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Method for accurately positioning chromosome deletion/repetition breakpoint

A chromosomal deletion and precise positioning technology, applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc., can solve the problems of complex operation, high cost, long detection cycle, etc., and achieve high efficiency and low cost. , the effect of design science

Pending Publication Date: 2021-08-10
CELULA CHINA MED TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method for precise positioning of chromosomal deletion / duplication breakpoints, which solves the problems of complex operation, long detection period and high cost in the prior art

Method used

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  • Method for accurately positioning chromosome deletion/repetition breakpoint
  • Method for accurately positioning chromosome deletion/repetition breakpoint

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This embodiment discloses a case of DMD gene No. 53-55 exon 53-55 exon deletion breakpoint roughly regional positioning, specifically:

[0041] 1. Use nucleic acid extraction or purification reagents from Nahai Hi-Tech Biotechnology Co., Ltd. to extract the patient's genomic DNA.

[0042] 2. Using restriction endonuclease (or sonication) to fragment the genomic DNA sample into fragments with an average length of about 150-250 bp. The fragment was then subjected to end repair and an "A" was added.

[0043] Fragmentation, end repair, and the conditions for adding A:

[0044] 37℃ 40min

[0045] 65℃ 30min

[0046] 4℃ hold

[0047] 3. Then connect adapters with tag sequences to both ends,

[0048] The connection condition is: 20°C 15min.

[0049] 4. Then, perform PCR to enrich the library, and the PCR conditions are:

[0050] Pre-denaturation at 95°C for 3 minutes

[0051] (denaturation at 98°C for 20s, annealing at 60°C for 15s, extension at 72°C for 30s) 7 cycles a...

Embodiment 2

[0063] This embodiment discloses an example of the accurate location of the breakpoint of exon 53-55 deletion of DMD gene, specifically:

[0064] 1. Through data analysis, the sample in Example 1 has exon 53-55 deletion, the upstream breakpoint range is chrX: 31856593-31863252, the downstream breakpoint range is chrX: 31954056-31955026, in the above suspected breakpoint area Upstream and downstream Design 13 upstream primers and one universal downstream primer; specifically:

[0065] DMD-1F GGGCAGCAAAGGGAAGATAAATA, as shown in SEQ ID NO:1;

[0066] DMD-2F TGTGATAAAAGTGCAAAAGAGTTCGC, as shown in SEQ ID NO:2;

[0067] DMD-3F GGCCCATCTGAATAGTCCATAAAATC, as shown in SEQ ID NO:3;

[0068] DMD-4F GTCCTTATAGTTTCTCATTTCTCTGC, as shown in SEQ ID NO:4;

[0069] DMD-5F ATATTCCTCCACAAGACATTCCAGTG, as shown in SEQ ID NO:5;

[0070] DMD-6F GATTTGTAATGCCCTATGGACCAGA, as shown in SEQ ID NO:6;

[0071] DMD-7F TTGCAGTCTTTTGTGTCAGTGTAGA, as shown in SEQ ID NO:7;

[0072] DMD-8F ACGGTAGGAGT...

Embodiment 3

[0104] This embodiment discloses the verification of the DMD gene exon 53-55 deletion breakpoint of embodiment 2, specifically:

[0105] According to the confirmed breakpoint position, primers were designed on the flanks, and primers were designed inside the deletion region to amplify the normal non-deleted sequence. The length of the product inside the breakpoint was 209bp, and the length of the product outside the breakpoint was 533bp. 100ng) genomic DNA was subjected to PCR and then run agarose gel electrophoresis,

[0106] PCR conditions:

[0107] Pre-denaturation at 94°C for 5 minutes

[0108] (denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 3min) 38 cycles and final extension at 72°C for 10min

[0109] Store at 4°C hold

[0110] Agarose gel electrophoresis conditions:

[0111] 3% agarose, electrophoresis at 50V for 120min.

[0112] Carriers should be two bands of different lengths, patients should be one longer band, and negative contr...

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Abstract

The invention discloses a method for accurately positioning chromosome deletion / repetition breakpoints, belongs to the technical field of biology, and solves the problems of complex operation, long detection period and high cost in the prior art. The method provided by the invention comprises the following steps: determining an approximate region of target gene deletion / repetition, and designing a continuous one-way specific primer; connecting the two ends of the fragmented DNA or free DNA of the sample to be detected with a linker sequence; adding a universal joint primer and a one-way specific primer to carry out multiple PCR amplification enrichment, and then purifying a PCR product; and connecting a sequencing linker with a tag sequence to the library subjected to semi-targeted amplification, then carrying out next-generation sequencing, and analyzing sequences capable of being compared to two ends of a breakpoint at the same time. The method is scientific in design, ingenious in conception and high in efficiency, and an accurate breakpoint can be obtained through one experiment; the method is high in applicability and is not influenced by the size of a breakpoint area; the method is low in cost and suitable for clinical popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for precise positioning of chromosome deletion / duplication breakpoints. Background technique [0002] Gene mapping refers to the linkage group or chromosome to which a gene belongs and the determination of the position of the gene on the chromosome. Gene mapping is a basic work in genetics research. The precise location of chromosomal deletion / duplication breakpoints is an important link in gene mapping. In the prior art, three methods are mainly used for accurate positioning: one is to use long-fragment PCR or inverse PCR for accurate positioning of breakpoints. This method has the advantage of low cost, but it needs to determine the range of bilateral breakpoints in a small range, and it needs to try the sequence near the breakpoint many times, the success rate of long-segment PCR is low, and inverse PCR needs to be used separately for amplification. The inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6827C12Q1/6869C12N15/11
CPCC12Q1/6827C12Q1/6869C12Q2537/143C12Q2531/113C12Q2535/122
Inventor 冯骏张海川吴镝李少军唐维琴
Owner CELULA CHINA MED TECH CO LTD
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