Template polynucleotide paired terminal sequencing method and kit

Abstract

The invention relates to the technical field of gene sequencing, in particular to a template polynucleotide paired terminal sequencing method and a kit. According to the invention, an extension primer for generating a complementary strand is specially designed to form an extension primer structure at least formed by connecting a first extension primer and a second extension primer into a whole, and a binding site of the first extension primer on template polynucleotide is designed to be located at the upstream of a binding site of the second extension primer. In an environment with strand displacement activity, the extension strand of the upstream extension primer displaces the extension strand of the downstream extension primer, and the displaced extension strand of the downstream extension primer is connected to the first extension primer through the second extension primer and then is combined to a template polynucleotide strand through the extension strand of the first extension primer, so that the template polynucleotide is obtained. The template polynucleotide chain is fixed on a solid support, so that the stability of the complementary chain in a cyclic sequencing environment is improved, and the sequencing quality is improved.

Description

technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a paired-end sequencing method and kit of template polynucleotides. Background technique [0002] Paired-end or paired-end sequencing techniques are well known in the field of analytical biology and allow the determination of two reads of sequence from two positions on a polynucleotide duplex. Since the paired-end sequences are not completely random, but are known to occur on a single double strand, they are connected or paired in the genome, which helps to combine the sequences of the whole genome into a consistent sequence, and improves the yield obtained by one sequencing. The length of the sequence information. [0003] Paired-end sequencing methods based on arrayed polynucleotide templates disclosed in the prior art include, as disclosed in WO2004 / 070005 implemented on a solid support, two or more primers are simultaneously hybridized to a target polynucleotide, Af...

AI Technical Summary

Problems solved by technology

During the implementation of this method, the immobilization of the formed complementary strand (second strand) is through the complementary hybridization of the part of the second strand near the 3-terminus with the template strand. This immobilization method is relatively poor in stability. It is easy to fall off during the process, reducing the sequencing signal and affecting the quality of sequencing

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