Increase the specificity of PCR:
1. Prime is ideal. Only primers that anneal to a single sequence on both sides of the target sequence and not other sequences must meet the following conditions
a. Long enough, 18-24bp, to ensure specificity. Of course, it does not mean that the longer the better, too long primers will also reduce specificity, and reduce yield
b. GC% 40% ~~~~ 60%
c. More GCs at the 5 end and intermediate sequences to increase stability
d. Avoid 3 end GC rich, don't have GC in the last 3 basses, or don't have GC in the last 3
e. Avoid the complementarity of 3 terminals, otherwise it is easy to cause DIMER
f. Avoid 3 terminal mismatch
g. Avoid internal secondary structure
h. The additional sequence (RT site, Promoter sequence) is added to the 5 end. It is not calculated when calculating the Tm value, but it is necessary to add them when detecting complementary and secondary structures.
i. When using merger primers, please refer to the codon usage table, pay attention to biological preferences, do not use merger primers at the 3 end, and use a higher primer concentration (1uM-3uM)
j. It is best to learn to use a design software. PP5, Oligo6, DNAstar, Vector NTI, Online desgin et al.
Another important parameter of the primer is the melting temperature (Tm). This is the temperature when 50% of the primers and complementary sequences appear as double-stranded DNA molecules. Tm is necessary to set the PCR annealing temperature. Under ideal conditions, the annealing temperature is low enough to ensure that the primers effectively anneal to the target sequence, and at the same time high enough to reduce non-specific binding. The reasonable annealing temperature is from 55 ℃ to 70 ℃. The annealing temperature is generally set to 5 ° C lower than the Tm of the primer.
There are several formulas for setting Tm. Some are derived from hybridization in high salt solutions and are suitable for primers of less than 18 bases.
Some estimates Tm based on GC content. The most reliable method to determine the primer Tm is the nearest neighbor analysis method. This method predicts the hybridization stability of primers from the sequence primary structure and the characteristics of adjacent bases. Most computer programs use nearest neighbor analysis.
Depending on the formula used and the primer sequence, Tm will vary greatly. Because most formulas provide an estimated Tm value, all annealing temperatures are only a starting point. The specificity can be improved by analyzing several reactions that gradually increase the annealing temperature. It starts to be lower than the estimated Tm by 5 ° C and gradually increases the annealing temperature in 2 ° C increments. Higher annealing temperatures will reduce the formation of primer dimers and non-specific products.
For best results, the two primers should have approximate Tm values. If the Tm difference of the primer pair exceeds 5 ° C, the primer will use a lower annealing temperature in the cycle and show a clear false start. If the Tm of the two primers is different, set the annealing temperature to 5 ° C lower than the lowest Tm or to improve specificity, you can first perform 5 cycles at the annealing temperature designed according to the higher Tm, and then at the lower Tm. Annealing temperature for the remaining cycles. This makes it possible to obtain a partial copy of the target template under more stringent conditions.
2. stability of primers
The standard purity of custom primers is sufficient for most PCR applications.
Primer yield is affected by the efficiency of synthetic chemistry and purification methods. Custom primers are shipped in dry powder form. It is best to re-dissolve the primers in TE so that the final concentration is 100 μM. TE is better than deionized water because the pH of water is often acidic, which can cause the hydrolysis of oligonucleosides. The stability of the primer depends on the storage conditions. The dry powder and dissolved primers should be stored at -20 ° C. Primers dissolved in TE at concentrations greater than 10 μM can be stored stably for 6 months at -20 ° C, but can only be stored for less than 1 week at room temperature (15 ° C to 30 ° C). Dry powder primers can be stored at -20 ℃ for at least 1 year and at room temperature (15 ℃ to 30 ℃) for up to 2 months.
3. optimize reactants concentration
a. magnesiom ions
The role of Mg ion is mainly that dNTP-Mg interacts with the nucleic acid backbone and can affect the activity of Polymerase. In general, the concentration of Mg is adjusted between 0.5-5mM. It is also important to remember that after adjusting the concentration of dNTPs Adjust the Mg ion concentration accordingly. For real-time quantitative PCR, use a 3 to 5 mM magnesium ion solution with a fluorescent probe
b. Other ions NH4 + K + will affect the PCR. Increasing the concentration of K + will affect the annealing temperature because it neutralizes the negative charge of the phosphate group on the nucleic acid backbone, thereby reducing the stringency of PCR. NH4 + also has The same effect. MBI TAQ enzyme provides two kinds of BUFFER, one is to add Mg and the other is already mixed with (NH4) 2SO4, of course, when the cation concentration is too high (KCL> 0.2M), DNA Denaturation does not occur at 94 degrees, and of course there is no way to talk about PCR.
c. polymerase
The enzyme efficiency of different companies is different. Operators need to master the appropriate enzyme concentration. Some high-fidelity efficiency is much lower than Taq polymerase, so the amount of enzyme may be larger. In addition, the general situation Under, the denaturation temperature can be used 90 ~ 92 degrees, the denaturation time can also be shortened, so as to ensure the activity of polymerase
d. template
50ul PCR SYSTEM
================================
human gDNA 0.1ug-1ug
E. Coli 10ng-100ng
LamadaDNA 0.5ng-5ng
Plasmid DNA 0.1ng-10ng
================================
4. termperature
a. denaturation
The normal is 94 degrees and 5 minutes, and the GC Rich's model is 95 degrees and 5 minutes. In addition to GC Rich, the conventional APPLICATIONS can shorten this part of the time to 1 to 2 minutes, or give a longer time at CYCLE 1 and cancel Initial denaturation
b. annealing
The important point is: under normal circumstances, it starts from 55 degrees. According to the situation, it can be adjusted with the Mg ion concentration. Conditional can do gradient pcr. Annealing time is 30-60S, shorter time can get better results. Because , polymerase will also have some activity during annealing time. So too long at AT will greatly increase the risk of non-specific amplification. In addition, for some difficult users, such as amplifying large fragments from gDNA, Two step PCR can also be used.
Dog Toys Tpr,Dog Squeaky Tpr Toy,Dog Toy Treat Tpr,Interactive Tpr Dog Toy
Dongguan King Pet Toys Co.,Ltd , https://www.kingpettoys.com