Multi-sample mixed sequencing method and kit

A hybrid sequencing and kit technology, used in biochemical equipment and methods, combinatorial chemistry, organic compound libraries, etc., can solve problems such as low mixing uniformity, and achieve the effects of increasing uniformity, shortening operation time, and simplifying steps.

Active Publication Date: 2011-09-14
HANGZHOU BERRYGENOMICS GENE DIAGNOSIS TECH
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  • Abstract
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  • Application Information

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Problems solved by technology

Sample mixing uniformity is also low

Method used

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  • Multi-sample mixed sequencing method and kit

Examples

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Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of the second-generation high-throughput sequencing library

[0036] The following is a brief description of the preparation steps of the second-generation multi-sample hybrid sequencing library:

[0037] DNA fragments that break to a specified range;

[0038] End filling and 5' end phosphorylation: jointly completed by the enzyme Klenow (New England Biolabs), T4 phosphorylase and DNA polymerase, and then the product was cleaned and purified;

[0039] Overhang A at the end: under the action of klenow ex-(New England Biolabs) (an improved Klenow enzyme, its 3'-5'exocutting activity is lost), the product of the previous step is overhanging the A base at the end of the double strand, The product is then cleaned and purified;

[0040] Connection: According to the different sources of samples and the planned channels to connect different adapters, T4DNA ligase is required, and different adapters have different tag sequences in Table 5 to distinguish ...

Embodiment 2

[0095]Embodiment 2: Kit for preparing multi-sample DNA mixed sequencing library

[0096] The kit for preparing a multi-sample DNA mixed sequencing library provided by the present invention mainly includes the following components:

[0097] Enzymes for repairing fragmented DNA ends and enzymes for 5' phosphorylation of DNA ends, including enzymes Klenow, T4 phosphorylase and DNA polymerase, as well as buffers required by various enzymes;

[0098] Enzymes that add adenine to the 3' end of DNA, such as klenow ex-(3'-5'exocutting activity loss), and dATP;

[0099] An enzyme such as DNA ligase that ligates the Y-linker to DNA;

[0100] Buffers suitable for various enzymes such as DNA ligase buffer, Klenow enzyme buffer and T4 DNA ligase buffer (NEB);

[0101] Y-type adapter mixture, including a variety of Y-type adapters, the tag sequences included in each Y-type adapter are different, such as various tag sequences listed in Table 5, including a Y-type adapter pair of a tag seque...

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Abstract

The invention provides a method for preparing a multi-sample deoxyribonucleic acid (DNA) mixed sequencing library, which comprises the following steps of: converting a DNA chain fragment of a sample into a DNA fragment which is hundreds of basic groups long; connecting upper Y-shaped joints at both ends of the DNA fragment; performing polymerase chain reaction (PCR) amplification of the DNA fragment connected with the upper Y-shaped joints; and mixing DNAs of various samples according to the arrangement of a computer sequencing channel and the difference between label sequences, wherein the Y-shaped joints comprise label sequences for distinguishing different samples; and a first primer for the PCR amplification and a second primer for the PCR amplification, besides sequences which are complementary to the Y-shaped joints, also comprise sequences which are complementary to two surface primers in a sequencing platform of the second generation respectively, so that the DNA fragments obtained by the amplification can hybridize with the surface primers complementarily in the sequencing process to start the process of sequencing while synthesizing.

Description

technical field [0001] The present invention relates to a method for multi-sample mixed sequencing, and more specifically, to a method for constructing a multi-sample high-throughput sequencing library and a kit used therefor. Background technique [0002] DNA sequencing technology has opened the door for humans to deeply study the genetic code of life, and it has played a vital role in advancing the development of molecular biology since its invention. [0003] Frederick Sanger invented the end-termination sequencing in the mid-1970s. The Sanger method became the mainstream of DNA sequencing at that time because it was simple and fast, and it was continuously improved. [0004] However, with the development of science, traditional Sanger sequencing can no longer fully meet the needs of research. For genome sequencing, sequencing technology with lower cost, higher throughput, and faster speed is needed, and second-generation sequencing technology has emerged as the times req...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C40B50/06C40B40/06
Inventor 周代星张建光高扬王珺刘卿
Owner HANGZHOU BERRYGENOMICS GENE DIAGNOSIS TECH
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