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A kit and method for detecting chromosome heterozygosity loss based on amplicon sequencing

A loss of heterozygosity, kit technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of chromosome deletion, non-disclosure and implication, high and low gene expression, etc. Achieve the effect of increasing the amount of DNA and reducing the cost of detection

Active Publication Date: 2019-11-19
艾普拜生物科技(苏州)有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene loci disclosed in prior art documents 1 and 2 are not all deleted on the chromosomes of different cells, but some cells have LOH, that is to say, the above-mentioned detected gene loci cannot determine whether the chromosome has occurred The state of complete deletion, but reflects the change of gene expression level
[0003] Any one of these prior art documents does not disclose or imply that amplicons are sequenced and the coverage (RAEDS number) of each segment to be detected on the chromosome is counted through the sequencing results and based on RAEDS (long arm) / RAEDS (short arm) or RAEDS(short arm) / RAEDS(long arm) ratio to determine the exact status of chromosomal loss of heterozygosity

Method used

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  • A kit and method for detecting chromosome heterozygosity loss based on amplicon sequencing
  • A kit and method for detecting chromosome heterozygosity loss based on amplicon sequencing
  • A kit and method for detecting chromosome heterozygosity loss based on amplicon sequencing

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Embodiment 1

[0025] Embodiment 1 kit of the present invention

[0026] The kit for detection of chromosomal heterozygosity loss based on amplicon sequencing of the present invention includes:

[0027] A primer pair set for determining the expression of 14 genes in the 1p36.32 region of the short arm of human chromosome 1: the primer pair set consists of SEQ ID NO: 1 and SEQ ID NO: 22, SEQ ID NO: 2 and SEQ ID NO: 23, SEQ ID NO: 3 and SEQ ID NO: 24, SEQ ID NO: 4 and SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 26, SEQ ID NO: 6 and SEQ ID NO: 27, SEQ ID NO : 7 and SEQ ID NO: 28, SEQ ID NO: 8 and SEQ ID NO: 29, SEQ ID NO: 9 and SEQ ID NO: 30, SEQ ID NO: 10 and SEQ ID NO: 31, SEQ ID NO: 11 and The primer composition of SEQ ID NO: 32, SEQ ID NO: 12 and SEQ ID NO: 33, SEQ ID NO: 13 and SEQ ID NO: 34, SEQ ID NO: 14 and the sequence shown in SEQ ID NO: 35, primer sequence see Table 1 The primer sequences of 14 genes in the 1p36.32 region of the short arm of human chromosome 1, and the concentration...

Embodiment 2

[0049] Example 2 Detection of 1p19q loss of heterozygosity in paraffin-embedded section samples from patients with glioma

[0050] 1. Detection method

[0051] 1.1 Extract DNA from paraffin sections.

[0052] 1.2 The components of the first PCR amplification (multiple targeted PCR amplification) are as follows:

[0053] components Final concentration primer mix 0.2μM / each GC stabilizer 1× PCR Enzyme Mix 1× FFPE DNA template 10ng wxya 2 o

to 25μL

[0054] Primer mixture preparation: 88 kinds of 1p36.32, 1q25.2, 19p13.2, 19q13.33 regions described in Table 1, Table 2, Table 3, and Table 4 described in Example 1, the concentration is 100 μM Add 3.5 μL of each primer solution to a centrifuge tube and mix thoroughly. When in use, take 10 μL and add it to the above PCR reaction system. The final concentration of each primer is 0.2 μM.

[0055] 1.3 The first PCR reaction procedure:

[0056]

[0057] 1.4 After the program runs,...

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Abstract

The invention discloses a kit and method for detecting chromosome heterozygosity loss on the basis of amplicon sequencing, belongs to the field of molecular biology nucleic acid detection, and relatesto the kit and method for detecting chromosome heterozygosity loss by means of amplicon sequencing. The kit contains a mixture of 14 groups of primer pairs used for detecting a 1p36.32 region of a human chromosome 1, 6 groups of primer pairs used for detecting a 1q25.2 region of the human chromosome 1, 14 groups of primer pairs used for detecting a 19p13.2 region of a human chromosome 19 and 7 groups of primer pairs used for detecting a 19q13.33 region of the human chromosome 19, 20 kinds of tag primers used for distinguishing samples, a GC stabilizer, a PCR enzyme mixture, purifying magneticbeads, a quality control standard substance and ddH2O. Through two-time PCR amplification and two-time product purification and sequencing, through a ratio of READS(1p) / READS(1q) to READS(19q) / READS(19p), the heterozygosity loss state of 1p19q in the samples is analyzed. By adopting the method, the variation situation of the 1p19q can be reflected comprehensively, truly and accurately, and gene variation of the 96 samples can be subjected to high-throughput detection and analysis simultaneously, so that the detection cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection in molecular biology, and relates to a kit and a method for detecting chromosome heterozygosity loss based on an amplicon sequencing method. Background technique [0002] Glioma originating from glial cells is the most common primary intracranial tumor, accounting for about 27% of all central nervous system tumors and about 80% of malignant tumors. The annual incidence of glioma in my country is (3-6.4) / 100,000, and the annual death toll reaches 30,000. The incidence rate of malignant glioma (Glioblastoma, GBM, WHO grade IV) is 5.8 / 100,000, and the 5-year mortality rate is second only to pancreatic cancer and lung cancer among systemic tumors, ranking third. Oligodendroglioma accounts for 5-18% of glioma, and is an independent type of glioma. Most oligodendroglial tumors have loss of heterozygosity (Loss of Heterozygosity, LOH) of the short arm of chromosome 1 (1p) and the long arm of chrom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/106
Inventor 龚建冯晓燕林挺于祥春穆珑丹
Owner 艾普拜生物科技(苏州)有限公司
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