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Multiplex amplification system for mouse short tandem repeats, and detection kit

A short tandem repeat and detection kit technology, which is applied in the field of biotechnology detection, can solve the problems of distinguishing, not ensuring each individual mouse, and insufficient resolution, etc.

Active Publication Date: 2020-09-29
广州赛库生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the resolution provided by 9 loci is not enough for a large number of mouse individuals, and it is not guaranteed to distinguish each individual mouse

Method used

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  • Multiplex amplification system for mouse short tandem repeats, and detection kit
  • Multiplex amplification system for mouse short tandem repeats, and detection kit
  • Multiplex amplification system for mouse short tandem repeats, and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Design of multiplex amplification system for mouse short tandem repeats

[0079] 1. Design of primer combination

[0080] First, based on the whole genome sequence of the mouse, we selected the four-base repeated core sequence among all the STR sites, which is highly polymorphic and species-specific. Use primer design tools such as PrimerPremier5 and NCBI Primer Blast to design primers, and based on previous design experience, try to ensure that the Tm value of each primer is within the range of (60±3)°C, the amplification efficiency is equivalent and the size of the amplification product of each pair of primers The difference was more than 20bp, and AutoDimer was used to analyze the interaction between primer dimers and primers. If the specificity of the primers is not high or a cross-amplification reaction occurs, it needs to be redesigned until it meets the requirements.

[0081] The DNA of NIH / 3T3 cells was selected as a template, and each STR site was amplified s...

Embodiment 2

[0133] The following is the specific implementation of our application of the present invention to detect 12 cases of mouse cells, 1 case of Chinese hamster cells, 1 case of rat cells, and 1 case of human cells.

[0134] 1. DNA extraction

[0135] Use a DNA extraction kit (full gold) for DNA extraction, operate according to the instructions, quantify with a UV spectrophotometer after DNA extraction, dilute it into a 2ng / μl solution, and immediately carry out the next step of detection or store it at -20°C for later use.

[0136] 2.PCR

[0137] 2.1 Reaction system:

[0138] Dissolve 19 pairs of primers respectively and make a stock solution with a concentration of 100 μM, and then make 500 μl of 10x primer mixture (10xPrimerMix) according to the volume ratio in Table 2:

[0139] Table 2 The volume ratio of each primer

[0140]

[0141]

[0142] Shake and mix each reaction reagent (Buffer, PrimerMix, Taq enzyme, etc.) and make a PCR reaction mixture according to the vol...

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Abstract

The invention discloses a multiplex amplification system for mouse short tandem repeats. The multiplex amplification system comprises 18 pairs of primers, and 18 mouse STR sites can be amplified at the same time. The invention also provides a detection kit formed by the multiplex amplification system. The multiplex amplification system can obtain a great amount of information through once amplification. An amplification product has fluorescence, and can be directly detected on a sequencer. The result is accurate; the repeatability is high; a standard reference database can be built by the data; the operation is simple and convenient; short time is required; and scale production and automation can be easily realized.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to a multiple amplification system and a detection kit for mouse short tandem repeat sequences. Background technique [0002] Shortly after the first laboratory-grown cell lines were created, there have been articles and reports about the problem of cross-contamination of cell lines. According to the test results, some authors estimated that as many as 20% to 33% of the cell lines used in the world are mislabeled or identified. Since 2015, many authoritative journals in the scientific community have successively required authors to provide identification reports on the cell lines used to prove that the cell lines used are not cross-contaminated or mislabeled. Afterwards, the issue of cross-contamination of cell lines has received the attention of the majority of scientific researchers. Recently, although the situation of using wrong cell lines has improved, it is only for hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2600/156C12Q2537/143C12Q2525/151C12Q2531/113
Inventor 梁轩毅秦庆添黄唯屹江婉婷陈雅欣
Owner 广州赛库生物技术有限公司
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