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SNP locus set used for genetic quality monitoring for BALB/cJ inbred strain mouse and primer combination and application of SNP locus set

A quality monitoring and primer combination technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc. Unable to provide alternative primers, etc., to achieve the effect of simple operation and cost reduction

Active Publication Date: 2019-10-22
GEMPHARMATECH CO LTD
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  • Summary
  • 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 BALB / cJ 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 locus set used for genetic quality monitoring for BALB/cJ inbred strain mouse and primer combination and application of SNP locus set
  • SNP locus set used for genetic quality monitoring for BALB/cJ inbred strain mouse and primer combination and application of SNP locus set
  • SNP locus set used for genetic quality monitoring for BALB/cJ inbred strain mouse and primer combination and application of SNP locus set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: SNP panel design

[0031] 1. Determine the inbred strains included in the SNP panel

[0032] Seven inbred lines were determined to establish the SNP detection panel of BALB / cJ inbred lines (Table 1). In follow-up experiments, the BALB / cJ strain SNP panel can be used for routine SNP detection to distinguish from 129S1 / SvImJ, A / J, C57BL / 6J, CBA / CaJ, DBA / 1J, FVB / NJ, NOD / LtJ strains, At the same time, it can be used for routine genetic quality monitoring of related mutant lines.

[0033] Table 1 SNP detection panel inbreeding series list

[0034] serial number Strain name 1 129S1 / SvImJ 2 A / J 3 C57BL / 6J 4 CBA / CaJ 5 DBA / 1J 6 FVB / NJ 7 NOD / LtJ

[0035] 2. Design the combination of specific loci for distinguishing BALB / cJ inbred lines from other lines

[0036] a) Design purpose: for screening the SNP loci of BALB / cJ strains that are different from the aforementioned other strains (129S1 / SvImJ, A / J, C57BL / 6J...

Embodiment 2

[0058] Example 2: BALB / cJ SNP site primer design

[0059] After the SNP site screening is completed, the 100bp sequences of the upstream and downstream sequences of the SNP site are pulled from the mouse genome sequence using programming tools; 5' end primer design: design 20-30bp primers at the upstream of the SNP site, and The ends of the primers are two mutated bases of the SNP, and a 20bp sequence that recognizes different signals, such as FAM and HEX signals, is added to the 5' end of the primer; the downstream primer is designed at the 3' end, and the length is about 18-28bp.

[0060] Tm (°C) is between 55-65°C, GC% is between 34%-60%, a total of 288 primers are designed, the primer sequences are shown in Table 6, and the three primers are simultaneously amplified by PCR. A signal indicates that the sample template contains the base mutation type.

[0061] Table 6 Site primer information

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] ...

Embodiment 3

[0069] Example 3: SNP combination typing test of BALB / cJ mice

[0070] 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, the primers for 96 loci were used to test the genotype of BALB / cJ, see Table 7 below for details; the results showed that BALB / cJ was 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 mice). This result verifies that the SNP primer combination used for BALB / cJ background detection in the present invention can be used for genetic quality monitoring of this strain .

[0071] Table 7 129S1 / SvImJ as the control BALB / cJ primer test results

[0072]

[0073]

[0074]

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Abstract

The invention belongs to the field of genetic background identification and genetic contamination detection for inbred strain mouse strains, and relates to a SNP locus set used for genetic quality monitoring for a BALB / cJ inbred strain mouse and a primer combination and application of the SNP locus set. SNP loci comprise 11 specificity identification loci for a BALB / C strain, 73 feature loci for identifying some strain except the BALB / c strain and 12 universal loci, by synthesizing results of the 96 SNP loci, the consistence degree of inbred strain backgrounds of a tested mouse individual anda specific mouse can be precisely reflected, genetic characters and variation conditions of a genome of the tested mouse individual are comprehensively detected, and the genetic quality state of the tested mouse and the stability quality of an experiment are monitored. According to the SNP locus set and the primer combination and application thereof, reaction conditions are optimized for SNP primers in later detection, and sensitive, specific and efficient detection is ensured.

Description

technical field [0001] The invention belongs to the field of genetic background identification and genetic pollution detection of inbred mouse strains, and in particular relates to a set of SNP sites and primer combinations for monitoring the genetic quality of BALB / cJ 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 are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156C12Q2600/16
Inventor 琚存祥赵静杨旭乐马秀英张明坤侯欢欢高翔
Owner GEMPHARMATECH CO LTD
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