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Improvement of RT-PCR Sensitivity for Fruit Tree Viruses by Small-scale dsRNA Extraction and Sodium Sulfite
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  • Improvement of RT-PCR Sensitivity for Fruit Tree Viruses by Small-scale dsRNA Extraction and Sodium Sulfite
저자명
Sin Ho Lee, Hyun Ran Kim, Jae Hyun Kim, Jeong Soo Kim
간행물명
The Plant Pathology Journal KCI
권/호정보
2004년|20권 2호(통권90호)|pp.142-146 (5 pages)
발행정보
한국식물병리학회|한국
파일정보
정기간행물|ENG|
PDF텍스트(0.41MB)
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영문초록

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiplestep protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications significantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

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