Method for multiplex amplification of pig microsatellite marker and special primer thereof

A technology of microsatellite labeling and compound amplification, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA preparation, etc., and can solve the problems of complicated operation, poor accuracy, and difficult to read the typed bands.

Inactive Publication Date: 2011-08-17
INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The typing method was first carried out by polyacrylamide gel electrophoresis combined with autoradiography or silver nitrate staining. This method is complicated to operate, and the typing bands are difficult to read, and the accuracy is poor; since the end of the last century, fluorescent markers Polyacrylamide gel electrophoresis and fluorescent-labeled capillary electrophoresis are gradually emerging and replacing traditional methods, which are easy to operate and have greatly improved accuracy

Method used

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  • Method for multiplex amplification of pig microsatellite marker and special primer thereof
  • Method for multiplex amplification of pig microsatellite marker and special primer thereof
  • Method for multiplex amplification of pig microsatellite marker and special primer thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Selection of microsatellite markers

[0028] Mark selection principle

[0029] Versatility: the microsatellite markers recommended by the American Pig Genome Collaboration Project for global pig genetic diversity research;

[0030] Wide distribution: 10 selected loci are distributed on 10 chromosomes;

[0031] Polymorphism: The number of alleles in a common pig herd is 5-22.

[0032] The selected microsatellite markers and fluorescent markers are shown in Table 1:

[0033] Table 1: Porcine microsatellite detection site information

[0034]

Embodiment 2

[0035] Example 2: Single-plex PCR amplification and genotyping of microsatellite marker sites

[0036] 1. DNA extraction and detection

[0037] Refer to the molecular cloning experiment guide and use conventional phenol extraction to extract DNA from Landrace pig ear tissue samples with genealogical records collected from Qingdao, Shandong. Dissolve the extracted DNA in TE and store at -20°C. Double check the purity and concentration of DNA with agarose gel electrophoresis and nucleic acid analyzer, and then dilute to 50ng / ul for use.

[0038] 2. PCR amplification

[0039] The total volume of the PCR reaction is 20μL, in which the template DNA is 100ng, contains 10×buffer (containing Mg2+, Takara), the final concentration of dNTP is 75μmol / L, the final concentration of primers is 0.3μmol / L, 2U Taq DNA polymerase (Takara), Insufficient part is made up with double distilled water.

[0040] Perform gradient PCR amplification of each pair of primers to find the appropriate annealing temp...

Embodiment 3

[0057] Example 3: Multiplex amplification and genotyping of microsatellite marker sites

[0058] 1. Experimental group

[0059] 1. Selection of microsatellite marker site combination

[0060] The 10-fold microsatellite loci compound amplification combination involved in the present invention was selected by the applicant through creative work, and the selection basis is as follows:

[0061] High polymorphism

[0062] No linkage between sites

[0063] The annealing temperature of each primer is similar

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Abstract

The invention discloses a method for multiplex amplification of a pig microsatellite marker and a special primer thereof. A primer composition provided by the invention consists of a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4, a primer pair 5, a primer pair 6, a primer pair 7, a primer pair 8, a primer pair 9 and a primer pair 10. Experiments prove that the data obtained by performing multiplex amplification and single PCR (Polymerase Chain Reaction) amplification by selecting a specific primer pair at a specific proportion are completely consistent, so that the accuracy and the feasibility of decuplet PCR provided by the invention are verified. The multiplex amplification method provided by the invention has great significance to paternity test, breeding and production of pigs.

Description

Technical field [0001] The invention relates to a method for compound amplification of pig microsatellite markers and its special primers. Background technique [0002] Microsatellite generally refers to a DNA tandem repeating sequence composed of short repeating units (generally 1 to 6 bases) in the genome. Microsatellites, also known as short tandem repeats (STRs) or simple sequence repeats, are simple repeats evenly distributed in the genome of eukaryotes, consisting of 2 to 6 Nucleotide is composed of tandem repeat fragments. Since the number of repeat units is highly variable and abundant among individuals, microsatellite markers are widely used. [0003] Microsatellite markers are currently widely used and have the following notable characteristics: 1) They are widely distributed in the genomes of higher animals. According to literature reports, there is a microsatellite marker every 10-50 kb; 2) Genetically stable and consistent Mendelian law of inheritance, high polymorph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 张颖崔文涛程文科黄宏刚裴黎凃政徐小玉
Owner INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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