Method and kit for constructing plasma DNA sequencing library

Abstract

The disclosure relates a method and a kit for constructing a plasma Deoxyribonucleic acid (DNA) sequencing library. The method provided by the disclosure includes: extracting a plasma DNA; making the plasma DNA ligate to a sequencing linker, and purifying a ligation product; performing Polymerase Chain Reaction (PCR) amplification for the purified ligation product, purifying the PCR amplification product, and obtaining the plasma DNA sequencing library, wherein, the method does not include the step of performing 5′-terminus phosphorylation for the plasma DNA. The kit provided by the disclosure includes: a reagent which ligates a plasma DNA to a sequencing linker, including the sequencing linker, a ligase and a ligation buffer; and reagents and instruments for purifying the ligation product; a reagent which performs PCR amplification for a purified ligation product, and reagents and instruments for purifying the PCR amplification product; wherein, the kit does not include the reagent which performs 5′-terminus phosphorylation for the plasma DNA. The disclosure simplifies the construction flow of a plasma DNA sequencing library, simplifies the experimental procedures, and makes the construction of the plasma sample library have lower cost, higher efficiency and faster speed, and is convenient for large-scale application.

Description

TECHNICAL FIELD OF THE APPLICATION[0001]The disclosure relates to a method and a kit for constructing a plasma Deoxyribonucleic acid (DNA) sequencing library, and in particular to a method and a kit for constructing a plasma DNA sequencing library for the second-generation high-throughput sequencing.[0002]Background of the application[0003]With the progress of science, the traditional Sanger sequencing cannot fully satisfy the needs of the research; the genome sequencing needs a sequencing technology which has lower cost, higher throughput and faster speed, so the second-generation sequencing technology is emerged at the right moment. The core idea of the second-generation sequencing technology is sequencing by synthesis, namely, to determine the DNA sequence by catching the mark of the newly-synthesized end; the existing technical platform mainly includes Roche / 454 FLX, Illumina / Solexa Genome Analyzer and Applied Biosystems SOLID system and the like. Taking Illumina product as an e...

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Benefits of technology

[0023]Based on the unexpected discovery that the terminus of DNA in plasma includes a monophosphate group, the disclosure omits the 5′-terminus phosphorylation which is necessary in the existing construction method of the plasma DNA sequencing library, and does not use the T4 PNK and the ATP; furthermore, on this basis, the disclosure performs the step of dA-overhang by using the normal Taq polymerase to replace the klenow ex-enzyme, thus the buffers of various reactions can be compatible; the steps of end-filling, dA-overhang and linker ligation can be perf

Problems solved by technology

The limitation of these methods above is that the mutation site is not common in the crowd, therefore, these methods are only suitable for a part of crowd.

The advantage of the dPCR is that it does not depend on any mutation site; however, the accuracy of dPCR is insufficient , and also requires a large amount of blood samples, which increases the difficulty in sampling.

But because of high sequencing cost, the technologies have not yet been widespread used at present.

Meanwhile, the way of detecting the change of local copy number of the embryonic chromosomes from the maternal blood is an unsolved problem.

There are some advantages to detect the change of copy number of the fetal chromosomes from maternal pl

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