The intrinsic potential of enzymatic mutation scanning for parallel identification of mutations can, in principle, be very high since the enzyme operates on numerous distinct mismatch-containing sequences on a molecule-tomolecule basis thus providing highly parallel mutation scanning. However, in the past the selectivity of the enzymes used and the endpoint detection method has limited the realization of this potential. Here we enabled Surveyor TM , an endonuclease that recognizes selectively mismatches formed by mutations and small deletions following 'cross-hybridized sequence' formation, to generate mutation-specific DNA fragments that are amplified and screened via differential melting curve analysis. The replacement of size-separation methods (capillary/gel electrophoresis, dHPLC) by real-time PCR technology as the endpoint detection platforms and the ability to scan more than one sequences in parallel result in a highly increased throughput for s-RT-MELT while retaining the ability to detect diverse mutations at low-levels. Cel I/II endonucleases have also been known to have exonuclease activity on 5 0 DNA-ends (26, 27) . For this reason, s-RT-MELT was designed to attach an oligonucleotide linker to the 3 0 -DNA ends via terminal transferase (TdT) instead of using the 5 0 -DNA ends. The exonuclease activity also tends to degrade the attached 5 0 -GC-clamps in s-RT-MELT, thereby eliminating their influence in reducing amplification of un-digested fragments. We found that if exposure of DNA 'cross-hybridized sequences' to Surveyor TM is limited to 15-20 min, the substantial degradation of 5 0 -GC-clamps is avoided. For multiplexing mutation detection using several PCR products simultaneously, the size of the GC-clamp on each PCR amplicon may need to be individually adjusted to ensure that mutations along all sequence positions of the PCR products included in the mixture can be screened at a single real-time PCR temperature and that undigested fragments do not amplify. The calculational tools developed in this work can be used to guide the individual design of GC-clamps. s-RT-MELT detects heterozygous SNPs as well as mutations. As with other mutation prescreening techniques, the presence of a SNP concurrently with a mutation might be difficult to identify without performing sequencing. Because SNPs occur at fixed positions, melting peaks originating from SNPs have a reproducible pattern and melting temperatures (46, 47) thus in many cases they should be distinguishable from mutations. Finally, it is noteworthy that s-RT-MELT is a general methodology that may also be applied to isolate mutations using mismatch-cutting enzymes other than Surveyor TM when enzymes with satisfactory properties for mutation detection become available. Detection platforms other than real-time PCR/melting (e.g. DNA microarraybased) may also be envisioned following enzymatic mutation selection.