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Composite amplification kit for 25 human chromosome loci and application thereof

A composite amplification and locus technology, applied in the field of composite amplification kits, can solve the problems of 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

[0009] In order to solve technical problems such as the limitation of the number of STR loci detected simultaneously by multiple amplification and the poor compatibility of the kits in the prior art, the present invention provides a multiple amplification kit capable of simultaneously detecting 25 chromosomal loci and its application

Method used

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  • Composite amplification kit for 25 human chromosome loci and application thereof
  • Composite amplification kit for 25 human chromosome loci and application thereof
  • Composite amplification kit for 25 human chromosome loci and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Development of multiplex amplification kit for 25 chromosome loci

[0073] 1. Design of primers for multiplex amplification of 25 chromosomal loci

[0074] For the composite amplification system, especially the composite amplification system of 25 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. Continuous amplification-optimization-amplification cycle experiments obtained 25 pairs of primers that specifically and efficiently amplified 25 loci, the specific sequences of which are shown in Table 1:

[0075] Table 1 Amplification primers of 25 loci

[0076]

[0077]

[0078] The above primers were fluorescently labeled with different colors according to the following grouping methods of the corresponding loci: the first group was D3S1358, CSF1PO, D2S441, D21S11, Penta E; the second group ...

Embodiment 2

[0086] Example 2 Establishment of multiple amplification system and program for 25 chromosome loci

[0087] For the amplification primers of 25 loci, the annealing temperature and cycle number were optimized to obtain the optimal reaction program for multiple amplification of 25 chromosomal loci: 95°C for 2 minutes; 94°C for 5 seconds; 60°C 45 seconds, heat preservation at 72°C for 45 seconds, this step runs 30 cycles; heat preservation at 60°C for 20 minutes; heat preservation at 4-10°C.

Embodiment 3

[0088] Example 3 Genotyping of 9948 Cell Line Using Multiplex Amplification Kit

[0089] The 9948 cell line was subjected to multiple amplification of 25 chromosomal loci by 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.

[0090] 1. Use the chelex-100 method to extract DNA (for specific methods refer to "Forensic DNA Protocol", HumanPress, 1998):

[0091] (1) Take 3 μl of cultured 9948 cell line in a 500 μl centrifuge tube.

[0092] (2) Shake and mix the chelex solution to suspend the chelex suf...

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Abstract

The invention relates to a composite amplification kit for 25 human chromosome loci and application thereof. The invention provides a composite amplification system of the 25 human chromosome loci. The system includes specific primers for amplifying the 25 loci including 23 autosomal STR loci, 1 gender recognition locus and a Y chromosome Indel locus. The 25 pairs of specific primers are subjectedto group fluorescence labeling by the six-color fluorescent labeling technology. Through design and optimization of the primer sequence and the working primer concentration, simultaneous efficient specific sensitive amplification of the 25 human 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 25 human chromosome loci and applications thereof. Background technique [0002] Short tandem repeats (STR for short), also known as microsatellites or simple sequence repeats (SSR), are a type of DNA tandem repeats widely present in eukaryotic genomes, with a core sequence of 2-6 base repeat units. The number of STR loci is large, the division is extensive, accounting for about 3% of the entire human genome, and the polymorphism is high. The polymorphism mainly comes from the difference in the number of core sequence repeats between individuals, and this difference is in the genetic process. Follow Mendelian laws of inheritance. Therefore, STR amplification detection technology is widely used in individual identification, kinship identification and population genetics research. [0003] At present, STR comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCY02A50/30
Inventor 不公告发明人
Owner BEIJING PEOPLESPOT TECH
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