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Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification

An extraction method, a large-scale technology, applied in the direction of recombinant DNA technology, DNA preparation, biochemical equipment and methods, etc., can solve the problems of large sampling volume, unsuitable for identification and molecular biological analysis, etc., to achieve convenient sampling and band The effect of uniformity and quality improvement

Inactive Publication Date: 2012-10-10
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sampling volume of these methods is relatively large, all above 50mL; and the extracted DNA can only be amplified by PCR of small gene sequences within 500bp, which is not suitable for large-scale genotype identification and molecular biological analysis

Method used

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  • Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification
  • Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification
  • Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Extraction and quality detection of DNA in milk

[0044] 1. Separation and enrichment of milk somatic cells

[0045] The above cryopreserved milk sample was thawed at 4°C, and centrifuged at 2500r / min, 4°C for 30min. Use a small spoon to scrape off the milk fat on the upper layer of the centrifuge tube, and use a straw to remove the milk protein in the middle layer, leaving the bottom layer of sediment. Add 600 μL of PBS to the bottom of the centrifuge tube, beat the bottom pellet to suspend and transfer it to a 1.5 mL centrifuge tube, centrifuge at 3500 r / min for 10 min at room temperature, discard the upper liquid, and keep the bottom pellet. Then add 60 μL of emulsifier and 540 μL of PBS to the bottom sediment, shake with an oscillator until the sediment is completely suspended, treat it in a constant temperature water bath at 40°C for 10 minutes to remove the milk fat around the somatic cells, centrifuge at 3500 r / min for 10 minutes at room temperatur...

Embodiment 2

[0054] Example 2: PCR amplification and genotype identification of bovine B2M gene B2M1F and B2M1R primers

[0055] 1. Selection of specific genes

[0056] The bovine B2 microglobulin gene B2M (beta-2-microglobulin) was randomly selected, and its gene sequence number in GenBank is NC_007308.

[0057] 2. Design and synthesis of primers

[0058] Primers were designed according to the bovine B2M gene sequence and synthesized by Shanghai Sangon Bioengineering Company:

[0059] Upstream primer (B2M1F): 5'CAT CTG TCT TTC CCT GCC GC 3'

[0060] Downstream primer (B2M1R): 5' CTA CAG CCT TCC TCA TCT CCC CT 3'.

[0061] 3. PCR reaction

[0062] (1) Carry out PCR amplification using bovine DNA as a template, and the 10 μL reaction system contains the following solutions or reagents:

[0063]

[0064] (2) Mix the above solutions, and after repeated exploration of the PCR annealing temperature, 65°C is finally determined as the annealing temperature. The PCR reaction conditions are...

Embodiment 3

[0071] Example 3: PCR amplification and genotype identification of bovine B2M gene B2M2F and B2M2R primers

[0072] 1. Design and synthesis of primers

[0073] Primers were designed according to the bovine B2M gene sequence and synthesized by Shanghai Sangon Bioengineering Co., Ltd., wherein:

[0074] Upstream primer (B2M2F): 5'GGC TTT CCC AGC ATC ACT AAC 3'

[0075] Downstream primer (B2M2R): 5' TCA CAG CAC CAC CAA ACT TAT CT 3'.

[0076] 2. PCR reaction

[0077] (1) Carry out PCR amplification using bovine DNA as a template, and the 10 μL reaction system is the same as in Example 2.

[0078] (2) Mix the solutions of the PCR reaction system. After repeated exploration of the PCR annealing temperature, 60°C is finally determined as the annealing temperature. The PCR reaction conditions are as follows:

[0079]95°C, pre-denaturation for 5min; 94°C, denaturation for 30sec, 60°C, annealing for 30sec, 72°C extension for 30sec, 30 cycles; 72°C extension for 10min.

[0080] (3)...

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Abstract

The invention discloses a method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification. Fresh milk is processed by improved technologies and methods such as a somatic cell enrichment technology, an SDS (Sodium Dodecyl Sulfonate) phenol splitting technology, a nucleic acid precipitation technology and the like, the acquisition quantity of the milk can be controlled between 10ml and 13ml, and the extracted genomic DNA has complete quality and better concentration and purity. At the same time, 500 bp even more than 1000 bp of long-segment gene sequences can be amplified in the extracted milk DNA, and large-scale genetic typing identification and subsequent molecular biology analysis can be carried out through the obtained sequence. The method has the characteristics of easiness in operation, low cost, high precision, convenient sampling, practicability and large scale, difficulties in blood and tissue sampling can be obviously avoided, stress response of cows can not be caused, and unnecessary loss brought to cow enterprises and farmers by other sampling methods is reduced. In addition, the method disclosed by the invention provides an advantageous theoretical foundation for controlling milk nutritional value, milk quality safety and milk production performance of the cows from a molecular level.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for extracting genomic DNA from milk suitable for large-scale genotype identification, which can extract genomes with complete quality, good concentration and purity from 10mL-13mL fresh milk DNA, using the DNA as a template for PCR amplification, can amplify a long-segment bovine genomic DNA sequence of more than 1000 bp, and then carry out genotype identification and molecular biological analysis. Background technique [0002] In recent years, molecular biology technology has become a favorable tool in dairy product quality inspection and molecular nutrition research. Obtaining high-quality genomic DNA is the basis for subsequent molecular biology research such as food nutritional function identification, food traceability, genetic variation in dairy cow populations, molecular marker-assisted selection breeding, parentage identification, and DNA Southern hy...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 刘永峰杨永芳李景景
Owner SHAANXI NORMAL UNIV
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