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Compound amplification system, kit and application of rapidly mutating y-chromosomal short tandem repeats

A compound amplification system and short tandem repeat technology, applied in the biological field, can solve the problems of individual recognition rate not reaching forensic science, few mutation sites, low mutation rate, etc. Strong inhibitory effect

Active Publication Date: 2021-11-05
SUZHOU MICROREAD GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of male sperm cell production, the Y chromosome will not recombine, so in general, it will be stably inherited to its male offspring. Therefore, in the genetic analysis of males in the same family or in the same region, the mutation rate is low, The traditional Y-filer locus with few mutation sites has obvious limitations, and the individual identification rate cannot meet the requirements of forensic science

Method used

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  • Compound amplification system, kit and application of rapidly mutating y-chromosomal short tandem repeats
  • Compound amplification system, kit and application of rapidly mutating y-chromosomal short tandem repeats
  • Compound amplification system, kit and application of rapidly mutating y-chromosomal short tandem repeats

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Using the compound amplification system of rapid mutation Y chromosome short tandem repeat sequence to amplify the samples of various inspection materials 1. Obtaining DNA

[0067] a. Genomic DNA was extracted by Chelex-100 method or magnetic bead method.

[0068] b. Direct amplification of whole blood

[0069] c. Direct amplification of buccal swabs

[0070] Use the Chelex-100 method to extract genomic DNA (refer to "Forensic DNA Protocol". HumanaPress, 1998); or take 0.5-5 μl of anticoagulated whole blood and place it in a 500 μl centrifuge tube, shake and mix the Chelex solution to fully suspend the Chelex, each tube Add 195 μl Chelex-100 (5%) solution and 5 μl proteinase K (20mg / ml) and shake and mix well, keep warm at 56°C for two hours or overnight, take out and shake for 2 minutes, heat in boiling water for 10 minutes, centrifuge at 13000rpm for 5 minutes, and pipette carefully Transfer 150 μl of supernatant to a new centrifuge tube.

[0071] 2. Reac...

Embodiment 2

[0086] Example 2 Distinguish family samples using the compound amplification system of rapidly mutated Y chromosome short tandem repeats

[0087] 1. Collect blood samples from the family (blood samples are provided by a genetic testing center)

[0088] 2. Take 0.5-5 μl of anticoagulated whole blood into a 500 μl centrifuge tube, shake and mix the Chelex solution to make the Chelex fully suspended, add 195 μl Chelex-100 (5%) solution and 5 μl proteinase K (20 mg / ml) to each tube and shake the mixture After incubating at 56°C for two hours or overnight, take out and shake for 2 minutes, heat in boiling water for 10 minutes, then centrifuge at 13,000 rpm for 5 minutes, and carefully transfer 150 μl of the supernatant to a new centrifuge tube.

[0089] 3. Perform PCR amplification according to the amplification conditions in Example 1.

[0090] 4. Detect on the genetic analyzer according to Example 1, the amplification effect is shown in Figure 7 , Figure 8 .

[0091] 5. The...

Embodiment 3

[0095] Example 3 Verification of anti-inhibition ability

[0096] A 1ng positive control was used for amplification, and different concentrations of humic acid were added to the system. Experiments have proved that the complex system can still be fully amplified when the system contains a high concentration of inhibitors, and most of the fragments are not affected except some fragments are inhibited. Therefore, the amplification system of the present invention has high anti-suppression ability, and is suitable for common case inspection materials.

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Abstract

The invention belongs to the field of biotechnology, and relates to a compound amplification system, a kit and an application of rapidly mutating Y chromosome short tandem repeat sequences. The compound amplification system includes 16 pairs of primers, which can simultaneously amplify 16 STR sites: DYS630, DYS464, DYF403S1b, DYF399S1, DYS518, DYF403S1a, DYS527, DYS713, DYS612, DYS626, DYS627, DYS526, DYF404S1, DYF387S1, DYS449, DYS547. Compared with the prior art, the present invention has the following advantages: 1. Contains 16 STRs with high mutation rate of human male Y chromosome, which is unique and novel, with large amount of information and good compatibility. 2. It has wide adaptability, strong directivity and high precision, and is suitable for forensic DNA analysis in all cases involving male cellular evidence. All biological samples containing male cells, such as blood stains, semen spots, saliva, hair, nails, cartilage and other human tissues, can be identified. 3. The system has good specificity and stability. After repeated verifications, no non-specific amplification products are produced, and the signal intensity is stable. 4. High sensitivity, the amount of DNA template as low as 15 pg can be accurately typed.

Description

technical field [0001] The invention belongs to the field of biotechnology and contains 16 high mutation rate Y-STR sites, 7 of which belong to multi-copy Y-STR sites, and the whole system can amplify 26 fragments. This system uses fluorescently labeled primers to simultaneously amplify multiple human male genome Y chromosome DNA short tandem repeats (STRs), efficiently and accurately perform STR typing, and then sensitively and efficiently identify forensic individuals and kinship identification, population genetic analysis, disease Gene mapping studies provide a basis for judgment. Background technique [0002] Human genome STR (short tandem repeat sequence) is a DNA genetic marker formed by tandem repeats with a few bases as the core unit. The distinction between different races, different populations and even different individuals is through the difference in core unit sequence and repeat number, which also constitutes the genetic polymorphism of STR. In the genome, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156C12Q2600/16
Inventor 于在亮杨凡陈初光庞蒙维郑玉
Owner SUZHOU MICROREAD GENETICS
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