DNA polymerase in PCR, have you chosen the right one?
CR (Polymerase Chain Reaction) is no stranger to laboratory dogs that run wild in laboratories all year round. So, have you chosen the right DNA polymerase, the core component of the PCR reaction?
Common DNA polymerases such as Taq, Phanta, Pfu, KOD, Primerstar, Klenow, Bst, Phi29, etc. According to heat resistance, it can be divided into heat-resistant enzymes and room-temperature enzymes. Thermostable polymerases such as Taq, Pfu, KOD, Primerstar, etc., and room temperature polymerases include Klenow, Bst, Phi29, etc. Divided according to fidelity, high-fidelity polymerases Pfu, KOD, Phanta, etc., and common polymerases Taq, etc.
Faced with so many choices, most freshmen who have just entered the laboratory silently express their fear of choice and commit...
So, the solution is to choose the appropriate DNA polymerase according to the purpose of the experiment. For example: for conventional PCR amplification and bacterial liquid identification, the fidelity requirements for fragments less than 5kb in length are not high, and Taq enzyme can be used to amplify them. Using the Mix format makes sample mixing simpler and operation more convenient. If the mixture contains a lot of foam and you want to eliminate the foam, it is recommended that you use Green Taq Mix. If you want to obtain higher yields, you can choose Taq Plus DNA polymerase, which has stronger amplification performance than Taq enzyme.
1. Choose hot start enzyme to reduce non-specific amplification
When using Taq enzyme for amplification, non-specific amplification sometimes occurs. Possible reasons can be checked one by one from the template, primer, system, and program. For example, whether the template is contaminated, how specific the primer is, whether the Mg2+ concentration is high, etc. One of the reasons that cannot be ignored is whether the type of DNA polymerase is appropriate. If there are still non-specific products after troubleshooting other reasons, you can choose a hot-start enzyme and re-experiment, which can effectively reduce or eliminate the accumulation of other products in the system. Commonly used hot-start enzymes are divided into two categories: one is a chemically modified hot-start enzyme, such as Ace Taq, which can release enzyme activity after pre-denaturation at 95°C for 5-10 minutes; the other is an antibody-modified hot-start enzyme. For example, Champagne Taq can release enzyme activity by pre-denaturing at 95°C for 30 seconds. Pay attention to the pre-denaturation time when using it to obtain satisfactory amplification results.
2. Select Lamp to amplify long fragments
How to amplify long fragments larger than 5Kb? If your experiment does not require high fidelity, then LAmp DNA Polymerase can be used. Its fidelity is 6 times that of Taq enzyme, and it can amplify genome and cDNA fragments exceeding 20Kb. It has a high success rate for templates with low copy, low purity, and different GC content.
3. Choose high-fidelity enzymes for rapid and efficient amplification
If the experiment requires high fidelity, you need to use high-fidelity enzymes when selecting DNA polymerase, such as Pfu, KOD, Primerstar, Phanta, etc. The first three are all first-generation high-fidelity enzymes and have been widely used in the market. The Phanta series of high-fidelity enzymes produced through vigorous transformation by Novozant's scientific research team have a fidelity that is 50 times that of Taq enzymes, and the amplification performance, amplification length, yield, and template adaptability have all been significantly improved. The fidelity of Phanta Max is 53 times that of Taq enzyme and 6 times that of Pfu. The effective amplification length of simple templates such as plasmids can reach 40kb, the effective amplification length of cDNA can reach 10kb, and the effective amplification length of genome can reach 20kb. It is suitable for Amplification of various GC contents can be used for direct PCR of a variety of samples such as plant leaves, whole blood, etc. And the amplification only takes 30s/kb, or even 2kb/s. So outstanding, you can’t put it down, right?
4. Select the direct amplification kit to amplify the crude product
Various direct amplification kits produced by xxx directly amplify crude products, greatly reducing the experimental work cycle. For example, One Step Mouse Genotyping Kit can be used directly for mouse genotyping, and Blood Direct PCR Kit V2 can be used directly for whole blood amplification.
