Mathematical Biology seminar

 Carl Wittwer
Pathology Dept., University of Utah
" Real Time PCR and DNA Melting Analysis"
April 6
3:05pm in LCB 121
Real-time polymerase chain reaction (PCR) monitors DNA during amplification. Separation of the two strands of DNA after PCR with heat (melting) is a convenient analytical tool for genetics, infectious disease, and cancer research. The accuracy and usefulness of both real time PCR and melting analysis depend on appropriate mathematical analysis. Conventional melting is performed after PCR on any real-time instrument to monitor product purity (dsDNA dyes) and sequence (hybridization probes). Recent advances include high-resolution instruments and saturating DNA dyes that distinguish many different species. For example, highly polymorphic HLA regions can be melted to establish sequence identity for transplantation matching. SNPs can be genotyped without probes and more complex regions can be typed with unlabeled hybridization probes. Mutation scanning (identifying heterozygotes) by melting is closed-tube and has similar or superior sensitivity and specificity compared to methods that require physical separation. Mutation scanning and genotyping with one or more unlabeled probes can be performed at the same time in the same tube, vastly decreasing or eliminating the need for resequencing in genetic analysis. High-resolution DNA melting is homogeneous, closed-tube, rapid (1-2 min), non-destructive and does not require covalently-labeled fluorescent probes.

References

1. Gundry CN, JG Vandersteen, GH Reed, RJ Pryor, J Chen, and CT Wittwer. Amplicon melting analysis with labeled primers: A closed-tube method for differentiating homozygotes and heterozygotes. Clin. Chem. 49:396-406, 2003.

2. Wittwer CT, GH Reed, CN Gundry, JG Vandersteen, and RJ Pryor. High-Resolution Genotyping by Amplicon Melting Analysis using LC Green, Clin. Chem., 49:853-60, 2003.

3. Willmore BS, Holden JA, Zhou L, Tripp S, Wittwer CT, Layfield LJ. Dectection of c-kit activating mutations in gastrointestinal stromal tumors by high-resolution amplicon melting analysis. Am. J. Clin. Path. 122:206-16, 2004.

4. Zhou L Vandersteen J, Wang L, Fuller T, Taylor M, Palais B, Wittwer CT. High-resolution DNA melting curve analysis to establish HLA genotypic identity. Tissue Antigens, 64:156-164, 2004.

5. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer CT. Genotyping of single nucleotide polymorphisms by high-resolution melting of small amplicons. Clin. Chem. 50:1156-64, 2004.

6. Zhou L, Myers AN, Vandersteen JG, Wang L, Wittwer CT. Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA Dye. Clin Chem. 50:1328-35, 2004

7. Reed GH, Wittwer CT. Sensitivity and specificity of SNP scanning by high-resolution melting analysis. Clin. Chem., 50:1748-54, 2004.

8. Wittwer CT and N Kusukawa. Real-Time PCR, in Molecular Microbiology: Diagnostic Principles and Practice. DH Persing, FC Tenover, J Versalovic, YW Tang, ER Unger, DA Relman, TJ White, eds., ASM Press, Washington, DC, 71-84, 2004.

9. Wittwer CT, N Kusukawa. Nucleic Acid Techniques, in Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed, Burtis C, Ashwood ER, and Bruns DE, eds, Elsevier Science, Philadelphia, in press, 2005.