Multiplex s-RT-MELT or OpenArray TM -based s-RT-MELT increases the throughput of mutation scanning A significant potential advantage of enzymatic mutation scanning is the ability to screen several sequences simultaneously for mutations. To demonstrate that s-RT-MELT can be used for parallel scanning of mutations in several PCR products, we mixed equimolar amounts of PCR products from p53 exons 5-9 containing mutations either in exon 8 or in exon 9. We then formed 'cross-hybridized sequences' and screened the mixture for mutations in p53 exons 5-9 in a single tube using s-RT-MELT, as depicted in Figure 1A . Following real-time PCR and melting curve analysis, the exon 8 or exon 9 mutants were clearly distinguished from the wild-type sample ( Figure 4A, curves 1-3) . Next, the mutant exon 8 DNA sample was first diluted 10-fold into wild-type exon 8 and the equimolar mixture of p53 exons 5-9 was prepared and screened again in a single tube via s-RT-MELT. The exon 8 mutation was again distinguished from the wild-type mixture of exons ( Figure 4B , curves 1-3). Since 480% of p53 mutations in human tumors are encountered in exons 5-9 (45), the multiplex single-tube s-RT-MELT reaction could be used to identify most p53 mutations encountered in clinical tumor samples. Combined with multiplex PCR directly from genomic DNA, this approach could result to a convenient, high-throughput method for mutation scanning.