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Fluorescence composite amplification system of 30 Y-chromosome STR (Short Tandem Repeat) loca and kit and application

A compound amplification system and Y chromosome technology, applied in the field of biological detection, can solve the problems of missing persons comparison, identification of unfavorable relatives, limited information amount, etc., and achieve stable signal intensity, good system specificity and stability, and information amount. big effect

Active Publication Date: 2018-05-22
SUZHOU MICROREAD GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing kits generally contain about 15 loci, and the amount of information provided is limited, which is not conducive to accurate kinship identification and missing population comparison

Method used

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  • Fluorescence composite amplification system of 30 Y-chromosome STR (Short Tandem Repeat) loca and kit and application
  • Fluorescence composite amplification system of 30 Y-chromosome STR (Short Tandem Repeat) loca and kit and application
  • Fluorescence composite amplification system of 30 Y-chromosome STR (Short Tandem Repeat) loca and kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Amplified FTA card and unextracted whole blood

[0059] 1. Get DNA

[0060] a. FTA card (made by applying fresh blood taken from a blood collection needle)

[0061] b. Fresh blood, etc. (blood samples are donated by volunteers)

[0062] 2. Reaction system

[0063] Shake and mix each reaction reagent (buffer, primer mixture, 1.0mm FTA card or 0.5ul unextracted whole blood) and make a PCR reaction mixture in the following way. The total volume of the amplification system is 25μl, including primers Mixture (5*Primer mix) 5μl, the concentration of primers is shown in Table 1, reaction buffer (5*Master Mix) 5μl, genomic DNA 2μl (extract template DNA, direct expansion test material, the test material is 1.0mm FTA card or 0.5 ul unextracted whole blood, etc.), add ddH 2 O complete the 25ul system.

[0064] 3. PCR reaction procedure

[0065] The PCR amplification program used in the multiplex amplification system involved in the present invention (Table 3).

[...

Embodiment 2

[0075] Embodiment 2 sensitivity, anti-inhibition ability verification

[0076] 1. Carry out gradient dilution with 1ng positive control and use this invention kit to amplify, select 0.05ng template amplification effect picture below (such as Figure 4 ). Experiments have proved that when 0.05ng of DNA template is used, the amplification effect is good, therefore, the amplification system of the present invention has high sensitivity.

[0077] 2. Amplify with 1ng of positive control, and add different concentrations of hematin (hematin) to the system, the amplification effect diagram (such as Figure 5 ). Experiments have proved that the complex system can still be fully amplified when the system contains a high concentration of inhibitors, and most fragments are not affected except for some fragments that are inhibited. Therefore, the amplification system of the present invention has high anti-suppression ability, and is suitable for common case samples.

Embodiment 3

[0078] Example 3 Mixed Sample Verification of Male and Female

[0079] Although the Y chromosome is unique to males, in practice we have found that due to the high homology between the Y chromosome and the X chromosome, some Y-STRs may also amplify female samples. In rape and murder cases, the police It is often possible to collect mixed samples of men and women. This requires that the primer design of Y-STR must have good specificity, so as to avoid the detection of non-target bands in the mixed spots with too high content of female samples, which will cause trouble for the DNA identification of criminal suspects.

[0080] Use 1ng of male samples mixed with female samples of different concentrations to amplify the effect diagram (such as Image 6 ). Experiments have proved that under the influence of ultra-high concentration of female templates, complete bands of male samples can still be amplified without non-specific amplification.

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Abstract

The invention belongs to the technical field of bioinstrumentation and relates to a fluorescence composite amplification system of 30 Y-chromosome STR (Short Tandem Repeat) loca and a kit and application. The following 29 STR loca on Y-chromosomes can be simultaneously amplified: DYS393, DYS570, DYS19, DYS392, DYS549, Y-GATA-H4, DYS391, DYS439, DYS481, DYS635, DYS448, DYS533, DYS456, DYS389I / II, DYS390, DYS438, DYS576, DYS460, DYS458, DYS437, DYS385a / b, DYS643, DYS587, DYS645, DYS531, DYS596, DYS593 and DYS443. In conclusion, the system has the advantages that firstly, the system comprises 30Y-chromosome STR loca of human females, including common sites and low-mutation sites, is unique and novel, large in formation quantity and good in compatibility; secondly, the system is wide in application range, good in directivity, high in precision and applicable to legal medical expert DNA (Deoxyribonucleic Acid) analysis for all material evidence cases with cells of males. By adopting the system, all bioinstrumentation materials with male cells, such as bloodstains, seminal stains, saliva, hair, nails, cartilages and other human body tissues, can be identified, and FTA cards, whole blood, hair with hair follicles, oral cavity swabs and the like can be directly amplified.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and is designed to detect the polymorphism of the short tandem repeat sequence locus in the human genome, specifically refers to a five-color fluorescent labeling system capable of analyzing 30 STR sites on the Y chromosome of a male individual, and is mainly used for The public security database can also be used as an auxiliary detection method for paternity testing. Background technique [0002] In recent years, with the discovery and technological development of short tandem repeats, it has become the most widely used DNA analysis technology. In most cases, the autosomal STR locus is widely used due to its high genetic diversity in the population, and then the Y chromosome due to its special non-recombination nature allows it to be applied to the study of kinship in the same paternal relationship . [0003] The short tandem repeat sequence specific to the human Y chromosome, nam...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156C12Q2600/16
Inventor 杨凡于在亮陈初光庞蒙维郑玉
Owner SUZHOU MICROREAD GENETICS
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