I believe that everyone will always encounter such or such problems when doing PCR reactions, but most of them can be classified into two main problems:

Too little amplification of the gene template (amplification);
Too much non-target gene amplification.
Using additives is one of the common strategies to solve these problems. Usually the role of additives has two aspects:
secondary structure of genes (secondary structure);
Reduce non-specific priming.
Today, the editor will briefly introduce to you the common additives in PCR reactions and their functions.
Additives that reduce secondary structure
sulfoxide (DMSO)
gene samples with high GC content . However, DMSO also greatly reduces Taq polymerase activity. Therefore, everyone has to balance the template accessibility and the activity of the polymerase. The editor suggests that you can try different concentrations of DSMO, such as from 2% to 10%, to find the concentration that suits your experiment.
Non-ionic detergents
Non-ionic detergents, such as 0.1-1% Triton X-100, Tween 20 or NP-40, usually reduce DNA secondary structure. Although this can increase the amplification of the template gene, it will also cause the trouble of non-specific amplification. So, these additives work well for low-yield PCR reactions without debris, but not so well for relatively impure PRC reactions. Another benefit of non-ionic detergents is the reduction of SDS contamination. Usually during the DNA extraction process, SDS will be brought to the PCR step, which greatly inhibits the activity of the polymerase. Therefore, adding 0.5% Tween-20 or Tween-40 to the reaction can neutralize the negative effects of SDS.
Betaine _
Betaine can improve DNA amplification by reducing secondary structure formation and is generally a “mystery” addition to commercial PCR kits. If you want to use betaine, you should put betaine or betaine mono-hydrate (Betaine or Betaine mono-hydrate), but not betaine hydrochloride (Betaine HCl), adjust to a final concentration of 1-1.7M. Betaine can also help improve specificity because it eliminates the base pair composition dependence of DNA melting/DNA denaturation.
Additives to reduce non-specific priming
Formamide
Formamide is a commonly used organic PCR additive. It can combine with major groove and minor groove in DNA, thereby reducing the stability of the master DNA double helix and lowering the melting temperature of DNA. The concentration of formamide used in PCR experiments is usually 1%-5%.
Tetramethyl ammonium chloride ( TMAC)
Tetramethylammonium chloride can increase the specificity of hybridization (hybridization specificity) and increase the melting temperature of DNA. Thus, TMAC can remove non-specific priming and reduce misbinding of DNA and RNA. If you use degenerate primers in the PCR reaction , remember to add TMAC, which is commonly used at a concentration of 15-100mM.
Other Common Additives
In addition to the two categories of additives mentioned above, there are many common additives in PCR reactions, although they have different functions, they are also very important.
Magnesium ion
Magnesium ion is an indispensable cofactor (cofactor) of polymerase, that is to say, without magnesium ion, polymerase is inactive. However, too much magnesium ions can also affect the efficiency of the polymerase. The concentration of magnesium ions in each PCR reaction will vary. Chelating agents (such as EDTA or citrate), the concentration of dNTPs and proteins all affect the concentration of magnesium ions. So, if you have problems with your PCR experiment, you can try to change different magnesium ion concentrations, for example, from 1.0 to 4.0mM, with an interval of 0.5–1mM in between.
It is worth noting that multiple freeze-thaw cycles can lead to concentration stratification of the magnesium chloride solution . Therefore, you must dissolve it completely before each use, and mix it well before using it.
Bovine serum albumin (Bovine albumin, BSA)
In molecular chemistry experiments, bovine serum albumin is a very common additive, especially in restriction enzyme digestion and PCR experiments. In PCR reactions, BSA is helpful in reducing contaminants such as phenolic compounds . And it is also said that it can reduce the adhesion of reactants to the wall of the test tube. In the PCR reaction, usually the concentration of BSA added can reach 0.8 mg/ml.
 
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