Human X-chromosome composite amplification system composed of 20 short serially-connected repetitive sequences and applications thereof

A compound amplification system and short tandem repeat technology, applied in the field of biotechnology and biotechnology, can solve the problems of genetic polymorphism, individual identification and lack of accuracy of detection, etc.

Active Publication Date: 2015-12-23
SUZHOU MICROREAD GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing X-STR kits and X-chromosome-related patents target a small number of STR loci, and there are certain deficiencies in genetic polymorphism, individual recognition and detection accuracy, so it is necessary to develop a site X-STR kit with a large number of points, high genetic polymorphism, and strong individual recognition

Method used

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  • Human X-chromosome composite amplification system composed of 20 short serially-connected repetitive sequences and applications thereof
  • Human X-chromosome composite amplification system composed of 20 short serially-connected repetitive sequences and applications thereof
  • Human X-chromosome composite amplification system composed of 20 short serially-connected repetitive sequences and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Design of multiplex amplification system for 20 short tandem repeat sequences of human chromosome

[0075] 1. Primer Design

[0076] The primers are designed using software such as PrimePremier5 and NCBIBlast. When designing the primers, it should be ensured that the Tm value of each primer is within the range of (60 ± 2) °C, the amplification efficiency is close, and the amplification products of each pair of primers are different in size. Sufficient to distinguish in capillary electrophoresis. After the design is completed, use software such as AutoDimer to analyze the interaction between the primer dimer and different primers. If there is an interaction that can produce non-specific products or dimers, it needs to be redesigned until the primer sequence that meets the requirements is obtained.

[0077] Select the human DNA template, use the above 20 pairs of primers to carry out single-plex amplification, place the amplified products on 1.5% agarose gel el...

Embodiment 2

[0085] Example 2 Genotyping detection of a certain sample

[0086] 1. Collection of blood samples (blood samples are donated by volunteers)

[0087] 2. DNA extraction

[0088]Use the Chelex-100 method to extract genomic DNA (refer to "Forensic DNA Protocol". HumanaPress, 1998). Take 0.5-5 μl of anticoagulated whole blood or (1-3mm)*(2-5mm) blood spots in a 500 μl centrifuge tube, shake and mix Homogenize the Chelex solution to fully suspend the Chelex, add 195 μl Chelex-100 (5%) solution and 5 μl proteinase K (20 mg / ml) to each tube, shake and mix, keep warm at 56 degrees Celsius for two hours or overnight, take it out and shake for 2 minutes, heat in boiling water for 10 Minutes later, centrifuge at 13000 rpm for 5 minutes, and carefully pipette 150 μl of the supernatant into a new centrifuge tube.

[0089] 3. Reaction system

[0090] Shake and mix each reaction reagent (buffer, primer mix, genomic DNA, etc.) and make a PCR reaction mixture in the following manner. The tot...

Embodiment 3

[0102] Embodiment 3 is aimed at the optimization experiment of enzyme reaction buffer (2.5*BufferD)

[0103] The concentration gradient of KCl in the enzyme reaction buffer is: 0.075M, 0.1M, 0.125M three concentration gradients. MgCl 2 The concentration gradient of the enzyme reaction buffer is: 1mM, 1.5mM, 2mM three concentration gradients. The concentration gradient of Brij58 in the enzyme reaction buffer is: 0.001mM, 0.0015mM, 0.002mM three concentration gradients. The concentration gradient of DTT in the enzyme reaction buffer is: 0.015M, 0.02M, 0.025M three concentration gradients. The concentration gradient of Betaine in the enzyme reaction buffer is: 0.05M, 0.1M, 0.15M three concentration gradients. The concentration gradient of DMSO in the enzyme reaction buffer is: 0.3%, 0.4%, 0.5% three concentration gradients. The concentration gradient of MRTaqⅡ in the enzyme reaction buffer is: 0.15U, 0.25U, 0.35U three concentration gradients. Orthogonal experiments with dif...

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Abstract

The invention relates to the technical field of biology, and relates to a human X-chromosome composite amplification system composed of 20 short serially-connected repetitive sequences and applications thereof. The composite amplification system comprises 20 pairs of primers and is capable of amplifying 19 STR sites: DXS6795, DXS6803, DXS6807, DXS9907, DXS7423, GATA172D05, DXS101, DXS9902, DXS7133, DXS9810, GATA31E08, DXS6800, DXS981, DXS10162, DXS6809, GATA165B12, DXS10079, DXS10135, and HPRTB, and one sex recognition site: Amelogenin. Five-color fluorescence labeling is adopted by the system, the size of amplification products is in a range of 85 to 450 bp, the operation is simple, and the individual recognition performance is high. The sites provided by the provided composite amplification system are highly compatible to the conventional commonly-used X-STR kits, moreover, sites with high polymorphism information are provided, and the provided composite amplification system can be applied to paternity identification related with X-chromosome, individual recognition, and auxiliary detection of X-chromosome sex-linked hereditary diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a compound amplification system of 20 short tandem repeat sequences of human X chromosome, in particular to a fluorescent detection reagent for performing compound amplification through polymerase chain reaction by using polymorphic genetic markers on the X chromosome The system can be applied to routine paternity testing related to the X chromosome, individual identification, and auxiliary detection of X-chromosome-linked genetic diseases. Background technique [0002] Human genome STR (short tandem repeat sequence) is a DNA genetic marker formed by tandem repeats with a few bases as the core unit. The distinction between different races, different populations and even different individuals is through the difference in core unit sequence and repeat number, which also constitutes the genetic polymorphism of STR. In the genome, there is an STR site every 15-20kb on average, accounting ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/156
Inventor 陈初光于在亮徐梅波
Owner SUZHOU MICROREAD GENETICS
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