Composite amplification kit for 38 human Y chromosome loci and application thereof

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

Active Publication Date: 2019-06-14
BEIJING PEOPLESPOT TECH
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  • Abstract
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  • Claims
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Problems solved by technology

[0008] In order to solve technical problems such as the limitation of the number of STR loci detected simultaneously by multiplex amplification and the poor compatibilit

Method used

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  • Composite amplification kit for 38 human Y chromosome loci and application thereof
  • Composite amplification kit for 38 human Y chromosome loci and application thereof
  • Composite amplification kit for 38 human Y chromosome loci and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 138

[0079] Example 1 Development of multiplex amplification kit for 38 Y chromosome loci

[0080] 1. Design of multiplex amplification primers for 38 Y chromosome loci

[0081] For the composite amplification system, especially the composite amplification system of 38 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, 38 pairs of amplification primers for specific and high-efficiency amplification of 38 loci were obtained, the specific sequences of which are shown in SEQ ID NO.1-SEQ ID NO.76 (Table 1).

[0082] The 38 pairs of primers shown in Table 1 were fluorescently labeled with different colors according to the following grouping methods of corresponding loci: the first group: IPC60, DYS392, DYS533, DYS390, DYS593, DYS448, DYS460, DY...

Embodiment 238

[0089] Example 2 Establishment of multiple amplification system and program for 38 Y chromosome loci

[0090] For the amplification primers of 38 loci, the annealing temperature and cycle number were optimized to obtain the optimal reaction program: 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

[0091] Example 3 Genotyping of 9948 Cell Line Using Multiplex Amplification Kit

[0092] The kit provided by the present invention is used to carry out multiple amplification of 38 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 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.

[0093] 1. Use the chelex-100 method to extract DNA (for specific methods refer to "Forensic DNA Protocol", HumanPress, 1998):

[0094] (1) Take 3 μl of cultured 9948 cell line in a 500 μl centrifuge tube.

[0095] (2) Shake and mix the chelex solution to suspend the chelex suff...

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Abstract

The invention relates to a composite amplification kit for 38 human Y chromosome loci and application thereof. The invention provides a composite amplification system of the 38 human Y chromosome loci. The system includes specific primers for amplifying the 38 loci including 35 STR loci and 3 Indel loci. The 38 pairs of specific primers are subjected to group fluorescence labeling by the six-colorfluorescent labeling technology. Through design and optimization of the primer sequence and the working concentration, simultaneous efficient specific sensitive amplification of the 38 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 38 human Y chromosome loci and applications thereof. Background technique [0002] Short tandem repeats (Short Tandem Repeat, STR), 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 bases Repeating sequences consisting of dozens to more than one hundred nucleotides of repeating units are currently commonly used genetic markers. The number of STR loci is large, widely distributed, and highly polymorphic. The polymorphism is mainly due to the differences in the number of core sequence repeats among individuals, and this difference follows the Mendelian law of inheritance in the genetic process. Therefore, STR amplification detection technology is widely used in individual identification, kinship iden...

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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