Method for detecting sheep FecB gene polymorphism by using micro-fluidic SNP chip

A gene polymorphism and chip detection technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of difficult high-throughput automatic determination, numerous procedures, and high cost. Achieve the effect of reducing manual operation process, easy interpretation and simplified operation

Inactive Publication Date: 2017-09-29
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI +1
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AI Technical Summary

Problems solved by technology

Most of the traditional FecB gene detection methods use PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) detection methods. These methods have low throughput, numerous procedures, and difficult Achieving high-throughput automated assays
At present, the newly developed high-throughput typing technologies such as TaqmanMGB and SnaPshot can realize fast and accurate high-throughput typing of the FecB gene, but there is a problem of high cost

Method used

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  • Method for detecting sheep FecB gene polymorphism by using micro-fluidic SNP chip
  • Method for detecting sheep FecB gene polymorphism by using micro-fluidic SNP chip
  • Method for detecting sheep FecB gene polymorphism by using micro-fluidic SNP chip

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Experimental program
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Embodiment 1

[0044]Embodiment 1 is aimed at the design of detection primer and probe of sheep FecB gene SNP site

[0045] A single nucleotide polymorphism A / G was carried out for the nucleotide of the 29382188bp site (NC_019463.1, based on the sheep genome sequence information version number Oar_v3.1, December 2012) on the No. 6 chromosome of sheep, and the designed The following primers and probe combinations were used to detect the polymorphism of sheep FecB gene by microfluidic SNP chip.

[0046] A total of 5 pairs of primers and probes were designed based on the polymorphic site sequence of the FecB gene, and fluorescent reporter groups of different colors were labeled on the 5' of the probes.

[0047] The nucleotide sequence of the primer 1 is as follows:

[0048] Forward primer: 5'-CCAGCTGGTTCCGAGAGACA-3'

[0049] Reverse primer: 5'-CTTATACTCACCCAAGATGTTTTCATG-3'

[0050] The nucleotide sequence of the probe 1 is as follows:

[0051] P-G: 5'-FAM-AAATATATCGGACGGTGTT-MGB-3'

[005...

Embodiment 2

[0078] Example 2 Method for detecting sheep FecB gene polymorphism using microfluidic SNP chip

[0079] 1. Experimental materials A total of 95 sheep produced by crossbreeding Small-tailed Han sheep as the female parent and other breeds as the male parent were selected as the test objects.

[0080] 2. Extraction of Genomic DNA 1ml of sheep blood was collected from the jugular vein and anticoagulated with EDTA. First, the red blood cell lysate is lysed to remove DNA-free red blood cells. The nucleus lysate lyses the cells to release genomic DNA. Then the protein precipitation solution selectively precipitates to remove the protein. The pure genomic DNA is precipitated by isopropanol and redissolved in the DNA solution.

[0081] 3. Microfluidic SNP chip for genotyping

[0082] The best detection primers and probes for the 29382188bp site on sheep chromosome 6 (NC_019463.1, based on the sheep genome sequence information version number Oar_v3.1, December 2012) screened in Example...

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Abstract

The invention provides a method for detecting sheep FecB gene polymorphism by using a micro-fluidic SNP chip. The method is capable of detecting loci A and G at high throughput and the result is accurate. Compared with other methods for high throughput detection of an FecB gene by using a Taqman MGB probe, the method provided by the invention has the advantages that samples capable of being detected every time are relatively flexible in quantity, the consumption of a reagent is low and the cost is significantly reduced. Nucleotide sequences of used primer and probe are as shown in SEQ ID NO.1-4, so that the specificity is high, the interference of other genes is avoided, the false positive risk caused by PCR product pollution is reduced and result interpretation is relatively intuitive. High throughput detection on the SNP loci of the FecB gene can be achieved, and the method has potential application value in large-scale molecular breeding of sheep.

Description

technical field [0001] The invention relates to a molecular marker detection technology, in particular to a method for detecting sheep FecB gene polymorphism with a microfluidic SNP chip. Background technique [0002] Livestock litter size is a quantitative trait with enormous economic value. The selection of lamb size is not only restricted by sex and age, but also the lamb size of sheep is a quantitative trait with low heritability, so it is difficult to improve the litter size trait with conventional breeding techniques. Marker-assisted selection can greatly improve the selection efficiency of such low-heritability traits by affecting the selection time, intensity and accuracy of selection, and finding molecular genetic markers linked to these quantitative trait loci is the key to realizing marker-assisted selection. Selected prerequisites. [0003] In the 1950s, the Seears brothers bred a group of Australian Merino (Booroola Merino, BM) that could give birth to multipl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12Q2563/107C12Q2565/629
Inventor 储明星刘秋月王姝杰狄冉魏丽任永红
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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