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United States Department of Agriculture

Agricultural Research Service

Research Project: METABOLOMIC AND MICROBIAL PROFILING OF TROPICAL/SUBTROPICAL FRUITS AND SMALL FRUITS FOR QUALITY FACTORS AND MICROBIAL STABILITY

Location: Citrus and Other Subtropical Products Research

Title: Application of real-time PCR to postharvest physiology – DNA isolation

Author
item Bai, Jinhe

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 11, 2012
Publication Date: N/A

Technical Abstract: Real-time PCR technology has been widely used in the postharvest plant physiology research. One of the difficulties to isolate DNA from plant martial and pathogen cells is the presence of rigid polysaccharide cell walls and capsules, which physically protect DNA from cell lysis. Many materials require additional lysis steps, such as mechanical disruption or sonication, enzymatic digestion or use of toxic chemicals. Co-purification of pectin and DNA has long been recognized. Pectins become apparent when they co-precipitate with DNA following the addition of alcohol during the DNA extraction process. A number of DNA extraction methods have been developed to avoid the co-precipitation of pectins, such as a high concentration of NaCl, modified cetyl trimethyl ammonium bromide (CTAB), phenol, ethylene glycol monoethyl ether and pectinase methods. Another common challenge for extracting quality DNA is from the contamination of the secondary metabolites and humic acid which can inhibit PCR reaction. Appropriate ion exchange columns or chelating agents can be used to remove contaminates. There were some successful applications to remove PCR inhibitors from apple, grape and water melon juices using Chelex treatment and Sephadex column filtration. However, these methods did not work in orange juice.

Last Modified: 8/22/2014
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