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Fluorescent labeled X-STR gene locus multiplex PCR method and application thereof

A fluorescent labeling and locus technology, applied in the field of genetic labeling, can solve the problems such as the inability to use paternity testing and personal identification of the Han population, the need for 3 to 4 weeks to arrive, and the limitation of wide application, and reach the scope of applicability of the test materials. Wide range of inspection materials and reagent consumables, high sensitivity

Inactive Publication Date: 2011-11-23
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Some loci in these three kits have low polymorphism in Chinese population, and the experimental data of other laboratories and the inventor's laboratory have all shown that the loci in these three kits are not in Chinese Han population. There is no obvious linkage disequilibrium, so the 4 haplotypes of this kit cannot be used for paternity testing and personal identification of the Han population
At the same time, foreign kits are expensive and inconvenient to purchase (it usually takes 3 to 4 weeks to arrive after ordering), which limits their wide application

Method used

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  • Fluorescent labeled X-STR gene locus multiplex PCR method and application thereof
  • Fluorescent labeled X-STR gene locus multiplex PCR method and application thereof
  • Fluorescent labeled X-STR gene locus multiplex PCR method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Children A (8 years old) and B (6 years old) accepted by the same orphanage are suspected to be full siblings and requested a paternity test.

[0038] experiment procedure:

[0039] 1. Collect hairs with follicles from A and B, clip 3 hair follicles and use Chelex-100 to quickly extract DNA, add 100 μL 5% Chelex-100 after centrifugation, incubate at 56°C for 30 minutes, incubate at 98°C for 10 minutes, 10000 rpm After centrifugation for 2 min, the supernatant was aspirated for PCR amplification.

[0040] 5×PCR Reaction mix 2 μL 10×STR Primer mixture 1 μL Hs Taq DNA polymerase (5 U / μl) 0.2 μL DNA template 1 μL Sterilized ultrapure water 5.8 μL

[0041] 5×PCR reaction mixture containing dNTP 200 mmol / L, MgCl 2 1.5mmol / L.

[0042] In the above primer mixture, the primer sequences and their fluorescent labels for each locus are shown in Table 1.

[0043] Table 1 Gene loci and their primer sequences and fluorescent marker...

Embodiment 2

[0058] The X-STR fluorescence-labeled multiplex PCR method was used to identify whether the two daughters Zhang and Wang, whose parents both died, were half-sisters of the same father.

[0059] experiment procedure:

[0060] 1. Collect oral swab samples of Zhang and Wang, extract DNA by Chelex-100 rapid extraction method, add 200 μL 5% Chele-100 after centrifugation, incubate at 56°C for 30 minutes, incubate at 98°C for 10 minutes, and centrifuge at 10,000 rpm2 min, the supernatant was aspirated for PCR amplification.

[0061] 2. PCR amplification: with embodiment 1.

[0062] 3. Capillary electrophoresis: the same as in Example 1.

[0063] 4. Genotyping: Same as Example 1.

[0064] 5. Simultaneously, 24 autosomal STRs were also typed using the autosomal STR multiplex amplification system PowerPlex® 16 kit (abbreviated as PP16) and STRTyper-10 kit (abbreviated as STRTyper-10). Typing methods were carried out according to the respective manuals.

[0065] Result analysis: ...

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Abstract

The invention discloses a fluorescent labeled X-STR locus multiplex PCR method and an application thereof; the system performs multiplex amplification analysis of 12 loci: GATA172D05, DXS6789, DXS10074, DXS10078, GATA165B12, DXS6797, DXS6803, DXS6804, GATA31E08, DXS7130, DXS9895, and DXS6810, wherein primers of the 12 loci are labeled respectively by four fluorescences of FAM, HEX, TAMRA, ROX. The method of the invention can be used to prepare a set of kit as an X-STR locus multiplex amplification kit with Chinese characteristics, and the kit is applicable to gene localization of paternity identification, individual identification, sex identification, and X-linked genetic diseases, and is especially applicable to paternity identification for sister recognition, half-blood half-sister recognition, generation-skipping recognition, etc.

Description

technical field [0001] The invention relates to the detection of polymorphic genetic markers in the human genome, in particular to a fluorescent label X-STR gene locus composite PCR method and its application through compound amplification of 12 STR loci. Background technique [0002] Short tandem repeat (short tandem repeat, referred to as STR) is a type of microsatellite DNA sequence formed by tandem repetition of 2-7 base pairs as the core unit, and its fragments can be amplified by PCR technology. The genetic polymorphism of the STR locus is mainly due to the change of the number of core repeat units, and its alleles can be typed by techniques such as silver staining, fluorescent labeling and autoradiography. In the human genome, there is an STR locus every 6-10kb on average, which provides a rich source of high-information genetic markers for forensic personal identification and paternity identification. [0003] The X chromosome STR (X chromosome short tandem repeat,...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 孙宏钰欧雪玲童大跃曾祥培任铮张兹钧
Owner SUN YAT SEN UNIV
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