Quantification of single nucleotide polymorphisms by automated DNA sequencing.

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.

[1]  P. Boutin,et al.  Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing. , 2002, BioTechniques.

[2]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[3]  N. Spurr,et al.  Making drug discovery a SN(i)P , 2000 .

[4]  Luquan Wang,et al.  Hepatitis C virus whole genome position weight matrix and robust primer design , 2002, BMC Microbiology.

[5]  T. Griffin,et al.  Single-nucleotide polymorphism analysis by MALDI-TOF mass spectrometry. , 2000, Trends in biotechnology.

[6]  M. Permutt,et al.  Assessing allele frequencies of single nucleotide polymorphisms in DNA pools by pyrosequencing technology. , 2002, BioTechniques.

[7]  D. Whitcombe,et al.  A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping. , 1998, Clinical chemistry.

[8]  Joakim Lundeberg,et al.  Monitoring Resistance to Human Immunodeficiency Virus Type 1 Protease Inhibitors by Pyrosequencing , 2001, Journal of Clinical Microbiology.

[9]  D. Nickerson,et al.  Comparative analysis of human DNA variations by fluorescence-based sequencing of PCR products. , 1994, Genomics.

[10]  L. Tsui,et al.  Erratum: Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA , 1989, Science.

[11]  M. Pericak-Vance,et al.  Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[12]  SNPCEQer: detecting SNPs in sequences generated by the Beckman CEQ2000 DNA Analysis System. , 2002, BioTechniques.

[13]  Manish S. Shah,et al.  A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes , 1993, Cell.

[14]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[15]  N Risch,et al.  The Future of Genetic Studies of Complex Human Diseases , 1996, Science.

[16]  L. Kruglyak.  Prospects for whole-genome linkage disequilibrium mapping of common disease genes , 1999, Nature Genetics.

[17]  D. Botstein,et al.  Construction of a genetic linkage map in man using restriction fragment length polymorphisms. , 1980, American journal of human genetics.

[18]  D. Nickerson,et al.  PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. , 1997, Nucleic acids research.

[19]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.