Real-time polymerase chain reaction (PCR) monitors DNA during
amplification. Separation of the two strands of DNA after PCR with
(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
Conventional melting is performed after PCR on any real-time
monitor product purity (dsDNA dyes) and sequence (hybridization
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
transplantation matching. SNPs can be genotyped without probes and
complex regions can be typed with unlabeled hybridization probes.
Mutation scanning (identifying heterozygotes) by melting is
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
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
require covalently-labeled fluorescent probes.
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.
2. Wittwer CT, GH Reed, CN Gundry, JG Vandersteen, and RJ Pryor.
High-Resolution Genotyping by Amplicon Melting Analysis using LC
Clin. Chem., 49:853-60, 2003.
3. Willmore BS, Holden JA, Zhou L, Tripp S, Wittwer CT, Layfield
Dectection of c-kit activating mutations in gastrointestinal stromal
tumors by high-resolution amplicon melting analysis.
Am. J. Clin. Path.
4. Zhou L Vandersteen J, Wang L, Fuller T, Taylor M, Palais B,
Wittwer CT. High-resolution DNA melting curve analysis to establish
genotypic identity. Tissue Antigens, 64:156-164, 2004.
5. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer
Genotyping of single nucleotide polymorphisms by high-resolution
of small amplicons. Clin. Chem. 50:1156-64, 2004.
6. Zhou L, Myers AN, Vandersteen JG, Wang L, Wittwer CT.
genotyping with unlabeled oligonucleotide probes and a saturating DNA
Clin Chem. 50:1328-35, 2004
7. Reed GH, Wittwer CT. Sensitivity and specificity of SNP
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
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,
C, Ashwood ER, and Bruns DE, eds, Elsevier Science, Philadelphia, in