How to remove PCR nonspecific bands?(1)

I realized that the non-specific amplification was an urgent and inevitable problem.
So how do you solve this problem?
Let's study together!

1. reaction components:
(1) The template
The importance of templates is not to be explained.
The concentration of the template can affect the results of PCR, because the higher the concentration of the template, the more the non-specific bands will be added, not only the dimer will be formed, but also the non-specific strips will be formed between the templates.
Therefore, in order to ensure the specificity of amplification, the initial concentration has certain requirements for the template of different sources.

To measure the concentration of the template, and then calculation should be diluted multiples, on the one hand, can reduce unnecessary waste, on the other hand can also reduce due to the template of nonspecific banding concentration was too high.

In addition, the purity of the template should be taken into consideration.
If the template itself is not pure and contains multiple sets of templates, there must be multiple non-specific bands.

(2)primer
Some say that the key to success in PCR is the primers.
From a certain point of view, can also be said to also primers the primers.
There are many aspects of the primers, and we'll explain them one by one.

A) the primers have a low specificity, leading to incomplete complementarity between primers and target sequences.
Careful primer design is the most important step in PCR.
The ideal primers are annealed for a single sequence on either side of the target sequence instead of the other sequence.
Poorly designed primers might be used to amplify other nonobjective sequences.
At the same time, the primers should be avoided to form the dimer themselves.
After the design is completed, BLAST detection is required, and the next experiment can be carried out when determining that there is no complementarity with other genes.

The typical primer is about 18 to 24 nucleotides.
The primers need to be long enough to ensure the uniqueness of the sequence and reduce the probability that the sequence exists in a non-target sequence site.
But primers longer than 24 nucleosides do not mean higher specificity.
Longer sequences can be hybridized with mismatched sequences, reducing specificity and slowing down production by crossing short sequences.

B) the content of G+C should be 40 to 60%.
G+C too few amplification effect, G+C too easy to appear non-specific bands.
ATGC is best randomly distributed to avoid the sequence of 5 or more purines or pyrimidine nucleotides.
It is possible to design the primers of G or C in the 5 'end and intermediate regions, which will increase the stability of the primers and the stability of the hybrid of the primers.
So why is there too much GC content in the primer for non-specific bands?
Because when high GC content in the primer, high melting temperature, to strip at a higher temperature solution chain, and in the chain fails to reach the solution temperature, the primer will not completely solution chain DNA as a template, nonspecific amplification of PCR, so easy to appear nonspecific banding.

C) the concentration of primers can affect specificity.
The optimum primer concentration is generally 0.1 to 0.5 mu M.
Higher concentration of primers leads to amplification of nonspecific products.

D) the standard purity of custom primers is sufficient for most PCR applications.
The yield of primers is influenced by the efficiency of synthetic chemistry and the purification method.
Custom primer is transported in dry powder form.
It is better to dissolve the primers in TE to the final concentration of 100 mu M.
TE is better than deionized water because the pH of water is often acidic, which causes the hydrolysis of oligonucleotides.

E) the stability of the primer depends on the storage conditions.
Dissolve powder and the primer should be stored under - 20 ℃.
When primer concentration is more than 10 microns, dissolved in the primer in TE - 20 ℃ can keep stable for 6 months, but at room temperature (15 ℃ to 30 ℃) can only save less than 1 week.
Dry powder primer can preserve at least 1 year in - 20 ℃, at room temperature (15 ℃ to 30 ℃) can save 2 months at most.
?
Does it just find that the various aspects of primers can affect PCR?

(3)Mg2+ concentration
Magnesium ion affects the primer annealing, which affects the specificity.
Higher free magnesium concentration can increase yield, but also increase nonspecific amplification.
Therefore, the Mg2+ concentration can be reduced as appropriate.

(4)DNTP excessive
The quality and concentration of dNTP are closely related to the PCR amplification efficiency.
In the PCR reaction, the dNTP should be 20 ~ 200 mu mol/L, and the low concentration will reduce the production of PCR products.
When concentration is too high, it can cause nonspecific bands.
In addition, dNTP can be combined with Mg2+ to reduce the free Mg2+ concentration.

(5) Enzyme
It is mainly divided into the quality and quantity of enzyme.
A) sometimes, the enzymes of different sources can cause different amplification results due to their different specificity.
We often use high fidelity enzyme to protect the authenticity, can cut off the wrong base.
Such as Pfu DNA polymerase 3 5 ´ ´ - circumscribed (correction) enzyme activity, when the polymerization base mismatch occurs, calibration of enzyme activity of mismatch can be the base of resection, nonspecific amplification can be greatly reduced (related products catalog E002, E031, E006, E035).
B) too much enzyme can sometimes cause nonspecific amplification, so we can properly reduce the amount of enzyme.

(6) PCR additive
Sometimes PCR additives are like ginseng, and the PCR response can be used to "replenish".
Optimization of enzymes, primers, and Mg2+ concentrations is sufficient to increase the high specificity of most templates. However, certain templates, including high GC content templates, require additional measures.
Additives that affect the temperature of DNA melt provide another way to increase the specificity and yield of the products.
The complete degeneration of the template is required for the best results.
In addition, the secondary structure blocks the extension of the primers and enzymes.
PCR additives, including formamide, DMSO, glycerine, betaine and PCR Enhancer (E050, E051, E055) can increase amplification.
PCR Enhancer has other advantages, too, when DNA polymerase is used together, with very little magnesium ion optimization.
Therefore, when we add the right amount of PCR additives, we can reduce the non-specific generation.

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