Topo1 connection method for improving compatibility and efficiency of PCR (Polymerase Chain Reaction) segment

Abstract

The invention discloses a Topo1 connection method for improving compatibility and efficiency of a PCR (Polymerase Chain Reaction) segment. The method comprises the following steps: putting a PCR amplification product without phosphorylation or modification at a 5'-end into a connection conversion system, and carrying out a reaction for 4-6 minutes at 20-30 DEG C, wherein the connection conversionsystem comprises a blunt terminalization factor. By adopting the Topo1 connection method for improving the compatibility and the efficiency of the PCR segment, one connecting system can be adopted tocarry out experiments no matter whether a PCR product is TA or a blunt end, the Topo Ta connection efficiency can be improved, the time can be shortened and the cost can be reduced for scientific researchers greatly, and the method can be applied to molecular biologics and first-generation sequencing.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a Topol ligation method for improving the compatibility and efficiency of PCR fragments. Background technique [0002] With the increasing development of molecular biology and molecular diagnosis, the traditional restriction enzyme digestion and T4 ligation have higher requirements for PCR products, the ligation reaction time is long, the positive rate is low, and the self-ligation rate is high, which will increase exponentially. valuable time and cost of researchers. In order to make up for this defect, a technology that utilizes vaccinia virus DNA topoisomerase 1 to connect DNA fragments, namely TOPO cloning technology, has been developed. [0003] There are only two sources of PCR fragments. One is the DNA fragment amplified by Taq enzyme. Since there is an extra A base at the 3' end, it can only be connected by TA; the other is the DNA fragment amplified by high-fidelity enz...

AI Technical Summary

Problems solved by technology

At present, there are two simple Topo ligation kits on the market, one is the Topo TA kit for the DNA fragments amplified by the above Taq enzyme, and the other is the Topo blunt ligation kit specifically for blunt; the two reagents At the same time, our experimental data found that because Topo1 has a very high selectivity for the structure of the dsDNA end, in the kit for preparing TA, due to the structure of the terminal DNA, the efficiency of the Topo TA ligation kit is very high. low, which is 20% of the original connection efficiency

[0004] Due to the different needs of each researcher, the source of the insert fragment, that is, the source of the PCR amplification product is diverse, so some researchers must use Taq enzyme for PCR amplification, while some researchers must use high-fidelity enzyme for PCR amplification. However, common TOPO cloning kits on the market, such as pEASY-TA ZeroCloning Kit (only for TA) and pEASY-Blunt ZeroCloning Kit (only for blunt ends), have their own limitations and cannot be compatible with both TA and peace end cloning

In many cases, scientific researchers can only try one by one when their samples are precious and do not know whether they are TA or blunt-ended, which greatly increases the experimental risk, time and cost of researchers.

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