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High-throughput library construction kit and library construction method for detecting thalassemia gene mutation

A technology for thalassemia and library construction, applied in the field of high-throughput library construction kits, to achieve long experimental procedures, simple operation steps, and cost reduction

Pending Publication Date: 2020-09-11
广州赛乐斯密医学科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these detection methods based on single-site PCR technology have certain limitations.

Method used

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  • High-throughput library construction kit and library construction method for detecting thalassemia gene mutation
  • High-throughput library construction kit and library construction method for detecting thalassemia gene mutation
  • High-throughput library construction kit and library construction method for detecting thalassemia gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Kit

[0069] 1) Sample information to be tested:

[0070] Using the kit and method provided by the present invention, the target gene fragment enrichment second-generation sequencing is performed on 3 α-thalassemia samples, 2 β-thalassemia samples, and 1 normal individual sample, and biological information analysis is performed on the sequencing results.

[0071] Table 1 Gene mutation information of five thalassemia samples

[0072] Sample number gender Illness Type of gene mutation CASE01 Female alpha thalassemia–– SE A / α WS

CASE02 Female alpha thalassemia–– SEA / -α 4.2

CASE03 male alpha thalassemia-α3.7 / α CS

CASE04 Female β-thalassemiaCD41–42(–CTTT) / –28(A> G) CASE05 male β-thalassemiaChinese(Aγδβ)0- / IVS–II–654(C> T) CONTROL male normal No mutation

[0073] 2) Kit components:

[0074] (1) DNA denaturation solution: buffer solution containing denaturant;

[0075] Preferred conditions: 0.6875×TE, pH8.0; 1.25×GC solution.

[0076] (2) Primer probe mixture...

Embodiment 2

[0094] Example 2. Experimental process

[0095] The library construction method for detecting thalassemia gene mutations provided by the present invention includes the following steps:

[0096] S1, provide a reaction system

[0097] The reaction system includes: a sample to be tested and components of the above kit;

[0098] among them:

[0099] The sample to be tested is a human nucleic acid sample, and the number of samples to be tested is multiple. In this case, 6 blood DNA samples of thalassemia patients were selected. The total amount of target nucleic acid fragments of the thalassemia gene in the sample is 300 ng. In this case, 54 pairs of primer probe sets targeting 28 thalassemia gene mutation sites were selected.

[0100] The 5'extension primer cannot be degraded by the 5'end direction exonuclease, but can be degraded by the 3'end direction exonuclease; the 5'end of the 5'extension primer has protection against exonuclease degradation Group.

[0101] The 3'block probe cannot ...

Embodiment 3

[0191] Example 3 Data analysis of experimental results

[0192] Sort the sequencing data according to different tag sequences to obtain the sequencing data of each sample. Determine the sequencing depth of each target and reference fragment by BLAST with the reference amplification sequence, and count the total sequencing amount of each sample, each target and reference fragment The sequencing depth and the enrichment efficiency of each sample. Sequencing of non-deletion mutation-specific amplified fragments READS first removes the primer probe sequence and the 3 base sequences near the primer probe on the 2 side of the amplified sequence, and then performs subsequent SNV / INDEL analysis. The sequencing depth data of non-deletion mutation-specific amplified fragments Used for copy number analysis of each gene fragment.

[0193] 1) SNV / Indel analysis: Sequencing data is analyzed by second-generation sequencing data analysis software such as BWA, GATK, ANNOVAR, etc. to obtain sequenc...

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Abstract

The invention relates to a high-throughput library construction kit for detecting thalassemia gene mutation. The kit comprises multiple pairs of primer probe sets corresponding to nucleotide sequencesin a sample to be detected, exonuclease, nucleic acid ligase, nucleic acid polymerase and other library building reagents. The invention also relates to a library construction method for detecting thalassemia gene mutation. The method comprises the following steps of: 1, providing a reaction system; step 2, performing hybridization; 3, conducting enzyme digestion and purification; 4, extending, connecting and purifying; and 5, enriching. The kit and the library construction method provided by the invention have the advantages of whole genome library construction, simplicity in operation, short experimental steps, low cost, high accuracy, enrichment efficiency up to 80% or above and the like.

Description

Technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a high-throughput library construction kit and a library construction method for detecting thalassemia gene mutations. Background technique [0002] Thalassemia (Thalassemia for short) is caused by a defect in the globin gene, which leads to the reduction or absence of the synthesis of the globin chain, and the imbalance of the α-chain / non-α-chain that forms hemoglobin. Genetic inheritance, hemolytic disease. Mild cases may have no clinical manifestations, and severe cases are mainly characterized by progressive anemia. According to the type of inhibition of globin peptide chain synthesis, thalassaemia can be divided into α-thalassaemia, β-thalassaemia, δ-thalassaemia, γ-thalassaemia, δβ-thalassaemia and εγδβ-thalassaemia. Clinically, thalassemia is divided into three types: light type (thalassemia gene carrier), intermediate type and severe type. [0003] Alpha-thalassemia i...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6858
CPCC40B50/06C12Q1/6858C12Q2535/122
Inventor 徐湘民滕祥云李琦黄少亚周姗阳治国陆世鑫
Owner 广州赛乐斯密医学科技有限公司
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