Fluorescence detection kit for simultaneously analyzing 16 loca of cattle genome DNA

A detection kit and genome technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc., can solve the problems that cannot meet the requirements of use, large differences between different cattle groups, and the impact of DNA detection technology application efficiency, etc. problem, to achieve high sensitivity and specificity

Inactive Publication Date: 2019-01-04
江苏苏博生物医学科技南京有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the process of realizing the invention, the inventor found that the existing technology has at least the following problems: there are only 11 bovine loci in the StockMarks Animal Genotyping System recommended by the American ABI company, and

Method used

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  • Fluorescence detection kit for simultaneously analyzing 16 loca of cattle genome DNA
  • Fluorescence detection kit for simultaneously analyzing 16 loca of cattle genome DNA
  • Fluorescence detection kit for simultaneously analyzing 16 loca of cattle genome DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation of Fluorescence-labeled Multiplex Amplification Reagent for 16 Loci of Bovine Genomic DNA

[0062] 1. Arrangement of multiple amplification system sites

[0063] The bovine genomic DNA 16 locus fluorescent marker composite amplification detection kit of the present invention includes TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA126, TGLA122, INRA023, CSSM66, ETH3, ETH225, BM1824, BM1818, BAmel, G18833 and BM720 .

[0064] The arrangement of the above STR sites is as attached figure 1 shown.

[0065] 2. Special primers for multiplex amplification system

[0066] Design composite amplification primers according to the above sites, primer sequences, primer concentrations and fluorescein labeling methods are shown in the table below.

[0067]

[0068]

[0069] 3. Preparation of fluorescence multiplex amplification verification system

[0070] The DNA verification reagent used for large-scale sample screening is the PCR amplification system, a...

example 2

[0077] Compound amplification and fluorescence detection of example 2 multiplex amplification detection kit

[0078] 1. Sample DNA extraction

[0079] The samples were bovine muscle tissue samples from two unrelated individuals, and the genomic DNA of the two bovine muscle tissue samples was extracted by Chelex-100 method.

[0080] Take muscle tissue the size of a grain of rice, put it into a 0.5ml centrifuge tube, add 1mL sdH 2 O in the tube, shake, centrifuge at 13,000rpm for 3 minutes, discard the supernatant, keep enough supernatant to cover the precipitate, do not stir the precipitate; add 200 μL of Chelex-100 and 5 μL of 5 mg / mL PK enzyme, put into 56 ℃ for more than 3 hours. Take out and shake, incubate at 100°C for 8 minutes, shake, centrifuge at 13,000 rpm for 3 minutes, and the supernatant is the extracted genomic DNA.

[0081] 2. Sample amplification

[0082] Add 24 μL of the DNA testing reagent prepared in Example 1 to 1 μL of the 0.4-1.0 ng / μL DNA extraction s...

Embodiment 3

[0089] Example 3 Application of 16 gene locus fluorescent labeling composite amplification detection kits to carry out paternity identification of cattle

[0090] The samples were related father, mother and offspring, DNA extraction, PCR amplification, genetic analyzer detection were carried out according to Example 2, and finally the typing results were obtained.

[0091] Results and Discussion: The basic principle of paternity testing is: according to Mendel's law of inheritance, the genotype of the parent determines the genotype of the offspring. Under the premise of no gene mutation and typing error: 1) A pair of alleles of the child must come from the father and one from the mother; 2) The child cannot carry an allele that neither parent has.

[0092] The result of this embodiment sees attached Figure 4-6 and Table 1, in the attached Figure 4-6 The corresponding test results of the father cow, cow cow and calf are listed in order.

[0093] Table 1:

[0094]

[00...

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Abstract

The invention discloses a fluorescence mark composite amplification detection kit for simultaneously analyzing 16 loca of cattle. The fluorescence labeling composite amplification detection kit is characterized by comprising the 16 loca including TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA126, TGLA122, INRA023, CSSM66, ETH3, ETH225, BM1824, BM1818, BAmel, G18833 and BM720, the loca are divided into four groups, and fluorescence marks of five colors are involved totally. The fluorescence mark composite amplification detection kit provides an oligonucleotide primer mixture used for conducting composite amplification on the 16 loca; in a system, the included locus used for identifying the sex of a DNA sample is a BAmel locus. The detection result of the system shows that the fluorescence mark composite amplification detection kit is high in polymorphism, good in balance, high in sensitivity and specificity, accurate in typing result, high in species specificity and capable of completelymeeting the demands for cattle individual identification, paternity identification and DNA database construction.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a fluorescent label composite amplification detection kit, in particular to a five-color fluorescent label composite amplification detection reagent for simultaneously analyzing 15 STR loci and DAmel loci of bovine genomic DNA through composite amplification box. Background technique [0002] Short tandem repeat sequence (STR for short), also called microsatellite DNA, is a kind of nucleotide repeat sequence widely present in prokaryotic and eukaryotic genomes. It generally consists of tens to more than one hundred nucleotide repeating sequences consisting of 2-6 nucleotides as repeating units. Different numbers of core sequences are arranged in tandem repeats, and the length is polymorphic. According to the conserved sequences at both ends, design primers, perform PCR, and then detect polymorphisms of STR genetic markers by polyacrylamide, agarose gel electrophoresis, or capillary el...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 李翔葛斌文陈拓
Owner 江苏苏博生物医学科技南京有限公司
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