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Gene polymorphism region sequencing library and preparation method thereof

A technology for gene polymorphism and sequencing library, which is applied in the field of sequencing library and preparation of gene high polymorphism region, can solve the problems of low recovery rate, large starting amount, long time consumption, etc., and achieves the effect of high specificity

Inactive Publication Date: 2016-08-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former is limited by the unbalanced conditions of amplification, which will cause deviations in the detection of the CDR3 library; while the latter, due to the random interruption of the fragments, takes a long time, has a low recovery rate, and requires a large initial amount

Method used

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  • Gene polymorphism region sequencing library and preparation method thereof
  • Gene polymorphism region sequencing library and preparation method thereof
  • Gene polymorphism region sequencing library and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 4

[0034] In Example 4, the Agencourt AMpure XP magnetic beads used were purchased from Beckman Coulter (product number: A63880). The composition and pH of the buffer solution used in this embodiment are:

[0035] NEB buffer 4 (10×buffer) is composed of 500mM potassium acetate, 200mM trishydroxyaminomethane acetic acid, 100mM magnesium acetate, 10mM DTT, and the pH value is 7.9;

[0036] 10×S1buffer is composed of 500mM sodium acetate, 2.8M sodium chloride, 45mM zinc sulfate, and the pH value is 4.5;

[0037] S1stop buffer consists of 300mM Tris, 50mM EDTA;

[0038] TE solution consists of 10mM Tris-HCl, 1mM EDTA, pH 8.0.

[0039] The preparation process of the gene polymorphism region sequencing library of the present invention is as follows: figure 1 shown.

Embodiment 1

[0040] Example 1: Extraction of total RNA

[0041] The method for extracting total RNA comprises the following steps:

[0042] Step 1. Using Lymphoprep from AXIS-SHIELD TM solution (product number: 1114544) to extract peripheral blood mononuclear cells (peripheral blood mononuclear cell (PBMC)) from human peripheral blood, specifically divided into the following 7 steps:

[0043] (1) Blood collection needles collect peripheral venous blood from healthy adults and add it to a glass tube filled with 1% heparin solution. The volume ratio of heparin solution to whole blood is 1:20;

[0044] (2) Add an equal volume of 0.9% NaCl physiological saline solution to the glass tube of step (1), mix well, and dilute the blood;

[0045] (3) In a new 15ml centrifuge tube, add 3ml of Lymphoprep solution at the bottom, then carefully add 6ml of diluted blood to the top of the solution, taking care not to disturb the liquid layer;

[0046] (4) Cover the lid, put it into a hanging basket cent...

Embodiment 2

[0060] Example 2: Synthesizing the second strand of cDNA of TCR

[0061] Synthesizing the cDNA second strand of TCR includes the following steps:

[0062] Step 1. Using the total RNA as a template, use the Target Switch method to reverse transcribe and synthesize the first strand of TCR full-length cDNA (the Target Switch method mainly uses the 3-base G cap that only the complete 5' sequence mRNA has, so that one with a complementary The primer with 3 G bases is used as a template, and is complementary to the first strand of the reverse-transcribed cDNA, allowing the reverse transcriptase to continue reverse transcription, so as to obtain a complete 5'-end cDNA with a specific fragment at the 5'-end One strand of cDNA.), specifically including the following 3 steps:

[0063] (1) Prepare the following mixed reaction solution:

[0064]

[0065] Mix the reaction solution (the total amount of RNA in the reaction solution is 1 μg, if the volume of 1 μg of RNA is less than 4 μl...

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Abstract

The invention relates to a gene polymorphism region sequencing library and a preparation method thereof and belongs to the field of molecular biology. The preparation method of the gene polymorphism region sequencing library comprises the following steps: firstly, designing a primer according to characteristics of a gene polymorphism region, and carrying out PCR (polymerase chain reaction) amplification, so that a DNA fragment containing a conserved region and a polymorphism region is obtained; secondly, adding exonuclease into the DNA fragment obtained through PCR amplification, controlling concentration of exonuclease and reaction time or controlling the reaction temperature and reaction time of exonuclease, and hydrolyzing the DNA fragment; thirdly, adding single-chain nuclease into hydrolysis products of the DNA fragment, and digesting protruded single chains of DNA, so that a mixed DNA fragment is obtained. The method for preparing the gene polymorphism region sequencing library has high specificity, is quick and can be used for sequence-based typing of high polymorphism regions.

Description

technical field [0001] The invention relates to a sequencing library and a preparation method thereof, in particular to a sequencing library of a highly polymorphic region of a gene and a preparation method thereof. Background technique [0002] There are many traditional methods for studying highly polymorphic regions, such as: (1) separating different biological DNA molecules by electrophoresis, and then hybridizing with them with labeled specific DNA probes, by autoradiography or non-isotope color development (2) use random primers (generally 8-10bp) to amplify DNA fragments non-specifically through PCR reaction, and then use gel electrophoresis to analyze the amplified DNA fragments. Randomly amplified polymorphic DNA (RAPD) that amplifies the polymorphism of the DNA fragment of the product; (3) The PCR labeling method of specific primers, which is designed for the DNA region of the known sequence to have a specific nucleotide sequence, which can be used in conventional ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C40B40/06
Inventor 徐安龙付永贵颜庆瑜
Owner SUN YAT SEN UNIV