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Fluorescence labeled composite amplification kit capable of simultaneously amplifying human autosome and Y-chromosome STR (short tandem repeat) loci and application thereof

A Y chromosome and autosomal technology, which is applied to a fluorescent labeling compound amplification kit for simultaneously amplifying human autosomal and Y chromosome STR loci and its application fields, can solve the needs of case investigation that cannot meet the needs of investigation, the investigation ability is limited, and the Restrictions and other issues to achieve the effect of saving manpower, material and financial resources, shortening detection time, and high sensitivity

Active Publication Date: 2018-07-06
GUANGDONG HUAMEI ZHONGYUAN BIOLOGICAL SCI & TECH +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent 201310364183.0 discloses the mixed detection technology of autosomal STR and Y-STR for the first time, but because the number of Y-STR loci is too small, and the five-color fluorescence technology is still used, the screening ability is limited, and the practical application is greatly restricted; the patent 201510918064.4 Although the latest six-color fluorescent technology is used, the amplified fragment is still within 500bp, and the detection of Y-STR sites still cannot meet the needs of case investigation

Method used

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  • Fluorescence labeled composite amplification kit capable of simultaneously amplifying human autosome and Y-chromosome STR (short tandem repeat) loci and application thereof
  • Fluorescence labeled composite amplification kit capable of simultaneously amplifying human autosome and Y-chromosome STR (short tandem repeat) loci and application thereof
  • Fluorescence labeled composite amplification kit capable of simultaneously amplifying human autosome and Y-chromosome STR (short tandem repeat) loci and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Determination of detection loci; determination of kit primer set, amplification system, and amplification method.

[0041] 1. Determination of genetic loci

[0042] On the basis of the 201110243734.9 scheme, the present invention adds two autosomal loci D19S433 and PentaE, so that the number of autosomal STR loci reaches 18, and through genotype detection of more than 10,000 unrelated individuals, according to the alleles of each locus Distribution frequency statistical analysis of individual discrimination (DP), heterozygosity (H), non-parent exclusion rate (PE) and other data, the results show that the DP values ​​​​of 16 A-STR loci are close to 0.9, and H is greater than 0.7. The PE value is above 0.5, which has good forensic application value. Although the genetic statistics of TH01 and TPOX loci are not as good as the above 16 A-STRs, they still belong to the recommended category of DNA database of the Ministry of Public Security. Maintaining the existi...

Embodiment 2

[0056] Example 2: Comparative test of novel fluorescent labels.

[0057] The present invention creatively extends the amplified fragment to 600bp in order to realize PCR single-tube amplification of 39 sites. For loci with larger amplified fragments (EA50 color DYS576, E11 color DYS533, 620 color DYS481), use traditional single fluorescent labeling, according to the technical scheme of Example 1, amplify a direct expansion blood card sample X1, and amplify the map See image 3 .

[0058] PCR is a complex molecular dynamics process. With the increase of the number of primers in the multiplex amplification system, the mutual interference between primers at different loci becomes more and more serious. At the same time, due to the larger amplification fragments and the longer fluorescence emission wavelength, the detection efficiency is lower. The locus of the fragment, using the traditional single fluorescent label, cannot meet the needs of amplification detection. The presen...

Embodiment 3

[0059] Example 3: Adjusting the PCR reaction program.

[0060] Because the kit of the present invention needs to meet the adaptability of different test materials and the tolerance of different annealing temperatures, it is necessary to test different amplification procedures to ensure that different test materials can obtain correct DNA analysis within a wide temperature range. type. First, the number of cycles test: 28 cycles, 29 cycles, 30 cycles, and 31 cycles were tested, and the experiment proved that the best cycle is 30; secondly, the annealing temperature test: 57 ° C, 58 ° C, 59 ° C were tested , 60°C, 61°C, 62°C, experiments have shown that the optimum temperature is 60°C and 58°C. The final amplification procedure was as follows:

[0061] Table 6 Amplification program

[0062]

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Abstract

The invention discloses a fluorescence labeled composite amplification kit capable of simultaneously amplifying human autosome and Y-chromosome STR (short tandem repeat) loci and application thereof.The kit is capable of simultaneously detecting 18 A-STR and 20 Y-STR and gender (Amel) loci and realizing simultaneous construction of two libraries of A-STR and Y-STR and can be applied to individualrecognition, paternity identification, family screening, family tree construction and the like as well as rapid screening of cases. The effects of shortening the detection time and simultaneously improving the efficiency of eliminating and determining criminal suspects are achieved. The 20 Y-STR loci have excellent regional and family name directionality for investigation of the cases, and irrelevant families can be eliminated to the greatest degree. The kit disclosed by the invention is capable of detecting trace male DNA under the background of a large amount of female DNA samples, and is astrong weapon of mixed stain detectors for detecting forcible rape crimes and the like. Moreover, when parent-child paternity identification is detected, since the Y-STR data are included, Y-STR experimental analysis does not need to be independently performed.

Description

technical field [0001] The invention belongs to the field of forensic physical evidence, and relates to a new type of fluorescent marker composite amplification inspection system with 39 loci, in particular to a six-color novel fluorescent marker composite amplification system for simultaneous analysis of human genome autosome and Y chromosome loci , the system can be applied to individual identification, father-son paternity identification, suspect family investigation, trace male sample detection in rape cases, autosome database and Y chromosome database construction. Background technique [0002] Short tandem repeat sequence (STR) is a molecular genetic marker commonly used at present. Its fragment is small and easy to amplify. It is suitable for testing trace and degraded biological samples. The amplification conditions of each locus are similar to achieve multiple amplification and automatic detection. , so it has the advantages of sensitivity, accuracy, speed, and larg...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6879C12Q1/6858
CPCC12Q1/6858C12Q1/6879C12Q1/6888C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 杜蔚安刘超李发院刘宏钱水柳勇张勇果王邦超赵鹏郑卫国
Owner GUANGDONG HUAMEI ZHONGYUAN BIOLOGICAL SCI & TECH
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