DNA(deoxyribonucleic acid) index library building method based on PCR (polymerase chain reaction)

A construction method and labeling technology, which is applied in the field of DNA library construction, can solve the problems of low PCR amplification efficiency, small number of labels, and high cost, and achieve the effects of increasing sequencing throughput, improving efficiency, and reducing sequencing costs

Active Publication Date: 2012-04-11
BGI TECH SOLUTIONS
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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
[0005] 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 for each label library, it needs to be cut and recovered, and then the glue is recovered. It is not only time-consuming and labor-intensive, but also expensive

Method used

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  • DNA(deoxyribonucleic acid) index library building method based on PCR (polymerase chain reaction)
  • DNA(deoxyribonucleic acid) index library building method based on PCR (polymerase chain reaction)
  • DNA(deoxyribonucleic acid) index library building method based on PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: The specific method of DNA tag experiment library construction

[0079] 1.1 DNA fragmentation

[0080] Interrupt 5ug of human whole blood genomic DNA using Covaris for 6 minutes (parameter setting: Duty cycle (duty ratio) -20%; Intensity (intensity)-5.0; Bursts persecond (pulse per second)-200; Duration ( Duration)-40 seconds; Mode-Frequency sweeping; Power-33-34W; Temperature-5.5 to 6℃) to make it the main bar displayed in agarose electrophoresis The band is concentrated around 200bp [5].

[0081] 1.2 End repair

[0082] Prepare the reaction mixture according to the following ratio:

[0083] DNA template 35μL

[0084] T4 DNA Ligase Buffer 50μL

[0085] dNTPs mixture 4μL

[0086] T4 DNA polymerase 5μL

[0087] Klenow DNA polymerase 1μL

[0088] T4 polynucleotide kinase 5μL

[0089]

[0090] Total volume 100μL

[0091] Adjust the comfort thermomixer to 20°C, react for 30 minutes, and then use QIAquickPCR purification kit for purification, ...

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Abstract

The invention designs 161 unique index sequences with a length of 8bp, and the indexes are embedded into DNA PCR (deoxyribonucleic acid polymerase chain reaction) primers to form DNA PCR index primers, thus being capable of importing the index sequences through PCR. The invention successfully establishes a method for building a DNA index library, and the method is applied to solexa DNA sequencing.

Description

Technical field [0001] The invention relates to the technical field of DNA library construction, in particular to the technical field of DNA tag library construction, in particular to a PCR-based DNA tag library construction method. In addition, the present invention also relates to labeling technology and a method for implementing library construction of multiple samples in the same reaction system. The method of the present invention is particularly suitable for second-generation sequencing technology, especially solexa sequencing technology. Background technique [0002] The Solexa DNA sequencing platform provided by Illumina can add four fluorescently-labeled nucleotides to one reaction at the same time, using Sequencing By Synthesis (SBS), which has a small sample volume, high throughput, and high throughput. Accuracy, with a simple and easy-to-operate automation platform and powerful functions [1-4]. The library construction first needs to repair the end of the target fra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B40/06C40B50/06
CPCC40B50/06C40B40/06C12Q1/6869C12N15/1065
Inventor 章文蔚于竞龚梅花张艳艳田方陈海燕周妍刘涛
Owner BGI TECH SOLUTIONS
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