Kit for multiplex amplification of 24 loci of human genome DNA

A compound amplification and locus technology, applied in the field of five-color fluorescent labeling compound amplification system, can solve problems such as male misjudgment and male Amelogenin-Y deletion.

Active Publication Date: 2014-03-05
AGCU SCIENTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a lack of Amelogenin-Y in mal...

Method used

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  • Kit for multiplex amplification of 24 loci of human genome DNA
  • Kit for multiplex amplification of 24 loci of human genome DNA
  • Kit for multiplex amplification of 24 loci of human genome DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] A kit for simultaneous analysis of fluorescently labeled multiplexed amplification of 24 loci of human genomic DNA, consisting of the following:

[0061] Table 4 Kit Composition

[0062] components

volume

Reaction Mix

100μL

Genomic DNA (9948Promega USA)

The content of 0.1~10μl is 0.125~5ng

Primers corresponding to the above 24 loci

5.0 μL

Hot start Taq enzyme (5U / μL)

0.5μL

wxya 2 o

Make up to 25.0 μL

[0063] The Reaction Mix mentioned in it is MgCl 2 7.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg / mL.

[0064] The primers and primer concentrations corresponding to the 24 loci are shown in Table 1.

[0065] The 24 loci are grouped, specifically: D3S1358, D13S317, D7S820, D16S539, Penta E, and DYS635 are the first group, and the fluorescent markers of the primers corresponding to this group of loci are 6-FAM, HEX, TEMRA, and ROX Any one of DYS456, TPOX, TH01, D2S1338, CSF1PO, Penta ...

Embodiment 2

[0086] Application of 24-loci fluorescence-labeled multiple amplification test system for paternity identification of single parent

[0087] 1. Collect blood samples in paternity testing cases: the samples in this experiment are provided by XX paternity testing agency. For DNA extraction, use the Chelex-100 method to extract genomic DNA from two whole blood samples: take 0.5-5 μl of whole blood into a sterilized 1.5ml centrifuge tube, add sdH 2Put O1ml in the tube, shake for a few seconds; place at room temperature for 10 minutes, shake for a few seconds, centrifuge at 12,000 rpm for 3 minutes, discard the supernatant, keep enough supernatant to cover the precipitate, do not stir the precipitate; add 200 μl of 5% Chelex-100, Shake for a few seconds; keep warm at 56°C for 30 minutes, shake for a few seconds; bathe in boiling water for 10 minutes, shake for a few seconds; centrifuge at 12,000rpm for 5 minutes, and the supernatant is the extracted genomic DNA. Genomic DNA was ex...

Embodiment 3

[0096] Application of four Y loci in the 24-loci fluorescent-labeled multiple amplification test system for case investigation

[0097] 1. Collection of different inspection materials in case investigation: The samples in this experiment were provided by the Guangzhou Municipal Bureau. 500 different biological samples including 180 blood samples, 140 semen spots, 140 saliva spots, and 40 tissues. Genomic DNA was extracted with reference to "GA / T383-2002 Forensic Science DNA Laboratory Inspection Specification".

[0098] 2. Amplification detection

[0099] Perform PCR amplification and genetic analyzer detection according to Example 1, and finally obtain the typing result.

[0100] 3. Conclusion

[0101] The results show (see Table 9), the test results of 200 samples from different sources show that the newly added four Ys can divide the 200 samples into 114 haplotypes; 300 samples taken randomly can be divided into 146 haplotypes. Plotype; 400 random samples can be divided...

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Abstract

The invention relates to a five-color fluorescence labeling multiplex amplification system for analysis of 24 loci of human genome DNA at the same time. 19 autosome loci, 4 Y chromosome loci and 1 sex determination locus are detected at the same time. The system divides the 24 loci into 4 groups, and relates to fluorescence labeling of 5 colors. The fluorescence labeling multiplex amplification system has high sensitivity, under condition that the DNA template amount is 0.12ng, all the 24 loci can be detected, and the system is suitable for direct amplification of blood filter paper and FTA card collecting samples.

Description

technical field [0001] The present invention relates to a fluorescent marker composite amplification inspection system for 24 loci, in particular to a five-color fluorescent marker composite amplification system for simultaneous analysis of human genome autosomal and Y chromosome loci. The system can be applied to individual identification and paternity testing. Background technique [0002] Short tandem repeat loci (STRs) are currently commonly used genetic markers. In the early 1990s, the polymorphism of the STR locus was discovered, especially the STR locus has a small fragment and is easy to amplify, which is suitable for testing trace and degraded samples, and the amplification conditions of each locus are similar and can be compounded and amplified, so it has the advantages of Sensitive, accurate, fast, large amount of information and other advantages. Especially in the establishment of DNA database, STR compound amplification technology has great advantages. Theref...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 郑卫国李发院葛斌文卢青刘超刘宏李越
Owner AGCU SCIENTECH
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