Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P–) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.
A 336 bp fragment of F-gene mRNA was reverse-transcribed and amplified from Sendai virus RNA isolated from persistently infected Vero cells. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit and the indicated number of viral genome copies. M: markers. (Data kindly provided by H. Rausch, Max Planck Institute for Biochemistry, Martinsried, Germany as part of the project "Experimental control of virological work at safety levels 2 and 3 in Bavaria," supported by the Bavarian Ministry of the Environment.)
A 439 bp fragment of transcription factor TAFII100 mRNA (GC content: 75%) was reverse-transcribed and amplified from HeLa-cell total RNA. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit with (+) or without (–) Q-Solution. Q-Solution ensures specific amplification of GC-rich templates. M: markers.
One-step RT-PCR was carried out using the indicated amounts of total RNA from HeLa cells and primers specific for α-catenin, amplifying a 690 bp product. All reactions were carried out following suppliers' instructions. M: 100 bp ladder.
One-step RT-PCR was performed using kits from the indicated suppliers over a range of annealing temperatures. A 1289 bp fragment from the human RCC1 gene was reverse transcribed and amplified from Hela RNA (arrow). M: markers. High levels of specific amplification without optimization were observed only with the QIAGEN OneStep RT-PCR Kit.
The QIAGEN OneStep RT-PCR Kit allows fast and easy RT-PCR setup. Whatever the application — virus detection, molecular diagnostics research, or gene expression analysis— just mix all components together in one tube and start the thermal-cycler program. The reaction mixture contains all of the reagents required for both reverse transcription and PCR; nothing needs to be added once the reaction has been started.