Joint connection-based deoxyribonucleic acid (DNA) polymerase chain reaction (PCR)-free tag library construction method

Abstract

The invention designs 161 unique tag sequences with the lengths of 8 bp. Tags are embedded into a deoxyribonucleic acid (DNA) joint so as to form a DNA polymerase chain reaction (PCR)-free tag joint. The invention also provides a DNA tag library which introduces the tag sequence by connecting the DNA PCR-free tag joint without a PCR and is applied to solexa DNA sequencing. After a library construction method is optimized, the DNA tag library is constructed without the PCR.

Description

technical field [0001] The invention relates to the technical field of nucleic acid sequencing, in particular to the technical field of DNA PCR-Free tag library based on linker ligation. In addition, the present invention also relates to labeling technology and a method for realizing library construction of multiple samples in the same reaction system. The method of the present invention is particularly suitable for the second generation sequencing technology, especially the solexa sequencing technology. Background technique [0002] The Solexa DNA sequencing platform provided by Illumina can simultaneously add four kinds of fluorescently labeled nucleotides in one reaction, and adopts Sequencing By Synthesis (SBS), which requires less sample volume and high throughput. High accuracy, with the characteristics of simple and easy-to-operate automation platform and powerful functions [1-4]. Library construction first needs to perform end repair on the target fragment, connect...

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Problems solved by technology

[0004] However, there are some defects in the method for preparing the tag library provided by Illumina: first, at present, Illumina only provides 12 tag sequences with a length of 6 bp, and the number of tags is small. Mixed sequencing of samples will be a huge defect; second, the current label library construction method provided by Illumina is to import the label sequence into the target fragment library through PCR reaction, which requires 3 PCR primers to amplify the target fragment (two Common primers and a PCR index primer, as shown in Table 1), and the PCR amplification efficiency is not high

Third, the linker in the label library construction method provided by illumina does not contain the label sequence. Each label library needs to pass a PCR reaction to import the label sequence, and then needs to cut the gel for each label library. Tagged target fragment libraries are then pooled, which is not only time-consuming, but also expensive

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