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A fluorescence-labeled multiplex amplification kit for simultaneously amplifying human autosome and y-chromosome str loci and its application

A technology for autosomes and Y chromosomes, which is applied to the fluorescent labeling composite amplification kit and its application field for simultaneously amplifying the STR loci of human autosomes and Y chromosomes. Limitation and other issues, to achieve the effects of saving manpower, material and financial resources, shortening detection time, and high sensitivity

Active Publication Date: 2021-06-22
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 autosomal STR and Y-STR hybrid detection technology 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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  • A fluorescence-labeled multiplex amplification kit for simultaneously amplifying human autosome and y-chromosome str loci and its application
  • A fluorescence-labeled multiplex amplification kit for simultaneously amplifying human autosome and y-chromosome str loci and its application
  • A fluorescence-labeled multiplex amplification kit for simultaneously amplifying human autosome and y-chromosome str loci and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0041] 1. Determination of the locus

[0042] On the basis of the scheme of 201110243734.9, the present invention adds two autosomal loci D19S433 and PentaE, so that the number of autosomal STR loci reaches 18. By performing genotype detection on 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 showed that 16 A-STR loci had DP values ​​close to 0.9, and H were all greater than 0.7. The PE value is above 0.5, which has good forensic application value. Although the genetic statistics of the TH01 and TPOX loci are not as good as those of the above 16 A-STRs, they still belong to the recommended category of the DNA database of the Ministry of Public Security. Maintaining the e...

Embodiment 2

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

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

[0058] PCR is a complex molecular dynamics process. With the increase of the number of primers in the composite amplification system, the mutual interference between primers at different loci becomes more and more serious. At the same time, due to the large amplified fragment and the longer fluorescence emission wavelength, the detection efficiency is lower, which leads to the lower detection efficiency. The locus of the fragment adopts traditional single fluorescent labeling, which c...

Embodiment 3

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

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

[0061] Table 6 Amplification procedure

[0062]

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Abstract

The invention discloses a fluorescent label compound amplification kit for simultaneously amplifying human autosome and Y chromosome STR loci and application thereof. The kit can simultaneously detect 18 A-STRs, 20 Y-STRs and gender (Amel) loci, realize the simultaneous construction of two libraries of A-STR and Y-STR, and can be used for individual identification, paternity testing, and family investigation And family tree construction, etc., can be used for rapid investigation of cases at the same time, shortening the detection time and improving the efficiency of excluding and identifying criminal suspects. The 20 Y-STR loci have great geographical and surname orientation for case investigation. Unrelated families can be excluded as much as possible. The invention can detect a small amount of male DNA in the background of a large number of female DNA samples, and is a powerful weapon for detecting mixed spot samples such as rape crimes. And when detecting paternity-child paternity, there is no need to do separate Y-STR experimental analysis because the Y-STR data is already included.

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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Patent Type & Authority Patents(China)
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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