The method of directly using the pcr product of the leaching solution of the dgge rubber tapping strip for sequencing

Abstract

The invention belongs to the field of microbial ecology. In order to overcome the defect that direct sequencing is inaccurate and even unsuccessful because a PCR product of DGGE tapping band leachate always contains background products except for target fragments, the invention provides a method for treating DGGE tapping band leachate. The method comprises the steps of firstly, enabling most of target fragments in the leachate and a small quantity of polluted fragments mixed into the target fragments to form heteroduplexes by using denaturalizing and annealing steps; and then, excising the heteroduplexes by using a mismatch enzyme to greatly eliminate the polluted fragments and not greatly lose the content of the target fragments. A template is purified, so that a single PCR product is obtained through template amplification, then, sequencing can be directly carried out, furthermore, complex cloning links are avoided, and therefore, the accurate sequence information of the fragment contained in a band can be obtained rapidly, simply and conveniently at very low cost.

Description

technical field [0001] The invention belongs to the field of microbial ecology, and relates to a method for sequencing rDNA fragments contained in DGGE rubber-tapping strips, in particular to a method for sequencing directly using PCR products of the leachate of DGGE rubber-tapping strips. Background technique [0002] DGGE (denaturing gradient gel electrophoresis) is a DNA fingerprinting technique, which can separate DNA fragments of the same length but different sequences, and has become an important means of studying the diversity of microbial flora. Compared with the traditional research methods of microbiology, the biggest feature of DGGE is that it is based on the total DNA (or RNA) of the sample, so it can detect a large number of non-culturable microbial flora in the ecosystem. At the same time, this technology also has the advantages of high reliability, high reproducibility, convenience and quickness, so it is widely used in the analysis of microbial ecology in var...

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the deficiency that the PCR product of the DGGE rubber tapping strip leachate often contains background products outside the target fragment, resulting in inaccurate or even failure of direct sequencing; the present invention provides a treatment method for the DGGE rubber tapping strip leachate, which can eliminate the Miscellaneous other fragments, so that the PCR products of the processed leachate can be directly sequenced

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