Microsatellite loci marker combination and application thereof

A microsatellite labeling and labeling technology, applied in the field of DNA labeling detection, can solve the problems of low repeatability, high price, complicated operation, etc., and achieve the effect of suitable size interval and easy simultaneous detection

Inactive Publication Date: 2010-05-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to improve the detection technology in view of the shortcomings of existing methods such as complex operation, time-consuming, low reproducibility and the shortcomings of existing commercial kits such as high price and high detection cost. Specifically select high-throughput, accurate and efficient 17 bovine microsatellite markers for amplification and typing. These markers are highly polymorphic, unlinked between markers, and have appropriate fragment size intervals, so they are easy to detect simultaneously

Method used

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  • Microsatellite loci marker combination and application thereof
  • Microsatellite loci marker combination and application thereof
  • Microsatellite loci marker combination and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Primer sequences for microsatellite loci

[0065] Synthesize oligonucleotide primers according to the following primer sequences, and dilute the primers to 5 μmol / L with sterilized deionized water.

[0066] Table 117 microsatellite marker primer sequences and fluorescent modifications

[0067]

[0068]

[0069]

Embodiment 2

[0070] Example 2: DNA Extraction

[0071] (1) DNA extraction from bovine blood

[0072] Genomic DNA was extracted from blood clots using the DP318 kit from Tiangen Biochemical Biotechnology Co., Ltd. The specific steps are as follows:

[0073] 1) Take out the sample, after melting, cut 200μL into a 2mL centrifuge tube;

[0074] 2) Add 600 μL cell lysate CL and shake well;

[0075] 3) Centrifuge at 10000rpm for 1min, discard the dark red supernatant;

[0076] 4) Repeat steps 2 and 3 once;

[0077] 5) Add 200 μL buffer GS, and fully suspend blood clot particles with a vortexer;

[0078] 6) Add 20 μL proteinase K, 220 μL buffer GB, shake well;

[0079] 7) Digest in an oven at 56°C for more than 3 hours. In the early stage of digestion, invert and mix several times until the solution becomes clear and free of blood clot particles;

[0080] 8) Add 200 μL of absolute ethanol, shake gently to mix, transfer to the adsorption column, centrifuge at 12000rpm for 30s, and discard th...

Embodiment 3

[0102] Embodiment 3: PCR amplification

[0103] PCR reaction system

[0104] Multiplex amplification set 1

[0105] Element

concentration

Mg 2+

2.0μmol / L

dNTP

200μmol / L

Primer

ETH10

0.25μmol / L

ETH225

0.25μmol / L

TGLA227

0.25μmol / L

BM1818

0.25μmol / L

TGLA126

0.50μmol / L

Taq DNA polymerase

1U

DNA template

20~50ng

[0106] Multiplex amplification set 2

[0107] Element

concentration

Mg 2+

2.0μmol / L

dNTP

200μmol / L

[0108] Element

concentration

Primer

BM1824

0.25μmol / L

INRA023

0.25μmol / L

TGLA53

0.25μmol / L

BM2113

0.25μmol / L

TGLA122

0.50μmol / L

TaqDNA polymerase

1U

DNA template

20~50ng

[0109] Multiplex amplification group 3

[0110] Element

concentration

Mg 2+

2.0μmol / L

...

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Abstract

The invention discloses a microsatellite loci marker combination which comprises 17 microsatellite loci markers, and simultaneously provides an efficient detection method based on the 17 microsatellite loci markers; the markers have high polymorphism, are not linked with each other and have proper fragments interval, and are easy to be detected simultaneously. The invention is improved in a detection technology, optimizes an experimental system by utilizing a multi-marker amplification technology and a fluorescent semi-automatic microsatellite typing method, thus establishing high flux, accurate and effective 17 cattle microsatellite marker compound amplification and a gene typing method.

Description

technical field [0001] The invention relates to the detection of DNA markers in the bovine genome, in particular to the specific combination and application of bovine microsatellite marker loci. Background technique [0002] Microsatellite (microsatellite) is a kind of DNA sequence with short length and high variability in the genome of higher organisms. Usually, the repeating unit consists of 2 to 5 nucleotides in the form of tandem repeats, so it is also called simple sequence repeat (Simple Sequence Repeat, SSR) or tandem repeat sequence (Short Tandem Repeat, STR). [0003] Microsatellite markers are currently widely used polymorphic DNA markers, and have the following notable features: 1) they are widely distributed in the genomes of higher organisms, and it is estimated that there is a microsatellite marker every 10-50 kb. The genetic map contains 3802 microsatellite markers; 2) High polymorphism and rich information content, many microsatellites have more than 5 allel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N27/447C12N15/11
Inventor 张毅张沅孙东晓王雅春俞英
Owner CHINA AGRI UNIV
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