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báo cáo hóa học:" Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR"

Tuyển tập các báo cáo nghiên cứu về hóa học được đăng trên tạp chí sinh học đề tài : Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR | Virology Journal BioMed Central Methodology Open Access Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR Steven B Kleiboeker Address Veterinary Medical Diagnostic Laboratory and Department of Veterinary Pathobiology College of Veterinary Medicine University of Missouri Columbia Missouri 65211 USA Email Steven B Kleiboeker - kleiboekers@missouri.edu Corresponding author Published II April 2005 Received 02 March 2005 Accepted 1 1 April 2005 Virology Journal 2005 2 29 doi 10.1186 1 743-422X-2-29 This article is available from http www.virologyj.cOm content 2 1 29 2005 Kleiboeker licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons.org licenses by 2.0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. Abstract Although PCR and RT-PCR provided a valuable approach for detection of pathogens the high level of sensitivity of these assays also makes them prone to false positive results. In addition to crosscontamination with true positive samples false positive results are also possible due to carry-over contamination of samples with amplicon DNA generated by previous reactions. To reduce this source of false positives amplicon generated by reactions in which dUTP was substituted for dTTP can be degraded by uracil DNA glycosylase UNG . UNG does not degrade RNA but will cleave contaminating uracil-containing DNA while leaving thymine-containing DNA intact. The availability of heat-labile UNG makes use of this approach feasible for RT-PCR. In this study real-time RT-PCR was used to quantify UNG degradation of amplicon DNA and the effect of UNG on RNA detection. Using the manufacturers recommended conditions complete degradation of DNA was not observed for samples containing 250 copies of amplicon DNA. Doubling the .