Nucleic acid capture probe for hla typing and its design method

A nucleic acid capture and probe technology, applied in biochemical equipment and methods, sequence analysis, recombinant DNA technology, etc., can solve problems such as optimization, large differences, and non-consideration, and achieve high-efficiency capture

Active Publication Date: 2020-02-21
上海仁东医学检验所有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the INDEL of small fragments has not been considered in the existing technology. The GC ratios of the Exon2 and Exon3 regions of the HLA genes are both above 60%, which is quite different from other regions on the human genome. The existing technology does not because of this difference. There are experimental optimizations

Method used

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  • Nucleic acid capture probe for hla typing and its design method
  • Nucleic acid capture probe for hla typing and its design method
  • Nucleic acid capture probe for hla typing and its design method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Design of nucleic acid capture probes for HLA typing

[0031] 1) According to the full lengths of the known subtypes of HLA genes (the length of most HLA genes is 3503bp), construct the sequence libraries of HLA-A, HLA-B, and HLA-C respectively.

[0032] 2) Use DECIPHER (from the R package of bioconductor) to perform multiple sequence alignments to find the main SNP sites on Exon2 and Exon3 of HLA-A, HLA-B, and HLA-C genes and within 100 bp upstream and downstream.

[0033] Taking Exon2 of the HLA-A gene as an example, the specific comparison steps are as follows:

[0034] ① Load the DECIPHER package——library(DECIPHER);

[0035] ②Use readDNAStringSet to read the fasta file of HLA-A——hla_A.fa;

[0036] ③Use the AlignSeqs function to align the read sequences;

[0037] ④Use writeXStringSet to write the aligned DNA sequence into a new fasta file, and the new fasta file is the result of MSA.

[0038] After alignment, a Multiple Sequence Alignment (MSA) alignmen...

Embodiment 2

[0065] Example 2 Nucleic acid capture effect verification

[0066] DNA was extracted from 23 normal tissue samples, and the probes designed in Example 1 above were used for hybridization and capture of HLA genes. The library construction kits Accel-NGS ® 2S Hyb DNA Library Kit (Cat. No. 23024 / 23096) and 2S Set A / B Indexing Kit (Cat. No. 26148 / 26248) from Swift Company were used for library construction.

[0067] The operation steps of hybrid capture are as follows:

[0068] 1. gDNA fragmentation and purification

[0069] 1) Take 500ng gDNA according to the concentration of Qubit, add water to make up to 100μl, add covaris 130μl interrupted tube, set the program: 50W, 20%, 200 cycles, 330s. Take 1 μl after the interruption, and use the Qsep100 automatic nucleic acid and protein analysis system to detect the fragment distribution, and the main peak is 150-200 bp.

[0070] 2) Transfer the interrupted product to a new 1.5ml centrifuge tube, add 1.4 times the volume of AMPure b...

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Abstract

The invention discloses a method for designing a nucleic acid capture probe for HLA typing. The method includes the steps: 1) constructing HLA-A, HLA-B, and HLA-C sequence libraries; 2) performing multiple sequence alignment, searching for SNP loci on Exon2 and Exon3 and upstream and downstream of each gene; 3) using a sliding window separation algorithm to select regions that cover a predetermined number of SNP loci as probe design candidate regions; 4) performing cluster analysis to obtain the representative sequences of the respective probe design candidate regions as candidate probes; and5) deduplicating the all candidate probes to obtain the respective capture probes of the three genes. The invention also discloses a probe designed by the above method, and its sequence is shown in SEQIDZNO: 9-88. The method designs the nucleic acid capture probes for all polymorphic loci in the Exon2 and Exon3 regions with a GC ratio of more than 60% in the HLA gene, thereby improving the effectof hybridization capture of the HLA gene probe.

Description

technical field [0001] The present invention relates to the field of gene sequencing, in particular to the design of probes, more specifically, to nucleic acid capture probes for HLA typing and a design method thereof. Background technique [0002] High-throughput sequencing is also known as next-generation sequencing. In recent years, with the advancement of high-throughput sequencing technology, the cost of sequencing has been decreasing, the objects of sequencing services and application segments have continued to expand, and the market size of high-throughput sequencing has continued to increase. With favorable policy conditions, the clinical application of high-throughput sequencing technology in reproductive health and tumor personalized medicine has entered the fast lane, with broad application prospects. [0003] The sensitivity of NGS (next-generation gene sequencing) detection technology is much higher than that of the current traditional detection technology. It c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B30/10C12Q1/6827C12N15/11
CPCC12Q1/6827G16B30/10C12Q2531/113
Inventor 赵国栋乔宗赟
Owner 上海仁东医学检验所有限公司
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