Composite amplification kit for 47 human autosome and Y chromosome loci and application thereof

An autosome and Y-chromosome technology, applied in the field of molecular genetics and biological detection, can solve the problems of poor kit compatibility and limited number of STR loci

Active Publication Date: 2019-05-14
BEIJING PEOPLESPOT TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] 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 test k...

Method used

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  • Composite amplification kit for 47 human autosome and Y chromosome loci and application thereof
  • Composite amplification kit for 47 human autosome and Y chromosome loci and application thereof
  • Composite amplification kit for 47 human autosome and Y chromosome loci and application thereof

Examples

Experimental program
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Effect test

Embodiment 147

[0069] Example 1 Development of multiple amplification kit for 47 human autosomes and Y chromosome loci

[0070] 1. Design of multiple amplification primers for 47 human autosomal and Y chromosome loci

[0071] For the composite amplification system, especially the composite amplification system of 47 loci described 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. , after continuous amplification-optimization-amplification cycle experiments, 47 pairs of primers for specifically and efficiently amplifying 47 loci were obtained, the specific sequences of which are shown in Table 1:

[0072] Table 1 Amplification primers of 47 loci

[0073]

[0074]

[0075] The above primers were fluorescently labeled with different colors according to the following grouping methods corresponding to the loci: the first group, ...

Embodiment 24

[0082] Example 2 Establishment of multiple amplification system and program for 47 chromosomal loci

[0083] The annealing temperature and cycle number were optimized for the amplification primers of 47 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

[0084] Example 3 Genotyping of 9948 Cell Line Using Multiplex Amplification Kit

[0085] The kit provided by the invention is used to carry out multiple amplification of 47 human autosome and Y chromosome loci on the 9948 cell line. 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 GeneMapperIDX 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.

[0086] 1. Use the chelex 100 method to extract DNA (for specific methods refer to "Forensic DNA Protocol", HumanPress, 1998):

[0087] (1) Take 3 μl of cultured 9948 cell line in a 500 μl centrifuge tube.

[0088] (2) Shake and mix the chelex solution to make the chelex...

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Abstract

The invention relates to a composite amplification kit of 47 human autosome and Y chromosome loci and an application thereof. The invention provides a composite amplification system of the 47 human autosome and Y chromosome loci, which comprises specific primers for amplifying the 47 loci, wherein the 47 loci comprise 19 autosome STR loci, 27 Y chromosome STR loci and 1 sex recognition locus. The47 pairs of specific primers are subjected to grouping fluorescence labeling by utilizing a six-color fluorescence labeling technology, and the simultaneous high-efficiency, specific and sensitive amplification of the 47 human autosome and Y chromosome loci is achieved through the design and optimization of primer sequences and working concentrations. The detection result of the composite amplification system has high individual identification capability and good data compatibility, and can be used for paternity identification and individual identification in practice, so that the detection cost of human DNA typing is effectively reduced, and the detection working efficiency is improved.

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 47 human autosomes and Y chromosome loci and applications thereof. Background technique [0002] Short tandem repeat locus (Short Tandem Repeat, STR) is a commonly used genetic marker. It widely exists in the genomes of prokaryotic and eukaryotic organisms, and is a kind of repeating sequence of tens to more than one hundred nucleotides composed of 2 to 6 nucleotides as repeating units. Different numbers of core sequences were arranged in tandem repeats, and their lengths showed genetic polymorphism. According to the conserved sequences at both ends, design primers, perform 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...

Claims

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Application Information

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