Method for extracting total DNA from milk

A technology for milk and milk samples, applied in the field of molecular biology of dairy cows, can solve the problems of low purity and concentration of DNA in milk samples

Inactive Publication Date: 2009-01-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As far as the current technology is concerned, the purity and concentration of the obtained milk sample DNA are relatively low, so it is more necessary to improve the method to obtain a better milk sample DNA sample

Method used

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  • Method for extracting total DNA from milk
  • Method for extracting total DNA from milk
  • Method for extracting total DNA from milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Total DNA extraction, including the extraction of total DNA from milk and the extraction of total DNA from cow blood;

[0045] (1) Extraction of milk sample total DNA, the steps are as follows:

[0046] 1) Preparation before sampling: Scrub the teats of cows with iodine, dry the iodine on the teats with a clean warm cloth, discard the first 2-3 handfuls of milk squeezed out, and then use a milking machine or artificial milking to obtain milk samples;

[0047] 2) Sampling: Add 1ml of somatic cell fixative and 0.5ml of 20% potassium dichromate in the centrifuge tube in advance as preservatives, put the collected milk sample into the centrifuge tube to 50ml, centrifuge at 2500 rpm, 4°C for 30 minute;

[0048] 3) Discard the upper milk fat and the middle emulsion of the centrifuge tube in step 2), keep the bottom sediment, add 1ml sterilized phosphate buffer to the bottom, beat the bottom sediment to suspend and transfer to a 1.5ml centrifuge tube, at 3500 rpm / min, ce...

Embodiment 2

[0080] This example has randomly taken the fresh milk sample of 5 dairy cows from the Wuhu Pasture of Wuhan Huierkang Yangzijiang Dairy Co., Ltd., Wuhan City, Hubei Province, extracted the total DNA in the milk sample according to the method of the present invention, and got 4 μ L of milk sample DNA and diluted 50 After doubling, the OD was measured on a Beckman-DU 800 UV spectrophotometer 260 / 280 value and DNA concentration. The concentration calculation formula used is: A260*dilution factor*50ug / ul. Get the data shown in Table 1:

[0081] Table 1 Milk sample DNA purity and concentration determination results

[0082] milk sample number

[0083] As can be seen from Table 1, DNA purity OD 260 / 280 If the value is within the range of 1.8-2.0, the DNA purity meets the requirements. Concentrations vary prominently between individuals due to differences in somatic cells contained in milk samples from different individuals.

Embodiment 3

[0085] PCR detection of DNA extraction and purification effect:

[0086] In this example, total DNA extracted from milk and total DNA extracted from blood were used as PCR templates to amplify exon 9 of the growth hormone receptor (GHR) gene, and the amplification effects were compared and detected.

[0087] 1. Primer sequence

[0088] Forward primer: 5'-TAGGAGTTCCTTTTTAGAGGATAGGTGC-3'

[0089] Reverse primer: 5'-GCCTTGTGGAGAAGTTGACAAA-3'

[0090] References for primers used Blott S, Kim J J, Moisio S, et al. Molecular Dissection of a QuantitativeTrait Locus: A Phenylalanine-to-Tyrosine Substitution in the Transmembrane Domain of the BovineGrowth Hormone Receptor Is Associated With a Major Effect on Milk Yield and Composition . J. Genetics, 2003, 163: 253-266.

[0091] 2. PCR reaction system

[0092]Add to the PCR reaction system with a total volume of 10 μl:

[0093] wxya 2 O 7.2 μl

[0094] 10*PCR buffer 1.0μl

[0095] 10mmol / L dNTPs 0.3μl

[0096] 2.5U Taq enzyme 0...

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Abstract

The invention belongs to the technology researching field of cow molecular, in particular to the technical field of preparing the total DNA of the cow. The invention discloses a method for extracting the total DNA from the cow. Because the cow belongs to an economic animal, in order to avoid the stress caused by blood sampling and collecting the tissue of a living cow as well as overcome the problem of containing higher protein pollution of the extracted total DNA of a milk sample, the invention proposes a more convenient source to obtain the DNA, researches out a key technology from purifying the somatocyte of the milk sample to extracting the DNA with high quality, prepares the reagent for purifying the somatocyte of the milk sample and a cracking mixture for extracting the DNA by test, then uses alcohol with different concentration grads to wash, then uses a phenol method to extract and remove the protein and other matters like amylase, finally uses absolute ethyl alcohol to wash and dissolves into TE after being dried. The invention is simple and convenient to sample; the quality of the sample of the total DNA that is extracted is higher; the method is economic, stable and convenient; the invention provides a novel method for the application of the cow molecular.

Description

technical field [0001] The invention relates to the technical field of dairy cow molecular biology. It specifically relates to a method for purifying somatic cells and extracting total DNA from milk samples. Background technique [0002] Obtaining genomic DNA is the basis for subsequent molecular biology research. Finding a suitable DNA extraction method is the first problem faced by most molecular biology experiments. Currently, obtaining cow DNA using a stress-free method is a challenge for researchers. Significant issues, especially in terms of animal welfare, are mainly due to the stress response caused by taking blood samples, which reduces the performance of dairy cows. At present, the sources of genomic DNA samples obtained from individual dairy cows mainly include: blood, muscle tissue, organ tissue, semen, etc. These sample sources use conventional extraction methods, and the extraction effect is better. At present, the use of milk as a source of high-quality DNA ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 张军伟易建明
Owner HUAZHONG AGRI UNIV
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