The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to the embryonic genetic program. Bruce Malcolm of Antiviral Therapeutics, Schering-Plough Research Institute for providing the cDNA plasmids and their assistance with the initial development of the Pyrosequencing assay. This technique proved effective in establishing the quantitation Acknowledgements
Through the use of engineered RNA molecules, different proportions of a wild-type and mutant SNP were prepared to mimic the various levels of heterozygosity that could be present in RNA pools. However, quantitation of SNPs from RNA pools using this technology is limited. Pyrosequencing has been shown to accurately quantitate SNPs from pooled DNA and is being accepted as a high throughput way of quantitating SNP in patient samples. 1C) and automated Sanger DNA sequencing (data not Discussion RT-PCR amplified the expected HCV RNA controls from concentrations of 1 × 10 6 down to 100 copies/reaction to form 214 bp target sequences as confirmed by both electrophoresis (Fig. Confirmation of DNA removal after DNase I treatments was demonstrated by PCR as no amplified DNA was present (Fig. The wild-type and mutant SNP control RNA standards generated by in vitro transcription were shown to contain a single band on an RNA gel (Fig. The HCV replicon plasmids containing WT or mutant SNP allele were amplified by PCR using a forward primer containing a 5 ′ T7 promoter overhang and a reverse primer, spanning the region containing the SNP site, to yield DNA fragments of 328 bp.
These plasmids were used to generate wild-type (WT) and mutant SNP control RNA molecules. HCV replicon cDNA plasmids used in a Pyrosequencing SNP analysis were provided by Antiviral Therapeutics, Schering-Plough Research Institute (Kenilworth, NJ). Section snippets Preparation of control RNA molecules