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Micro-satellite marker and specific primer for identifying female and male individuals of pseudobagrus ussuriensis and application

A technology of microsatellite marking and microsatellite, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as wrong judgment of gender, and achieve easy distinction, fast detection speed, and high resolution high effect

Active Publication Date: 2017-06-09
HUAIYIN TEACHERS COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still deficiencies in this method, because the marker is to identify the sex by judging whether an individual has an amplified band at the corresponding site. If a point mutation occurs in the flanking sequence of the primer, the individual will There is no amplified band in the middle, that is, there is a null allele (Null Allele), then using this marker to judge the sex of such individuals may be wrong

Method used

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  • Micro-satellite marker and specific primer for identifying female and male individuals of pseudobagrus ussuriensis and application
  • Micro-satellite marker and specific primer for identifying female and male individuals of pseudobagrus ussuriensis and application
  • Micro-satellite marker and specific primer for identifying female and male individuals of pseudobagrus ussuriensis and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Screening of microsatellite sites

[0034] The specific microsatellite marker between female and male of Ussuri puma in the present invention is obtained through two rounds of strict screening. The technicians constructed the microsatellite enrichment library of Ussuri breamus by the magnetic bead enrichment method, and obtained the sequence containing microsatellite repeats by cloning and sequencing. From the sequences containing microsatellites, select the sequences whose core repeats more than 5 times and meet the requirements of primer design, and use Primer Premier5.0 to design primers. The main parameters are set as follows: primer length 20-25 bp, PCR product fragment length range 120-350 bp, optimum annealing temperature 50-60°C. The GC content is generally between 40% and 60%, and the secondary structure should be avoided as much as possible. Finally, 61 microsatellite markers of P. ussurii that can be expanded stably were successfully obtained.

...

Embodiment 2

[0037] Example 2 Composition and preparation of PCR system for identification of female and male-specific microsatellite markers in Ussuri pufferfish

[0038] A). PCR system composition:

[0039] dNTPs (10 mM) are products of Beijing Kangwei Century Company; Taq DNA polymerase (5 U / µL), 10×PCR Buffer are products of Dalian TaKaRa Company;

[0040] B). Composition of 10×PCR Buffer:

[0041] Tris-HCl (pH8.3) 100 mM, KCl 500 mM, MgCl 2 15mM;

[0042] C). PCR reaction system preparation:

[0043] Total volume 13 µL, containing 30-50 ng template DNA, 0.5 U Taq DNA polymerase, 1.3 µL 10×PCRBuffer, 0.5 µL dNTP (2.5 mM), 0.5 µL forward and reverse primer mix (2.5 µM each), 9.6 µL Sterilized ultrapure water;

[0044] D). Polyacrylamide gel preparation:

[0045] 30% (w / w) acrylamide 9 mL, 5×TBE 5 mL, 10% (w / w) ammonium persulfate 0.5 mL, TEMED 10 µL, distilled water 12 mL.

Embodiment 3

[0046] Example 3 Identification of female and male individuals judged morphologically by specific microsatellite markers between females and males of Ussuri pufferfish

[0047] A). Take 80 individuals to be identified as female and male according to their morphological characteristics, and use the phenol-chloroform method to extract the genomic DNA from the caudal fin tissue of each individual, and dilute the DNA to a concentration of 30-50 ng / µL.

[0048] B). Using the genomic DNA mentioned in step A) as a template, prepare a PCR reaction mixture according to the system in step C) of Example 2.

[0049] C). Amplify on a PCR machine, pre-denaturation at 95°C for 5 minutes; (denaturation at 94°C for 40 seconds, annealing at 54°C for 40 seconds, extension at 72°C for 40 seconds) for 36 cycles; final at 72°C Extend for 7 minutes and store the PCR product at 4°C.

[0050] D). The PCR products were separated by polyacrylamide gel electrophoresis prepared according to the system in...

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Abstract

The invention belongs to the technical field of colony identification in aquaculture, and discloses a micro-satellite marker and specific primer for identifying female and male individuals of pseudobagrus ussuriensis, and application. One sex-specific micro-satellite site of the pseudobagrus ussuriensis is provided, and has a nucleotide sequence shown by any one of SEQ ID NO:1-2. The invention also provides a primer designed from the micro-satellite site, and the primer has a nucleotide sequence shown by SEQ ID NO:3-4. One micro-satellite site is screened out of a micro-satellite enriched library of the pseudobagrus ussuriensis, and a specific primer is designed according to flanking sequences at two ends of a micro-satellite, so that an amplification result has sex specificity, the female and male individuals of the pseudobagrus ussuriensis can be effectively identified, and the specific primer can also be used for performing real-time monitoring on sex structures of colonies in natural water areas and breeding places. The invention also discloses a kit for identifying the female and male individuals of the pseudobagrus ussuriensis. The identification method provided by the invention has the advantages of high accuracy, high resolution ratio and high speed.

Description

technical field [0001] The invention belongs to the technical field of group identification in aquaculture, in particular to a microsatellite marker and a specific primer for identifying the sex of the Ussuri catfish and its application in the identification of female and male individuals of the Ussuri catfish. Background technique [0002] Microsatellites, also known as Simple Sequence Repeats (SSR), refer to simple tandem repeat DNA sequences consisting of 1-6 nucleotides. It has been found in all biological species studied so far, and its distribution density is very large. Because microsatellites are widely distributed in the genome, they have the characteristics of high density, rich polymorphism, high heterozygosity and good stability, co-dominant inheritance following Mendelian segregation law, and easy PCR amplification. It is an eye-catching new type of DNA marker, and is widely used in many research fields such as family genealogy authentication of biological re...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6879C12Q2600/156C12Q2600/172
Inventor 朱传坤潘正军王辉常国亮丁怀宇聂孝燕余祥胜吴楠
Owner HUAIYIN TEACHERS COLLEGE
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