Method for rapidly detecting copy number variation of alpha-globin gene cluster

A copy number variation and globin technology, applied in the field of biochemistry, can solve the problems of easy contamination, complicated operation, long time-consuming, etc., and achieve the effects of avoiding inconsistent amplification efficiency, high accuracy and specificity, and simple operation.

Inactive Publication Date: 2012-07-25
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The common disadvantages of the existing methods for detecting the copy number of specific sites include complex operation, open detection platform is easy to cause cross-contamination between samples, high requirements for instruments, etc.
Taking the currently widely used MLPA method as an example, the disadvantages of this method are: 1. The open detection platform is easy to cause pollution; 2. The long probe designed by it cannot be synthesized by ordinary chemical methods, and needs to be synthesized by M13 carrier Obtained by cloning method; 3. The hybridization step takes too long (16 hours)
But multiplex PCR has its own problems
Mismatch amplification between different primer pairs, inconsistent amplification efficiency of different target sequences, resulting in the inhibition of low-efficiency targets by targets with higher amplification efficiency, which will affect the quantitative analysis of copy number

Method used

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  • Method for rapidly detecting copy number variation of alpha-globin gene cluster
  • Method for rapidly detecting copy number variation of alpha-globin gene cluster
  • Method for rapidly detecting copy number variation of alpha-globin gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] The detection of 96 cases of DNA samples with known genotypes includes the following steps, wherein the standard sample gene is used as an external standard, which is a normal human genome sample with 2 copies of known α1 / α2:

[0106] 1) Using the gene of the sample to be tested and the gene of the standard sample as templates, add three pairs of tailing primers to perform the first round of nested fluorescent quantitative PCR. The reaction system is: 30 ng DNA template, 50 nM three pairs of tailing primer mixture (α1 -FT / α1-RT, α2-FT / α2-RT, β-actin-FT / β-actin-RT, and the amount of each upstream and downstream tailing primers are the same), 20 mM Tris–HCl (pH8.4 ), 50 mM KCl, 2.0 mM MgCl 2 , 1.0 U TaqHS polymerase, 250 μM dNTPs, made up to 20 μL with ultrapure water; the reaction program was: 95°C for 5 min; 94°C for 45 s, 62°C for 45 s, cycle 2 times; 92°C for 30 s, 70°C for 15 s, cycle 15 times; Obtain the first round of PCR products;

[0107] 2) Using the first-rou...

Embodiment 2

[0112] Select the genotype as αα / αα, -α 3.7 / αα, ααα anti3.7 / αα, -α 4.2 / αα, ααα anti4.2 / αα One case of each of the five DNA samples was used to test the repeatability of this method.

[0113] The standard sample (30ng) with known genotype αα / αα was used as a reference template, and a negative control without template was set up at the same time. Each sample was repeated 5 times, and the detection was carried out according to the steps in Example 1, and the experimental results of the detection were counted. , so as to evaluate the repeatability of this method, and the statistical analysis results are shown in Table 2. It can be seen from the table that the genotype coincidence rate was 100%.

[0114]

[0115] The standard deviation SD of the experiment in this example is 0.038-0.095, and the repeatability coefficient of variation CV% is 6.09%-12.77%, indicating that the detection repeatability of this method is good.

[0116] Southern Medical University

[0117] ...

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Abstract

The invention discloses a method for rapidly detecting the copy number variation of alpha-globin gene cluster, which comprises the following steps: detecting one pair of universal primers, three kinds of TaqMan probes and three pairs of tailing primers, and detecting a sample: adding the three pairs of tailing primers by taking a gene to be detected and a standard gene as templates, carrying out the first round of nested fluorescent quantitative PCR (polymerase chain reaction) to obtain a first round of products of PCR, adding the universal primers and three kinds of TaqMan probes by taking the first round of products of PCR as templates, carrying out the second round of fluorescent quantitative PCR, collecting fluorescent signals after finishing each cycle of reaction, and quantifying the copy numbers of both the alpha1-globin gene and the alpha2-globin gene according to the collected fluorescent signals. Due to the adoption of the method, the reaction efficiency for amplification of a plurality of target segments in the same reaction pipe is consistent, and enotypes of the alpha-globin gen can be distinguished accurately. The method is easy to operate and has high throughput and requires a two-step PCR cycle reaction of 2h or shorter.

Description

technical field [0001] The invention belongs to the field of biochemistry and relates to a method for rapidly detecting the variation of the copy number of α-globin gene cluster. Background technique [0002] Copy number variation (CNV) of the α-globin gene is very common in the human genome, and some studies believe that this phenomenon is related to the natural selection of malaria during the evolution of humans. Both deletion and multiple copies of the α-globin gene can lead to decreased or increased expression of the encoded α-globin, resulting in a series of biological traits: gene deletion leads to decreased expression of α-globin and α-thalassaemia, and multiple copies of the gene lead to Increased expression of α-globin aggravates β-thalassemia symptoms. In addition, in the α-globin gene cluster, the α1 and α2 genes have high GC content and are highly homologous, and their encoded polypeptide chains are identical. In the gene structure, the DNA sequences of the X, Y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 周万军徐湘民
Owner SOUTHERN MEDICAL UNIVERSITY
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