Method for targeted capture and sequencing of HLA gene sequences

A gene sequence and targeted capture technology, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problems of artificially synthesized probe sequences, low resolution and accuracy, and high cost, and achieve the realization of The effect of high abundance and high diversity

CN109913539AInactive Publication Date: 2019-06-21ZHEJIANG UNIV
7 Cites 6 Cited by

Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2019-06-21
Estimated Expiration
Not applicable · inactive patent

Smart Images

  • Figure 1
    Figure 1
  • Figure 2
    Figure 2
  • Figure 3
    Figure 3
Patent Text Reader

Abstract

The invention relates to the field of biotechnology, and particularly provides a method for targeted capture and sequencing of HLA gene sequences. The method comprises the following steps: 1) hybridizing a denatured nucleic acid sample with a nucleic acid probe library fixed on a solid-phase carrier under a hybridization condition to form a target nucleic acid molecule-probe-solid-phase carrier membrane complex; 2) cleaning the 'probe-solid-phase carrier membrane complex' bound with target nucleic acid in step 1) by using a cleaning solution, eluting the probe-solid-phase carrier membrane complex bound with the target nucleic acid from a solid-phase carrier membrane by using an eluting solution, purifying, enriching or constructing to obtain a nucleic acid library; and 3) taking the nucleic acid library obtained by enrichment or constructed after enrichment in step 2) for high-throughput sequencing. A nucleic acid probe which can rapidly generate high-fold coverage and single-base displacement and is used for enriching HLA genes can be immovably fixed on the solid-phase carrier membrane. The method has characteristics of simplicity and convenience in operation, flexible probe acquisition and low cost, is favorable for improving the HLA genotype identification accuracy, and is rapid and convenient.
Need to check novelty before this filing date? Find Prior Art

Description

technical field

[0001] The invention relates to the field of biotechnology, and specifically provides a method for targeted capture and sequencing of HLA gene sequences. Background technique

[0002] Human Leucocyte Antigen (Human Leucocyte Antigen, HLA), also known as Major Histocompatibility Complex (MHC), this complex is the most complex human gene system known so far, located on the short arm of chromosome 6 , about 4000Kb in length, containing 224 loci, dozens of loci, and co-dominant expression, is the most complex genetic polymorphism system known to the human body. At the same time, each gene locus has dozens of alleles, such as A gene, B gene, and C gene related to immune rejection in the HLA-I family, which contain 3657, 4459, and 3290 alleles, respectively. HLA is expressed on the surface of human cell membranes and represents the antigen specificity of each individual. Therefore, HLA is closely related to the body's immune system, and the main cause of immune r...

Examples

Embodiment 1

[0098] Example 1 Obtaining the double-stranded nucleic acid library after fragments and quality inspection

[0099] 1. Probe performance analysis

[0100] 1. Perform similarity analysis on the cDNA sequences of the selected 14 genes and the corresponding HLA gene sequences in the IMGT HLA database (version 3.26).

[0101] 2. Comparing the similarity in units of exons, the minimum similarity is 85%. This result shows that the cDNA sequences of these 14 genes in the database can all be captured. The results are shown in the appendix figure 2 .

[0102] 2. Preparation of double-stranded nucleic acid library:

[0103] 1. Mix 14 kinds of nucleic acid solutions according to the same number of molecules, including HLA-I family genes and HLA-II family genes, wherein the HLA-I family genes are HLA-A*01:01:01, B*08:01:01, C*16:01:01, E*01:01:01, G*01:01:02, HLA-II family genes are DPA1*02:01:01, DPB1*01 :01:01,DQA1*01:03:01,DQB1*03:02:01,DRA*01:01:01,DRB1*13:03:01,DRB3*01:01:02,DRB...

Embodiment 2

[0110] Example 2 Construction of DNA sample library

[0111] 1. The DNA samples, a total of 31 samples, were applied from the China Bone Marrow Bank. The DNA samples were samples with known HLA genotypes, and the method used was PCR-SBT.

[0112] 2. Take 500ng of DNA sample and fragment the DNA with an ultrasonic instrument (Bioruptor). The conditions are 30s on / 90s off, 5 cycles, and the range of fragments generated is 300bp-600bp;

[0113] 3. Take 5 μl of the sonicated solution, add 1 μl of 6x loading Buffer, and check the size of the sonicated fragment by agarose gel electrophoresis, and the size is in the range of 300bp-600bp;

[0114] 4. For the remaining samples, use Ultra TM DNA Library Prep Kit for Illumina (E7645, NEB) was used to construct a sample DNA library, and the insert size range was 300-600bp.

Embodiment 3

[0115]Example 3 Enrichment of HLA gene sequences

[0116] 1. Preparation of single-stranded nucleic acid probe library:

[0117] 1. The preparation source of the single-stranded nucleic acid probe library is the double-stranded nucleic acid library in Example 1;

[0118] 2. Use alkaline denaturation method to denature the above double-stranded nucleic acid library and prepare at room temperature:

[0119] 24μl 500ng / μl DNA library

[0120] 12 μl 3M NaCl

[0121] 1.44 μl 10N NaOH

[0122] 3. Leave it at room temperature for 15 minutes to obtain a single-stranded nucleic acid probe library.

[0123] 2. Transfer the single-stranded nucleic acid probe to the solid-phase carrier membrane, so that the probe can be firmly combined with the positively charged nylon membrane:

[0124] 1. Cut about 0.3cm with scissors 2 Positively charged nylon membrane (NG0312, RPN3038, GE);

[0125] 2. Use a pipette gun to transfer the denatured solution containing the single-stranded nucleic a...