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Method for detecting 22q11.2 copy number deletion

A copy number and gene copy number technology, applied in the field of molecular biology, to achieve the effect of reasonable program setting, reduced cost input and low cost

Active Publication Date: 2017-01-11
SHANGHAI CHILDRENS MEDICAL CENT AFFILIATED TO SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the method for detecting 22q11.2 copy number deletion using limited dNTP competitive PCR combined with HRM technology with high sensitivity, specificity, stability and accuracy of the present invention has not been reported yet.

Method used

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  • Method for detecting 22q11.2 copy number deletion
  • Method for detecting 22q11.2 copy number deletion
  • Method for detecting 22q11.2 copy number deletion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 detection method

[0034] 1. Primer design:

[0035] According to the characteristics of 22q11.2 copy number deletion disease, genes located between different low copy repeats (LCR) in the chromosome 22q11.2 region were selected (LCR22A-B: CLTCL1; LCR22B-C: KLHL22; LCR22C-D: PI4KA / SNAP29) as detection object ( figure 1 ), design four pairs of PCR amplification primers, which are respectively used to amplify fragments in the above genes, and simultaneously select CFTR genes located on different chromosomes, and design primers as reference sequences. When designing primers, the length of the PCR product is controlled at 50-120bps, and the melting curve and Tms value of the product are predicted at the same time, and the Tm difference between the target sequence and the reference sequence is set between 2°C and 10°C. The specificity of the target and reference primers was confirmed by searching the human genome database, and the amplified fragments were kep...

Embodiment 2

[0042] Verification of embodiment 2 detection method

[0043] Material

[0044] Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (Qiagen), KlenTaq enzyme (Ab Peptides), Plus dye (BioFire Defense), primers (BGI Biotechnology Co., Ltd.), human genomic DNA samples (extracted from 22q11.2 microdeletion patients and normal control populations).

[0045] method

[0046] In order to verify the detection method in Example 1, we used the established system to detect 99 patients with copy number deletion in the 22q11.2 region and normal control samples. Each sample was repeated three times and amplified with multiple junction probes Technology (Multiplex Ligation-dependent Probe Amplification, MLPA) to verify the results.

[0047] result

[0048] In this study, a rapid detection method for 22q11.2 copy number deletion was established by using limited dNTP competitive PCR combined with HRM technology. This system used three competitive PCR reactions to detect four genes ...

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Abstract

The invention provides a method for detecting 22q11.2 copy number deletion by limited dNTP (deoxyribonucleotide triphosphate) competitive (polymerase chain reaction) in combination with an HRM (high resolution melt) technique. The method comprises the step of detecting CLTCL1, SNAP29, KLHL22, PI4KA and CFTR genes. The invention also provides a primer combination and kit of the method. The method is simple and quick to operate, and only comprises the processes of PCR reaction and subsequent HRM analysis, thereby shortening the detection period. The method implements closed pipe operation; and since fluorescent dyes are added in the PCR, HRM curve analysis can be performed without other treatments after the detection segment amplification is completed, thereby effectively avoiding the pollution. In the reaction, only conventional PCR reagents and small amounts of fluorescent dyes are needed, and special detection and analysis apparatuses are not needed. The detection on 99 patients and normal person control samples indicates that the sensitivity and specificity respectively reach 100%, and the stability and accuracy of the system are confirmed.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for detecting 22q11.2 copy number deletion by using limited dNTP competitive PCR combined with HRM technology. Background technique [0002] 22q11.2 microdeletion syndrome is the most common clinical genetic syndrome, which is caused by deletion of 22q11.21-q11.23 microfragment near the centromere in the long arm of chromosome 22. Its core region is located between the low-copy repeat sequence LCR22-A to LCR22-D on the 22q11.2 region, with a size of 3Mb. About 90% of the syndrome patients show an overall deletion of one copy of this fragment, and some patients have different Combinations of deletions among low-copy repeats. According to the clinical manifestations and characteristics, patients with this syndrome can be initially diagnosed, and the detection of the 22q11.2 deletion fragment is an important basis for the diagnosis of the disease. [0003] At pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2537/143C12Q2563/107C12Q2527/107C12Q2537/161
Inventor 傅启华张晓青杨海鸥
Owner SHANGHAI CHILDRENS MEDICAL CENT AFFILIATED TO SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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