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Method for extracting grape genome DNA from wine

A wine and genome technology, applied in the biological field, can solve the problems of lack of commercial kits, inapplicability, molecular biological analysis that affects the quality of DNA extraction, and achieve the effect of high extraction and purification efficiency, good integrity and good purity

Inactive Publication Date: 2015-03-25
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of these DNA extraction methods are suitable for grape DNA extraction in wine
[0003] Wine is a highly processed product, and its production process has gone through many links such as fermentation, bottle transfer, aging, clarification and filtration. The DNA content in the finished wine is very small, and a large amount of polyphenols (tannins), Complex chemical substances such as polysaccharides and organic acids seriously affect the quality of DNA extraction and downstream molecular biological analysis. There is no report of a wine DNA extraction method that can reach the application level, and there is no mature commercial kit.

Method used

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  • Method for extracting grape genome DNA from wine
  • Method for extracting grape genome DNA from wine
  • Method for extracting grape genome DNA from wine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Extraction and purification of DNA in commercially available red wine

[0043] 1. Extraction and purification of DNA from commercially available red wine

[0044] 1), DNA enrichment

[0045] Take 30mL of wine into a 50mL centrifuge tube, add 220μg of glycogen, 0.5% β-mercaptoethanol (V / V) and 0.7 times the volume of isopropanol in sequence, mix thoroughly, and place in a -75℃ refrigerator for 1h. Then move to -18°C for precipitation for about 12h to precipitate the DNA in the wine, and then centrifuge at 7,000g for 40min at 4°C to collect the precipitate.

[0046] 2) Removal of polysaccharides

[0047] Add 0.8 mL of pre-cooled polysaccharide-removing buffer [80mmol / L Tris-HCl (pH8.0), 6mmol / L EDTA-Na 2 , 0.25mmol / L NaCl, 1.5% (w / v) PVP-40T], after all the precipitates on the centrifuge tube wall were washed down by blowing and blowing with a pipette gun, they were transferred to a 2mL centrifuge tube as much as possible, and placed in an ice bath for 5min. ...

Embodiment 2

[0062] Example 2 Extraction and purification of DNA in commercially available white wine

[0063] 1. Extraction and purification of DNA from commercially available white wine

[0064] 1), DNA enrichment

[0065] Take 20mL of wine into a 50mL centrifuge tube, add 180μg of glycogen, 1.2% β-mercaptoethanol (V / V) and 1 times the volume of isopropanol in turn, mix thoroughly, and place in a -65℃ refrigerator for 1.5h , and then moved to -20°C overnight (about 24h) to precipitate the DNA in the wine, and then centrifuged at 6,000g for 30min at 4°C to collect the precipitate.

[0066] 2) Removal of polysaccharides

[0067] Add 1 mL of pre-cooled polysaccharide-removing buffer [120mmol / L Tris-HCl (pH8.0), 4mmol / L EDTA-Na 2 , 0.2mmol / L NaCl, 2.5% (w / v) PVP-40T)], after blowing and blowing with a pipette gun to wash all the precipitates on the centrifuge tube wall, transfer it to a 2mL centrifuge tube as much as possible, and put it in an ice bath for 8min. Mix by inverting once dur...

Embodiment 3

[0082] Embodiment 3 (the extraction result of embodiment 1, 2 is detected)

[0083] 3.1, DNA concentration detection

[0084] Use NANODROP1000 to measure the concentration of DNA extracted from wine, and the results are shown in Table 1 below:

[0085] Table 1 DNA extraction results

[0086]

[0087] 3.2, VvNCED2 gene amplification analysis

[0088] The primers and probes of the VvNCED gene were selected for the establishment of the fluorescent quantitative PCR system, and the primers and probes were synthesized by TaKaRa Biotechnology Co., Ltd. The amplified product of the pair of primers is 94bp.

[0089] Its sequence is:

[0090] F: 5'-ATGGCGACGGTATGGTTCA-3',

[0091] R: 5'-CGCTCCTGGACCAATCTCTG-3',

[0092] Probe: Fam-AGTCACCCCTTAATGGCGGTTC-Tamra.

[0093] The VvNCED2 gene amplification analysis was performed according to the PCR reaction system and conditions shown in Table 1 below.

[0094] Table 1 Fluorescent quantitative PCR reaction system (20μL)

[0095] ...

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Abstract

The invention provides a method for extracting a grape genome DNA from wine. The method comprises the following steps: 1) DNA enrichment; 2) removal of polysaccharides; 3) crude extraction of DNA; 4) DNA purification; and 5) DNA recovery, thus obtaining the grape genome DNA. The DNA solution extracted by adopting the method is clear, the extraction and purification efficiency is high, and the obtained DNA has high concentration, good purity and good integrity.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for extracting grape genome DNA from wine. Background technique [0002] The molecular identification of wine includes two parts: the extraction of grape DNA in wine and downstream molecular biological analysis, in which high-quality grape DNA obtained from wine is the basis for downstream molecular biological analysis. Plant genomic DNA extraction methods are relatively mature. Based on traditional methods such as CTAB method and SDS method, DNA extraction methods suitable for different types of plant materials have been widely used, and even developed into commercialized plant DNA extraction kits. However, none of these DNA extraction methods are suitable for grape DNA extraction in wine. [0003] Wine is a highly processed product, and its production process has gone through many links such as fermentation, bottle transfer, aging, clarification and filtration. The DNA co...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 刘津高苏娟刘青卢丽张隽陈源树李婷顾瑜娟何日荣高东微
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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