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Quantitative detection method for gene copy number

A quantitative detection method and gene copy number technology, applied in the field of molecular biology, can solve problems such as inability to accurately analyze the difference in Ct values

Inactive Publication Date: 2012-08-01
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] According to the above rules, combined with the currently widely used multiplex TaqMan real-time quantitative PCR technology, through system optimization and adjustment, the difference in Ct value that can be achieved in its analytical sensitivity is 0.8-1.0, so it can accurately detect 0, 1, and 2 copies of functional genes, but The 3 copies whose Ct value difference is only 0.27 cannot be accurately analyzed, so the multiplex TaqMan real-time quantitative PCR technology also has certain limitations

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  • Quantitative detection method for gene copy number
  • Quantitative detection method for gene copy number
  • Quantitative detection method for gene copy number

Examples

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Embodiment 1

[0045] Example 1 Detection of mouse SRY plasmid vector gene copy number

[0046] This embodiment selects the mouse SRY gene plasmid carrier as the detection object, applies the method of the present invention to simultaneously detect gene deletion and gene multiple copies, detects the SRY gene copy number, and observes that this method is used for detection by analyzing the gene copy number results. The feasibility and effectiveness of target gene deletion and multiple copies, especially the observation of the blocking effect of the blocking probe.

[0047] Selection basis and representativeness:

[0048] Using gene cloning technology, a section of about 400bp mouse SRY gene sequence and a section of about 400bp mouse housekeeping gene β-Actin were connected to the circular DNA sequence of the T vector, which were used as the detection objects of the target gene and reference gene respectively . After the constructed T vector is transformed, enriched, separated and purif...

Embodiment 2

[0074] Example 2 Detection of gene copy number of human α-globin gene

[0075] In this embodiment, the human α-globin gene is selected as the detection object, and the invented method of simultaneously detecting gene deletion and multiple copies of the gene is used to detect its copy number. By analyzing the results of the gene copy number, it is observed that this method is used for detection purposes The feasibility and effectiveness of gene deletion and multiple copies, especially the accuracy and stability of the method.

[0076] Selection basis and representativeness:

[0077] CNVs of the α-globin gene are common in the human genome, and some studies have suggested 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 expr...

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Abstract

The invention discloses a quantitative detection method for a gene copy number. The method comprises the following steps of: respectively designing primers and fluorescence probes for target gene and reference gene amplification sequences, and designing a closed probe for the target gene amplification sequence; detecting a sample: performing multiple TaqMan fluorescent quantitative polymerase chain reaction (PCR) on target genes and reference genes by using the DNA sample to be detected as a template, and respectively recording Ct values by using a DNA sample with known target gene copy number as a reference sample; and analyzing the result. By the method, the copy number of the functional genes can be directly and quantitatively detected, and deletion, deletion number and multiple copies (CNVs) of the detected genes are directly reflected, so that synchronous quick detection of gene deletion and multiple gene copies is realized; and the method is high in sensitivity, the synchronous quick detection of the gene deletion and the multiple gene copies can be finished only by a common fluorescent quantitative PCR instrument, and experiments prove that the method is accurate and reliable.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a quantitative detection method of gene copy number. Background technique [0002] Recent studies have found that gene copy number variation (copy number variants, CNVs), like single nucleotide polymorphisms (SNPs), is an important genetic marker of molecular evolution and genetic polymorphism, and is also the pathogenesis of certain diseases. At present, more and more CNVs have been discovered and submitted to the corresponding molecular biology databases. [0003] In terms of molecular mechanism, the essence of CNVs is a copy number change of a DNA sequence. In a set of genomic DNA of an individual, under normal circumstances, the copy number of an allele is 2. If the gene is missing on both chromosomes, the copy number is 0. If one of the chromosomes is missing, the copy number is It is 1, if there is another segment of this gene sequence on one of the chromosomes, th...

Claims

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Application Information

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