Monitoring the cDNA synthesis of dengue-2 virus by RT PCR.
Reverse transcriptase polymerase chain reaction (RT PCR) was utilized to observe the complementary DNA (cDNA) synthesis from the 10723 base dengue-2 virus template by in vitro reverse transcription. The PCR product amplified from 5'-end of the genome (PCR primers N1A-E1) indicates the completeness of the cDNA synthesis because the cDNA primer D8B was located at 3'-end and the cDNA synthesized encompassed the entire RNA template. The integrity of the dengue virus RNA was also determined by the cDNA primer extension from 3'-end and the PCR amplification at various regions from 5'-end of the RNA template. In conclusion, RT PCR could be applied to monitor cDNA synthesis by in vitro reverse transcription and measure the integrity of viral RNA template. Moreover, it is demonstrated that at least 10.7 kilobases of the cDNA could be synthesized from dengue-2 virus RNA by in vitro reverse transcription.
Liu
H S
HS
Department of Microbiology, National Cheng Kung University, Tainan, Taiwan, R.O.C.
Tzeng
H C
HC
Chen
C C
CC
D000330:Aedes / Q000821:virology; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics*; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics*; D003716:Dengue Virus / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic; D014773:Virology / Q000379:methods
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral