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Composite amplification kit for 44 human Y chromosome loci and application thereof

A Y-chromosome and compound amplification technology, applied in the field of compound amplification kits, can solve problems such as poor compatibility of the kits, limited number of STR loci, etc.

Active Publication Date: 2019-06-14
BEIJING PEOPLESPOT TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve technical problems such as the limitation of the number of STR loci detected simultaneously by multiplex amplification and the poor compatibility of the kits in the prior art, the present invention provides a multiplex amplification kit capable of simultaneously detecting 44 chromosomal loci and its application

Method used

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  • Composite amplification kit for 44 human Y chromosome loci and application thereof
  • Composite amplification kit for 44 human Y chromosome loci and application thereof
  • Composite amplification kit for 44 human Y chromosome loci and application thereof

Examples

Experimental program
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Effect test

Embodiment 144

[0074] Example 1 Development of multiplex amplification kit for 44 chromosome loci

[0075] 1. Design of multiplex amplification primers for 44 chromosomal loci

[0076] For the composite amplification system, especially the composite amplification system of 44 loci in the present invention, the requirements for the specificity of the primer sequence, the secondary structure, the stability of binding to the target sequence, and the amplification efficiency are extremely high. Through continuous amplification-optimization-amplification cycle experiments, 44 pairs of primers for specifically and efficiently amplifying 44 loci were obtained, and their specific sequences are shown in Table 1:

[0077] Table 1 Amplification primers for 44 loci and 2 IPCs

[0078]

[0079]

[0080] The above primers were fluorescently labeled with different colors according to the following grouping methods corresponding to the loci: the first group, IPC60, rs199815934, DYS456, DYS549, DYS439...

Embodiment 24

[0087] Example 2 Establishment of multiple amplification system and program for 44 chromosomal loci

[0088] The annealing temperature and cycle number were optimized for the amplification primers of 44 loci, and the optimal reaction program was obtained: 95°C for 2 minutes; 94°C for 5 seconds; 60°C for 45 seconds; 72°C for 45 seconds Minutes, this step runs 30 cycles; 60°C for 20 minutes; 4-10°C for heat preservation.

Embodiment 3

[0089] Example 3 Genotyping of 9948 Cell Line Using Multiplex Amplification Kit

[0090] The 9948 cell line was subjected to multiplex amplification of 44 Y chromosome loci using the kit provided by the present invention. The template DNA derived from 9948 cell line was extracted by Chelex 100 method. The amplification reaction was carried out on the ABI 9700 thermal cycler, the electrophoresis and detection were carried out on the ABI 3130 genetic analyzer, and the data analysis was performed using GeneMapper IDX v1.2 software. Reagent materials such as the allelic ladder (ladder) used can be obtained from commercial sources and are conventional materials commonly used by those skilled in the art.

[0091] 1. Use the chelex 100 method to extract DNA (for specific methods refer to "Forensic DNA Protocol", HumanPress, 1998):

[0092] (1) Take 3 μl of cultured 9948 cell line in a 500 μl centrifuge tube.

[0093] (2) Shake and mix the chelex solution to make the chelex fully s...

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Abstract

The invention relates to a composite amplification kit for 44 human Y chromosome loci and application thereof. The invention provides a composite amplification system of the 44 human Y chromosome loci. The system includes specific primers for amplifying the 44 loci including 41 Y chromosome STR loci and 3 Y chromosome Indel loci. The 44 pairs of specific primers are subjected to group fluorescencelabeling by the six-color fluorescent labeling technology. Through design and optimization of the primer sequence and the working concentration, simultaneous efficient specific sensitive amplification of the 44 human Y chromosome loci is achieved. A detection result of the composite amplification system has high individual recognition capability and good data compatibility, and can be used in practice for paternity testing and individual recognition, thereby effectively reducing the detection cost of human DNA typing and improving the detection working efficiency.

Description

technical field [0001] The invention relates to the technical fields of molecular genetics and biological detection, in particular to a composite amplification kit for 44 human Y chromosome loci and applications thereof. Background technique [0002] Short tandem repeat locus (Short Tandem Repeat, STR) is a genetic marker commonly used at present, which widely exists in prokaryotic and eukaryotic genomes, and is a kind of several repeating units composed of 2 to 6 nucleotides. Ten to more than one hundred nucleotide repeats. Different numbers of core sequences are arranged in tandem repeats, while the lengths are polymorphic. According to the conserved sequences at both ends, design primers for PCR, and then detect polymorphisms of STR genetic markers by polyacrylamide, agarose gel electrophoresis or capillary electrophoresis. Compared with other genetic markers, STR markers have small fragments and are easy to amplify, and are more suitable for trace and degraded samples....

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCY02A50/30
Inventor 不公告发明人
Owner BEIJING PEOPLESPOT TECH
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