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DMD (Duchenne muscular dystrophy) gene capture probe and application thereof in DMD gene mutation detection

A gene capture and capture probe technology, which is used in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc. Inefficiency, etc.

Active Publication Date: 2017-06-13
MYGENOSTICS (CHONGQING) GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The DMD gene is huge, the mutation rate is high, and the mutation types are diverse. According to statistics, there are as many as 4000 to 5000 kinds of mutations, which brings great difficulties to the clinical detection of DMD genes.
[0003] Traditional DMD gene mutation detection methods require different detection techniques for different mutation types. For point mutations, Sanger sequencing needs to be used for detection, and for exon duplication or deletion mutations, microarray comparative genomic hybridization technology (a -CGH), MLPA or multiplex PCR and other technologies to detect, the above-mentioned detection strategy and method detection efficiency is low, and the precise site and fragment size of the exon deletion cannot be determined, therefore, this area needs to be able to detect multiple mutation types of DMD simultaneously A more accurate and efficient method of

Method used

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  • DMD (Duchenne muscular dystrophy) gene capture probe and application thereof in DMD gene mutation detection
  • DMD (Duchenne muscular dystrophy) gene capture probe and application thereof in DMD gene mutation detection
  • DMD (Duchenne muscular dystrophy) gene capture probe and application thereof in DMD gene mutation detection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the preparation of DMD gene capture probe

[0064] 1. Sequence design and preparation of single-stranded subprobes

[0065] The sequence of the DMD gene used is chrX:31137345-33357726 of the human reference genome version Hg19 (updated on August 6, 2015). Design the sequence of the DMD gene capture single-stranded sub-probe according to the DMD gene sequence, the length of each single-stranded sub-probe is 60bp, and the sequence of each single-stranded sub-probe is as follows:

[0066] The sequence of the first single-stranded subprobe is the 1-60 position of the DMD gene (ie chrX: 31137345-31137404);

[0067] The sequence of the second single-stranded subprobe is the 58th-117th position of the DMD gene;

[0068] The sequence of the third single-stranded subprobe is the 115th-174th position of the DMD gene;

[0069] ...

[0070] The sequence of the nth single-stranded subprobe is [57(n-1)+1]-[57(n-1)+60] of the DMD gene;

[0071] ...

[0072] The seq...

Embodiment 2

[0087] Embodiment 2, establishment of DMD gene mutation detection method

[0088] 1. Construction of whole genome library of samples to be tested

[0089]1.1 Ultrasound fragmentation: Dilute the whole genome DNA of the sample to be tested with an initial amount of 0.5-3 μg to 30 ng / μL with 1×low TEBuffer (Thermo). Covaris S2 ultrasonic instrument was used for ultrasonic fragmentation, and the value of Covaris system was set according to the standard, 6 cycles × 60s, water bath temperature: 5°C, duty cycle: 20%, intensity: 5, mode: Frequencysweeping, to obtain fragmented DNA.

[0090] 1.2 End filling: Take 100 μL of fragmented DNA from step 1.1, 8 μL of dNTPs, 2 μL EndPolishing enzyme I (10 U / μL, Agilent) and 16 μL End Polishing enzyme II (5 U / μL, Agilent), add water to a total volume of 200 μL After incubation at 25°C for 30 min, the DNA was purified using the PureLink PCR Purification Kit (Invitrogen) to obtain end-filled DNA.

[0091] 1.3 Ligation of P1 and P2 adapters: T...

Embodiment 3

[0118] Embodiment 3, utilize the DMD gene capture probe of embodiment 1 and the DMD gene mutation detection method of embodiment 2 to detect the DMD gene mutation of clinical samples

[0119] After informed consent, DNA samples from 5 patients with exon deletion / repeat expansion mutations in the DMD gene were selected (verified by MLPA detection, Figure 2-Figure 11 ) as a sample to be tested, and a DNA sample negative for MLPA detection was used as a normal control (negative control), and the DMD gene mutation detection method of Example 2 was used to detect the DMD gene mutation. The kit used by MLPA to detect whether there is exon deletion / repeat expansion mutation in DMD gene is MLPA P052 detection kit (HRC-Holland).

[0120] The results showed that the high-throughput sequencing images of 5 cases of MLPA-positive samples were shown in figure 1 , see Table 1 and Table 3 for data analysis results. figure 1 In , the negative control refers to the high-throughput sequencing...

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Abstract

The invention discloses a DMD (Duchenne muscular dystrophy) gene capture probe and application thereof in DMD gene mutation detection. A preparation method of the DMD gene capture probe disclosed herein comprises: preparing N sub-probes, connecting the sub-probes to obtain the DMD gene capture probe; N is a natural number greater than or equal to 2; the N sub-probes can cover whole sequences of DMD gene; any two adjoining sub-probes on the DMD gene meet the case where there is one or more downstream nucleotide to the corresponding upstream sub-probe, which overlap with the upstream of the corresponding downstream sub-probe; the length of each of the N sub-probes is 50-150 bp. By detecting the DMD gene with the DMD gene capture probe, it is possible to detect whether the DMD gene experiences amplification mutation deletion / repetition or not, and it is also possible to precisely locate a break point region and the size of fragments and to detect DMD gene site mutations of a sample under detection.

Description

technical field [0001] The invention relates to a DMD gene capture probe and its application in the detection of DMD gene mutation in the field of biotechnology. Background technique [0002] The DMD gene is the causative gene of pseudohypertrophic muscular dystrophy syndrome (DMD / BMD). It is located in the Xp21.2 region of the X chromosome. It is the largest human gene known so far. It contains 79 exons and has a high probability of gene mutation. , reaching about 1:3500 in newborn boys. The DMD gene contains a variety of mutation types, single or multiple exon deletion mutations are the most common, accounting for about 60-70%, single or multiple exon duplication mutations, accounting for about 5-10%, point mutations include single Or several nucleotide substitutions, deletions or insertions, etc., account for about 25% to 35% of all mutations in DMD. The DMD gene is huge, the mutation rate is high, and the mutation types are diverse. According to statistics, there are a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2600/166C12Q2565/519C12Q2535/122
Inventor 伍建林朋
Owner MYGENOSTICS (CHONGQING) GENE TECH CO LTD
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