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Fluorescent mark multi-amplification kit of 27 STR loci of human Y-chromosome and application of kit

A technique of fluorescent labeling and multiple amplification, applied in the field of molecular genetics, can solve the problems of low polymorphism, low cumulative individual recognition rate, and small number of loci, and achieve strong amplification specificity, amplification specificity and The effect of high amplification sensitivity and low application threshold

Active Publication Date: 2015-12-23
AGCU SCIENTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

commonly used in forensic science The number of loci is small, and the cumulative individual recognition rate is low, Have been identified as having very low polymorphisms in certain populations

Method used

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  • Fluorescent mark multi-amplification kit of 27 STR loci of human Y-chromosome and application of kit
  • Fluorescent mark multi-amplification kit of 27 STR loci of human Y-chromosome and application of kit
  • Fluorescent mark multi-amplification kit of 27 STR loci of human Y-chromosome and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Screening of Y chromosome STR loci

[0050] 通过对DYS549、DYS522、DYS389I / II、DYS439、DYS447、DYS392、DYS576、DYS19、DYS388、DYS391、DYS635、DYS460、DYS456、DYS533、DYS520、DYS438、DYS527a / b、DYS449、DYS709、DYS622、DYS481、DYS437、 The study of gene polymorphism and mutation rate of 38 Y chromosome STR loci including DYS390, DYS630, DYS448, H4, DYS458, DYS607, DYS393, DYS552, DYS570, DYS385a / b, DYS593, DYS444 and DYS643, finally screened out DYS392 、DYS389I / II、DYS447、DYS438、DYS527a / b、DYS522、DYS391、DYS456、DYS19、DYS388、DYS448、DYS385a / b、DYS481、DYS437、DYS390、DYS460、DYS533、DYS458、DYS393、Y_GATA_H4、DYS439、DYS635、DYS444 , DYS643, a total of 27 Y chromosome STR loci. The corresponding information for each locus is shown in Table 4:

[0051] Table 4. DatabaseY27 locus information

[0052]

[0053]

[0054] Embodiment 2.27 Y chromosome STR locus arrangement

[0055] According to the above 27 loci, a unique locus arrangement and chemical fluorescent dye labeling method are designed: D...

Embodiment 327

[0056] Example 3. Design of specific primers for 27 Y chromosome STR loci and adjustment of different primer concentrations

[0057] 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, and the kinetics of the reaction system become more and more complex, so it is necessary to design a large number of primer sequences for complex testing And explore the specific concentration ratio between the primers in the multiplex amplification system, and finally ensure that more STR loci are compounded without reducing the specificity and sensitivity of the kit.

[0058] (1) Specific primer design

[0059] Before designing primers, it is necessary to analyze the properties of the target sequence to be tested, and select highly conserved regions with uniform base distribution for primer design.

[0060] a. Primer Tm value: Due to different algorithms, primers designed by ...

Embodiment 4

[0066] Embodiment 4. Adjust PCR reaction mixture

[0067] In the PCR system, Mg 2+ Concentration test 0.5mM, 1.0mM, 1.5mM2.0mM, 2.5mM, 3.0mM6 gradients, dNTPs concentration test 0.1mM, 0.15mM, 0.2mM, 0.25mM, 0.30mM, 0.35mM6 gradients, hot start Taq enzyme The content was tested in 5 gradients of 1.0U, 1.5U, 2.0U, 2.5U and 3.0U respectively, the concentration of Tris-HCl was set at 10mM, and the concentration of KCl was set at 40mM. By designing an orthogonal experiment, the Mg 2+ The concentration was set at 2.0mM, the concentration of dNTPs was set at 0.25mM, the content of hot-start Taq enzyme was 2.0U, the concentration of Tris-HCl was set at 10mM, and KCl40mM. The above materials were used to prepare a reaction mixture ReactionMix, which was added to the PCR system. The components of the final PCR system are shown in Table 5.

[0068] table 5:

[0069]

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Abstract

The invention discloses a fluorescent mark multi-amplification kit of 27 STR loci of a human Y-chromosome and an application of the kit. The 27 low-mutation-rate STR loci on the Y chromosome can be amplified for multiple times in a single reaction. According to the fluorescent mark multi-amplification kit of the 27 STR loci of the human Y-chromosome and the application of the kit, a unique locus distribution mode and a specific primer sequence are designed, the 27 loci are divided into four groups, and different fluorescent marks are marked for each group. The multi-amplification system is one of the Y-STR kits which can detect the maximum number of the loci on the market, and the contained loci are all low-mutation-rate Y-STR loci, so that the kit is more suitable for checking of a male family. The multi-amplification system is good in primer specificity, wide in temperature tolerance range and high in check-material adaptability.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and in particular relates to a fluorescent marker composite amplification kit for 27 STR loci of human Y chromosome and its application, which can be applied to database construction and investigation, paternity identification, DNA family tree construction and the like. Background technique [0002] STR genetic markers, also known as microsatellite DNA, widely exist in the genomes of prokaryotic and eukaryotic organisms, and are a kind of repeating sequence consisting of tens to more than one hundred nucleotides consisting of 2 to 6 nucleotides as repeating units . Different numbers of core sequences are arranged in tandem repeats, while the lengths are polymorphic. According to the conserved sequences at both ends, design primers, perform PCR, and then detect polymorphisms of STR genetic markers by polyacrylamide, agarose gel electrophoresis, or capillary electrophoresis. [0003] The Y chrom...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 郑卫国李科杰陈拓梅兴林郭育林
Owner AGCU SCIENTECH
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