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DNA molecular label technology and DNA incomplete interrupt policy-based PCR sequencing method

A sequencing and primer labeling technology, applied in recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of inability to detect PCR products and obtain DNA sequence information

Active Publication Date: 2010-12-22
BGI GENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Taking Illumina GA (Illumina's Genome Analyzer sequencer, Illumina GA for short) as an example, the current maximum sequencing length of Illumina GA is 200bp. When the length of the PCR product is greater than 200bp, Illumina GA sequencing is used to directly adopt the method of PCR sequencing. Detect the entire PCR product and fail to obtain the entire DNA sequence information of the detected PCR product
The short sequencing read length limits the application of second-generation sequencing technology. In addition to gradually improving the sequencing technology to obtain longer actual sequencing read length, the development can overcome the existing sequencing read length of the second-generation DNA sequencer in the application of PCR sequencing. New technologies that are insufficient in the field have also become a top priority

Method used

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  • DNA molecular label technology and DNA incomplete interrupt policy-based PCR sequencing method
  • DNA molecular label technology and DNA incomplete interrupt policy-based PCR sequencing method
  • DNA molecular label technology and DNA incomplete interrupt policy-based PCR sequencing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Sample extraction

[0050] DNA was extracted from 95 blood samples with known HLA-SBT typing results (Chinese Hematopoietic Stem Cell Donor Database (hereinafter referred to as "Chinese Bone Marrow Bank")) using KingFisher automatic extractor (Thermo Company, USA). The main steps are as follows: Take out 6 deep-well plates and 1 shallow-well plate matched with Kingfisher automatic extractor, add a certain amount of reagents according to the instructions and mark them, and place all well plates with reagents as required At the corresponding position, select the program "Bioeasy_200ul Blood DNA_KF.msz" and press "star" to execute the program for nucleic acid extraction. After the end of the program, collect about 100ul of the elution product in the plate elution is the extracted DNA.

Embodiment 2

[0052] PCR amplification

[0053] Different PCR tag primers are made by synthesizing PCR primers with different primer tags at the 5'end, so that different PCR tag primers can be used for different samples. The PCR primers are for HLA-A / B 2, 3, 4 PCR primers for exon number and HLA-DRB12 exon. Afterwards, primer tags were introduced at both ends of the PCR product through PCR reaction, thereby specifically marking PCR products from different samples.

[0054] 95 sets of PCR tag primers were used to amplify 95 DNA samples. Each set of PCR tag primers consists of a pair of bidirectional primer tags (Table 1) and exons 2, 3, 4 and HLA-A / B. The PCR primers of HLA-DRB12 exon (Table 2) consist of a pair of primer labels connected to the 5'end of each forward PCR primer, and a pair of primer labels connected to the 5'end of the reverse PCR primer. Reverse primer label to primer label. The primer tag is directly added to the 5'end of the PCR primer when the primer is synthesized.

[0055...

Embodiment 3

[0077] PCR product mixing and purification

[0078] Take 20ul each of the remaining PCR products from the 96-well HLA-P-A2 (except the negative control) and mix them in a 3ml EP tube, labeled HLA-A2-Mix, and do the same for the other 6 96-well plates , Respectively marked as HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix and HLA-D2-Mix, shake and mix, from HLA-A2-Mix , HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix and HLA-D2-Mix each take 200ul and mix in a 3ml EP tube, mark For HLA-Mix, 500ul DNA mixture is taken from HLA-Mix and purified by Qiagen DNA Purification kit (QIAGEN) (see the instructions for specific purification steps). The 200ul DNA purified is purified by Nanodrop 8000 (Thermo Fisher Scientific Company) determined the HLA-Mix DNA concentration to be 48ng / ul.

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Abstract

The invention provides a PCR sequencing method. The length of a PCR product which can be completely measured by a sequencer exceeds the measuring length of the sequencer through the combination of a primer label, a DNA incomplete interrupt policy and second-generation sequencing technology (Pair-End sequencing technology) and by taking full advantage of the characteristics of high flux and low cost of a second-generation sequencer at the same time, so that the application range of the method is greatly widened. At the same time, the invention also provides the primer label for the PCR sequencing method and the application of the method to gene typing, particularly to HLA analysis.

Description

Technical field [0001] The present invention relates to the field of nucleic acid sequencing technology, in particular to the field of PCR sequencing technology. On the other hand, the present invention also relates to the technical field of PCR-index / barcode. The method of the present invention is particularly suitable for the second-generation sequencing technology, especially the Pair-end sequencing technology in the second-generation sequencing technology, and can also be used for HLA genotyping. Background technique [0002] The PCR sequencing method is to obtain the target gene DNA fragments by PCR, and then to obtain the target gene DNA sequence information through the DNA sequence detection of the obtained target gene DNA fragments. It is intuitive and accurate. The characteristics of it have long been widely used in the fields of gene mutation detection and genotyping. [0003] PCR-index / barcode technology, by adding a primer index sequence at the 5'end of PCR primers, a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2537/149C12Q2537/159
Inventor 李剑刘涛赵美茹张现东
Owner BGI GENOMICS CO LTD
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