Biochemical and Biophysical Research Communications
Quantification of single nucleotide polymorphisms by automated DNA sequencing
Section snippets
Preparation of control RNA molecules
HCV replicon cDNA plasmids used in a Pyrosequencing SNP analysis [18] were provided by Antiviral Therapeutics, Schering-Plough Research Institute (Kenilworth, NJ). These plasmids were used to generate wild-type (WT) and mutant SNP control RNA molecules. 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.
Preparation of HCV control RNA standards
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. 1A). Confirmation of DNA removal after DNase I treatments was demonstrated by PCR as no amplified DNA was present (Fig. 1B). RT-PCR amplified the expected HCV RNA controls from concentrations of 1 × 106 down to 100 copies/reaction to form 214 bp target sequences as confirmed by both electrophoresis (Fig. 1C) and automated Sanger DNA sequencing (data not
Discussion
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. However, quantitation of SNPs from RNA pools using this technology is limited. 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. This technique proved effective in establishing the quantitation
Acknowledgements
We thank Jackie Wright-Minogue, Dr. Fred Lahser, and Dr. 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.
References (21)
- et al.
Single nucleotide polymorphism analysis by MALDI-TOF mass spectrometry
Trends Biotechnol.
(2000) - et al.
Making drug discovery a SN(I)P
Drug Discov.
(2000) - Huntington’s Disease Collaborative Research Group, A novel gene containing a trinucleotide repeat that is expanded and...
- et al.
Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA
Science
(1989) - et al.
Apolipoprotein E: high-avidity binding to β-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease
Proc. Natl. Acad. Sci. USA
(1993) - et al.
Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by Pyrosequencing
J. Clin. Microbiol.
(2001) - et al.
The future of genetic studies of complex human diseases
Science
(1996) Prospects for whole-genome linkage disequilibrium mapping of common disease genes
Nat. Genet.
(1999)Pharmacogenetics
Hum. Mol. Genet.
(2001)- et al.
Construction of a genetic linkage map in man using restriction fragment length polymorphisms
Am. J. Hum. Genet.
(1980)
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