Differential amplifying RT-PCR: a novel RT-PCR method to differentiate mRNA from its DNA lacking intron.
A major problem with reverse transcription-polymerase chain reaction (RT-PCR) is that the products amplified from RNA and DNA are not distinguishable. The use of a primer set targeted for an intron-containing sequence or RNA extract without contaminating DNA has been established to overcome this problem. However, an intron sequence does not always exist in the target region and complete removal of DNA during RNA extraction is not practical. For these reasons, we developed differential amplifying RT-PCR (DART-PCR) based on differences in reaction temperatures between RT and PCR, as well as in architectures between RNA and DNA. DART-PCR was designed to differentiate products amplified from RNA and DNA regardless of the presence of introns. DART-PCR can be readily applied for diagnosis of active infection of human cytomegalovirus by detection of glycoprotein B mRNA, which gene lacks introns. Other advantages of DART-PCR were as follows: there is no need for separation of RNA from DNA, simultaneous/differential detection in a single tube and possibility of determination of relative amounts of RNA and DNA.
Joo
Chul Hyun
CH
Department of Microbiology, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Republic of Korea.
Lee
Heuiran
H
Kim
Eunok
E
Lee
Boyoung
B
Cho
Young Keol
YK
Kim
Yoo Kyum
YK
D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D006801:Humans; D007438:Introns; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013698:Templates, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics*
D017931:DNA Primers; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus