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Y-STR compound amplification detection kit marked by novel fluorescent labeling method and use method thereof

A detection kit and composite amplification technology, applied in the biological field, can solve problems such as difficulty in sensitivity, and achieve the effects of fast amplification speed, strong amplification specificity, and good balance

Active Publication Date: 2017-06-13
JIANGSU SUPERBIO LIFE SCI CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, ROX dye labeling is more than FAM labeling, the same amount of product, the signal intensity is only one-fourth to one-eighth, under such a large difference in fluorescence efficiency, better balance between the various fluorescent channels and a better High sensitivity becomes difficult

Method used

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  • Y-STR compound amplification detection kit marked by novel fluorescent labeling method and use method thereof
  • Y-STR compound amplification detection kit marked by novel fluorescent labeling method and use method thereof
  • Y-STR compound amplification detection kit marked by novel fluorescent labeling method and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: Preparation of Y-STR composite amplification reagent

[0063] 1. Arrangement of sites in the Y-STR multiplex amplification detection system

[0064] The Y-STR kit of the present invention contains 20 low mutation rate loci commonly used at present: DYS393, DYS19, DYS392, Y_GATA_H4, DYS460, DYS458, DYS481, DYS635, DYS448, DYS533 DYS456, DYS389I / II, DYS390, DYS431, DYS39 , DYS439, DYS437 and DYS385a / b; and seven rapidly mutating loci: DYS570, DYS576, DYS627, DYF387S1a / b, DYS449 and DYS518.

[0065] The arrangement of the above STR sites is as follows figure 1 shown.

[0066] 2. Special primers for Y-STR multiplex amplification system

[0067] The composite amplification primers, primer sequences, primer concentrations and fluorescein labeling methods were designed according to the above sites as shown in Table 2.

[0068] 3. Preparation of Y-STR fluorescence multiplex amplification verification system

[0069] The kit includes the following components: ...

Embodiment 2

[0078] Example 2. Application of Y-STR compound amplification verification reagent

[0079] 1. Sample amplification

[0080] Add 24 μL of the DNA test reagent prepared in Example 1 to 1 μL of 0.5 ng / μL male DNA standard M4615 for PCR amplification. DNA standard M4615 was purchased from Suzhou Yuewei Gene Technology Co., Ltd.

[0081] The PCR amplification program is: 95°C for 3 minutes; 94°C for 5 seconds, 60°C for 1 minute, 27 cycles; 60°C for 10 minutes final extension; 4-16°C hold.

[0082] 2. Detection of PCR products

[0083] After the amplification reaction was completed, the reaction tube was taken out, and electrophoresis and detection were performed with an ABI3100 genetic analyzer.

[0084] 1) Take (0.5 μL molecular weight internal standard + 10 μL deionized formamide) × (number of samples) to make a mixed solution, mix well and dispense, 10 μL per tube.

[0085] 2) Then add 1 μL of the amplification product or allele ladder (ladder), and centrifuge briefly to co...

example 3

[0087] Example 3. Identification of male individuals in mixed male and female plaques by Y-STR multiple amplification verification reagent

[0088] Add 24 μL of the DNA test reagent prepared in Example 1 to 1 μL of the male and female DNA mixture, wherein, the male DNA sample M M46150.1ng, the female DNA sample 9947A2ng. Amplification and detection were performed according to the sample volume and amplification procedure in Example 2. Its test results are as Figure 4 As shown, the above mixed sample can detect the STR typing of the complete male DNA sample, and there is no amplification for the female sample.

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Abstract

The invention discloses a Y-STR compound amplification detection kit marked by a novel fluorescent labeling method and a use method thereof. The Y-STR compound amplification detection kit performs compound amplification of 27 Y-STR loca with a compound amplification primer by adopting a polymerase chain reaction; the amplification product is detected by means of a gene sequencing instrument of a single channel or multichannel capillary tube; 27 Y-STR loca comprise 20 conventional low mutation rate loca and 7 fast mutant loca. The novel fluorescent labeling method comprises the steps of: marking a first alkaline group at the 5' end of the primer by a conventional dye; and marking the sixth alkaline group with a short excitation wavelength fluorescent dye to realize fluorescent energy transfer. The signal strength generated by the method is 3-11 times higher than that of the conventional method, and an amplification result which is more stable and balanced and higher in sensitivity can be obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Y-STR composite amplification detection kit marked by a novel fluorescent labeling method and a use method thereof. Background technique [0002] Short tandem repeat sequence (STR for short), also called microsatellite DNA, is a kind of nucleotide repeat sequence widely present in prokaryotic and eukaryotic genomes. It generally consists of tens to more than one hundred nucleotide repeating sequences consisting of 2-6 nucleotides as repeating units. Different numbers of core sequences are arranged in tandem repeats, and the length is 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 genetic polymorphism of the human Y chromosome has many research advantages, such as no recombination wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6888C12Q2600/156C12Q2600/16C12Q2563/107C12Q2537/143C12Q2525/151
Inventor 葛斌文陈拓李翔
Owner JIANGSU SUPERBIO LIFE SCI CO LTD
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