SNP loci group for genetic quality monitoring of CBA/CaJ inbred mice, and primer combinations and application of SNP loci group

A technology of quality monitoring and primer combination, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of inability to determine the SNP detection site in a targeted manner, the specificity of the detection result and the detection signal cannot be guaranteed. Specific sites can not provide alternative primers and other problems, to achieve the effect of large throughput, clear typing, and sensitive detection

Active Publication Date: 2019-04-12
GEMPHARMATECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chinese patent ZL2018104752119, a SNP rapid detection method for monitoring the genetic quality of inbred lines and SNP sites and primers, the patent provides a SNP rapid detection method, but this method cannot specifically determine a certain strain (such as CBA / CaJ mouse) SNP detection site; in addition, there can be many different primers for the same site, even if the site is determined, it cannot guarantee that the subsequent primers can be used normally, let alone the specificity of the detection results and the detection signal Ok, so the subsequent reaction conditions for getting the primers also need to be further optimized
In addition, no alternative primers are available for specific loci for specific strains

Method used

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  • SNP loci group for genetic quality monitoring of CBA/CaJ inbred mice, and primer combinations and application of SNP loci group
  • SNP loci group for genetic quality monitoring of CBA/CaJ inbred mice, and primer combinations and application of SNP loci group
  • SNP loci group for genetic quality monitoring of CBA/CaJ inbred mice, and primer combinations and application of SNP loci group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: SNP site screening and specific primer design

[0023] 1. Determine the inbred mouse strains that need to be identified by the combination of SNP loci

[0024] Select CBA / CaJ from the CBA inbred mouse strain as a representative, and integrate the locus information in the published mouse SNP databases such as NCBI, Sanger, BroadInstitute, etc., and screen and determine 8 inbred strains to participate in the comparison. SNP detection combinations for CBA / CaJ inbred strains were established on a genome-wide scale (Table 1).

[0025] Table 1 SNP detection panel inbreeding series list

[0026] serial number

Strain name

1

DBA / 1JNju

2

C57BL / 6JNju

3

A / JNju

4

BALB / cJNju

5

FVB / NJNju

6

CBA / CAJ / 1JNju

7

NOD / ShiLtJNju

8

129S1 / SvImJNju

[0027] 2. Design the combination of specific loci for distinguishing CBA / CaJ inbred lines from other lines

[0028]a) Design purpose: for s...

Embodiment 2

[0050] Embodiment 2: The relevant primer design of the SNP locus of genetic quality monitoring of CBA / CaJ strain

[0051] After the above SNP site combination design is completed, primer design is carried out at about 100 bp upstream and downstream of each site sequence in the mouse genome: 20-30 bp primers are designed upstream of the SNP site, and the 3' ends of a pair of primers are respectively SNP Two different mutated bases; a sequence of about 20 bp is added to the 5' end of the primer to generate different recognition signals, such as FAM and HEX signals; the 3' downstream primer is designed with a length of about 18-28 bp.

[0052] Primers were designed for the SNP sites, and the Tm (°C) of the primers was controlled between 55-65°C, and the GC% was between 34%-60%. For each SNP site, design 3 primers as a group, and design multiple sets of primer combinations for specific sites for primer testing and result verification. There are 288 designed primers. The primer seq...

Embodiment 3

[0065] Embodiment 3: SNP combination typing test of CBA / CaJ mice

[0066] High-throughput genotyping and data analysis, use IntelliQube fluorescence detection for reading, use IntelliScore for data analysis after PCR, and automatically export genotype for analysis. Using 129S1 / SvImJ mice as a control strain, 96 loci of CBA / CaJ were tested for genotyping, as shown in Table 8 below; the results show that CBA / CaJ is the same as the locus registered on NCBI among the 96 loci The information is all consistent, and there is a significant difference between the characteristic loci and the control strain (129S1 / SvImJ mouse 129S1 / SvImJ mouse). This result verifies that the SNP primer combination used for CBA / CaJ background detection in the present invention can be used in this Genetic quality monitoring of lines.

[0067] Table 8 129S1 / SvImJ as the control CBA / CaJ primer test results

[0068]

[0069]

[0070]

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Abstract

The invention belongs to the field of genetic background identification and genetic pollution detection of inbred mouse strains, and relates to an SNP loci group for genetic quality monitoring of CBA / CaJ inbred mice, and primer combinations and application of the SNP loci group. The SNP loci include 10 specific recognition loci for a CBA strain, 70 recognition loci for strains other than CBA, and16 universal loci. The results of 96 SNP loci can accurately reflect the degree of coincidence of backgrounds of subject mouse individuals with specific inbred mouse strains, comprehensively detect the genetic traits and variation of the genome of the subject mouse individual, and realize the monitoring of mouse genetic quality status and quality of experimental stability. Reaction conditions areoptimized for the SNP primers in the subsequent detection to ensure that the detection is sensitive, specific and efficient. In addition, multiple sets of primers are designed for 10 specific loci tofacilitate selection according to different detection requirements and specific detection conditions.

Description

technical field [0001] The invention belongs to the field of genetic background identification and genetic pollution detection of inbred mouse strains, and particularly relates to a group of SNP sites and primer combinations for monitoring the genetic quality of CBA / CaJ inbred mice. Background technique [0002] There are mainly three types of methods for the quality control detection of the genetic background of experimental animals: biochemical marker analysis, microsatellite DNA, and SNP (single nucleotide polymorphism) detection. The current international genetic detection method is biochemical marker analysis, which is to detect changes in isoenzymes or isomeric proteases to infer corresponding gene changes; using this method for detection has low accuracy, low sensitivity, and detection There are methodological defects such as limited loci and limited genetic profiles. Molecular genetic markers can conduct more detailed genetic analysis of experimental animals, and ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 赵静琚存祥马秀英陶然张冬冬杨旭乐李永兵裴喆侯欢欢高翔
Owner GEMPHARMATECH CO LTD
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