How to remove PCR nonspecific bands?(2)

Let's talk about another important factor that affects PCR - reaction conditions

2. reaction conditions

Let's start with a review of the typical PCR response, which has three steps: denaturation, annealing, extension, and multiple cyclic reactions to obtain the target segment.

And we're going to talk about them separately.

(1)Degeneration

In general, PCR, we set up the denaturation temperature between 90 ~ 98 ℃.

So, before reaching the denatured temperature, does the DNA polymerase capture the brief time in the heating process, sneak up on its own enzymes, and amplify some of the non-purpose strips?

The answer is YES.

DNA polymerase is "Till the end of life a silk worm keeps spinning silk. Till burning itself out a candle goes on lighting us." dedication, effort to play their own value, though, looks like we don't want this process...

In this case, a kind of elixir called thermal activation enzyme came into being.

You may have heard about the name of the hot starter enzyme, but do you really know how it works?

In the development history of PCR, the discovery of hot stable Taq enzyme is an important factor restricting the development of PCR.

The enzyme that was initially applied to PCR is the Klenow fragment of e. coli DNA polymerase I, and the Klenow enzyme needs to be rejoined every time. The process is cumbersome.

Since then, scientists have successfully extracted from aquatic thermophilic bacillus DNA polymerase to heat, the enzyme reaction under 90 ℃ for 2 hours, still have 70% of the activity, this will avoid each loop to enzymatic process, improve the efficiency of amplification, so widely used in the PCR amplification.

As we learn about biochemical primer, we have learned that temperature has a dual effect on enzyme activity, high temperature loss and low temperature suppression.

Ordinary Taq polymerase optimum temperature is 72 ℃, the activity of the enzyme best, less than 72 ℃, the enzyme has a weak activity (note: weaker indicates no activity!!!!!

).

In temperature rise gradually, to achieve the process of denaturation temperature, the activity of the enzymes play a weaker, system mismatch easily or the formation of primer dimers, especially when designing primers or the 3 'end is G C, mismatch chain is difficult to solve.

So to improve the specificity of the PCR amplification and decrease the rate of reaction mismatch, can artificially control the activity of enzyme, before 72 ℃ for enzyme activities, don't play this avoids the reaction system (or configuration) in the process of heating up chain mismatches, thus ensuring the specificity of the amplification.

Inshore warm start enzyme (E017 product article number, E018 E027 E028) is to use chemical modification methods of closed enzyme active center: low temperature chemical small molecules and enzyme activity center, enzyme activities, no temperature to 94 ℃, both from, enzyme activity center, began to guide system amplification, greatly increase the amplification of the specificity and sensitivity.

Compared with the enzyme that is sealed by the antibody, the enzyme activity of the chemical modification is more stable (the antibody closure is a dynamic balance) and there is no foreign DNA contamination.

Due to the hot start enzyme can quickly specific amplification of any known DNA fragments, therefore has been widely applied in various fields of molecular biology, augmented by warm start method is also a common way to improve the PCR specificity.

In addition, warm start enzyme also has high specificity and high sensitivity is widely applied to various aspects: constructing cDNA library, produce a large number of DNA sequencing, mutant analysis of constructing, gene isolation, genetic disease diagnosis, forensic identification, etc.

(2) The annealing

We have also said that the primers are also primers, and the primers' Tm values are not to be underestimated.

So what does annealing have to do with it?

In the range of Tm values, the selection of high annealing temperature can greatly reduce the non-specific binding between the primer and the template, and improve the specificity of PCR response.

The annealing time is generally 30 ~ 60sec, enough to combine the primer with the template.

Tm is necessary to set the annealing temperature of PCR.

Under ideal conditions, the annealing temperature is low enough to ensure that the primers are effectively annealed with the sequence of the target, and high enough to reduce nonspecific binding.

Proper annealing temperature from 50 ℃ to 70 ℃.

Annealing temperature is generally set 5 ℃ lower than the primers of Tm.

At the same time, the annealing temperature determines the PCR specificity and yield.

High temperature specificity, but too high primers can not be combined with the template, and the efficiency of DNA amplification decreases.

Low temperature yield is high, but too low can cause the primer and template mismatch, and non-specific products increase.

Therefore, we can set a series of gradient experiments to determine the optimal annealing temperature of a specific reaction.

By Tm first commonly - 5 ℃, 2 ℃ for incremental, gradually improve the annealing temperature.

At this point, it should be noted that the two primers should have approximate Tm values for optimal results.

Primers Tm differences if more than 5 ℃, will primers in a loop using low annealing temperature and showed obvious mistakes.

If two different primers Tm, to set the annealing temperature is 5 ℃ lower than the minimum of Tm.

Extension 3.

This is easier to understand.

Stretching temperature, such as ordinary Taq polymerase and the commonly used Pfu DNA polymerase, generally set to 72 ℃, the high fidelity of other enzymes, such as a KOD - plus is 68 ℃, the nature of the enzyme and the kinds of itself determines its temperature.

In general, the conventional DNA polymerase amplification rate is 1kb/ 60. When we amplify a 3400bp size fragment, the extended time can be set to 3min30s.

Duration = product length/extension speed, depending on the type of enzyme and the length of the extended segment, and then leave some redundant time.

In addition, when we extend the low concentration template, we can extend the extension time slightly.

The slow speed of extension (1kb/60s) inconveniences many users.

Now novoprotein paper improves the traditional DNA polymerase, new polymerase has characteristics such as rapid, high sensitivity, strong anti-jamming capability, can greatly shorten the expansion time, and let us have more time to read a paper / / doing experiments with the boss to discuss the scientific research.

Again, when we set the extension time, it should be determined based on the length of the extended segment, if the extended time is too long, it will lead to non-specific amplification.

For example, there used to be a friend with a rapid expansion of 2 x Super TaqMaster Mix (E034), extended Super fast speed, this enzyme is true to its claims to 1 KB / 15 s, amplification fragment of 700 bp, the junior partner For extension of time and with enough insurance in case one thousand, will extend the time set to 2 min, and sure enough, he witnessed the miracle of success, the electrophoresis results below, nonspecific banding and more bright.

So, when we expanded the DNA fragment within 1 KB, even the Taq enzyme and the Pfu enzyme in the face of "tortoise speed" would be enough to extend the time for 1min!

Do not doubt the strength of the DNA polymerase, or you may suspect that you have a few more markers...

(4) Number of cycles

The cycle of PCR determines the degree of amplification.

Generally, we set the number of cycles between 25 and 40 times.

The number of cycles depends on the concentration of the template DNA, and on the other hand, it is determined by the purpose of amplification.

When we need to expand the fragment to build the clone, we can set the number of loops to 30 or so to ensure the fidelity of the fragment.

When we only identify for PCR, we can set the number of loops to about 35.

Yes, I think you should find that when we don't have to think about the fidelity of the product, we can increase the number of cycles properly.

As the number of reaction cycles increases, the product increases, and the nonspecific products increase.

In addition, the DNA polymerase is not a hundred - dense, there is a certain error rate, the cycle is too high, so the chance of error in the replication process is higher.

(5) Operating environment

Here's operating environment is not allow you to read before doing the experiment a few sound "oh god, bless me, let me experiment done" this (though I often do), but when we add sample faster, and need to be done on the ice.

To do so on the one hand is to reduce the purpose of the template or other degradation products, on the other hand is also in order to reduce the nonspecific amplification, the possibility of reducing enzyme secretly work at room temperature (see the previous hot start enzyme).

(6) PCR instrument

Sometimes, the PCR instrument itself has some problems due to its aging and instability.

Sometimes the same device will have different results under the same conditions, so please remember to change an instrument when you perceive that the situation seems to be wrong.

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