Quantification of single nucleotide polymorphisms by automated DNA sequencing

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Abstract

Single nucleotide polymorphisms (SNPs) are linked to phenotypes associated with diseases and drug responses. Many techniques are now available to identify and quantify such SNPs in DNA or RNA pools, although the information on the latter is limited. The majority of these methodologies require prior knowledge of target sequences, normally obtained through DNA sequencing. Direct quantitation of SNPs from DNA sequencing raw data will save time and money for large amount sample analysis. A high throughput DNA sequencing assay, in combination with a SNP quantitative algorithm, was developed for the quantitation of a SNP present in HCV RNA sequences. For a side-by-side comparison, a Pyrosequencing assay was also developed. Quantitation performance was evaluated for both methods. The direct DNA sequencing quantitation method was shown to be more linear, accurate, sensitive, and reproducible than the Pyrosequencing method for the quantitation of the SNP present in HCV RNA molecules.

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.

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