BMC Cancer 2008, 8:59 http://www.biomedcentral.com/1471-2407/8/59 ResoLight dye (Roche Diagnostics) with PCR grade water adjusted to a total volume of 10 µl. The reaction condition included an activation step at 95°C for 10 minutes fol- lowed by 55 cycles of 95°C for 10 seconds, a touch down of 65°C to 55°C for 10 seconds (1°C/cycle) and 72°C for 30 seconds. Before the high resolution melting step the products were heated to 95°C for 1 minute. The HRM was carried out over the range from 72°C to 95°C rising at 1°C per second with 30 acquisitions per degree. All reac- tions were performed in duplicate in a 96-well microtiter plate. HRM Analysis Upon completion of the run (approximately 2 hours), HRM curve analysis was performed using the LightCycler 480 Software version 1.3. supplied with the LightCycler 480. The melting curves were normalised and temperature shifted to allow samples to be directly compared. Differ- ence plots were generated by selecting a negative control, converting the melting profile to a horizontal line and normalising the melting profiles of the other samples against this sample Significant differences in fluorescence from the horizontal baseline were indicative of muta- tions. Differences were judged as significant if the repli- cates fell outside the range of variation seen in the wild type samples. Results We tested DNA from the peripheral blood of individuals heterozygous for known mutations in the three regions containing the Ashkenazi founder mutations predispos- ing to breast and ovarian cancer as well a series of wild type controls (Table 2). As in our previous publications on mutation detection [13,14], we found that difference curves, where the melting profile of one of the control samples is converted to a horizontal line and the melting profiles of the other samples are normalised against this line, allowed the best visualisation of mutations. For BRCA1 exon 2 (Figure 1a), we tested DNA from 4 indi- viduals with the Ashkenazi mutation 185delAG as well as 1 individual with the mutation 188del11 and 2 individu- als with the mutation 185insA. The mutations were all readily differentiated from the wild type. They were also readily differentiated from each other giving characteristic Table 2: List of mutations tested melting curves as is readily seen from the figure. It can also readily be seen that biological replicates had near identi- cal melting patterns. Similarly for the amplicon including BRCA1 exon 20 (Fig- ure 1b) and for the amplicon including the region around the 6174delT mutation in BRCA2 exon 11 (Figure 1c), all the mutations were readily identified, displayed character- istic melting curves and were clearly differentiated from the wild type and from each other. The mutations tested in addition to the 5382insC (6 individuals) were the 5396+1G>A (4 individuals) and the 5331G>A (5 individ- uals). The mutations tested in addition to the 6174delT (3 individuals) were the 6024delTA (2 individuals) and the 6293C>G (2 individuals). Biological replicates again had near identical melting patterns. Discussion DNA sequencing is regarded as the gold standard for BRCA1 and BRCA2 mutation detection. However, in prac- tice, most testing laboratories perform a preliminary scan of exons and splice junctions using methods such as the protein truncation test (PTT), denaturing gradient gel elec- trophoresis (DGGE) and/or denaturing high pressure liq- uid chromatography (DHPLC) to reduce cost and increase throughput. Any region that is found to potentially con- tain a mutation by scanning is then characterised by sequencing. Scanning methods are particularly relevant to studies involving large numbers of individuals. A recent Cana- dian study [15] used a combination of PTT, DGGE and DHPLC to scan the BRCA1 and BRCA2 genes, followed by DNA sequencing to screen for BRCA1 and BRCA2 muta- tions. Although very sensitive, PTT, DGGE and DHPLC do not detect all mutations. In particular, PTT, which is used for screening of the large exons comprising approximately half of the total sequence of the BRCA1 and BRCA2 genes, does not detect missense alterations. Both DHPLC and DGGE are both operator and interpreter sensitive. It has been shown that DHPLC, which is currently considered as the gold standard of screening methods, is superior to DGGE for the detection of BRCA1 and BRCA2 mutations [16]. BRCA1 exon 2 Mutation Number of samples BRCA1 exon 20 Mutation Number of samples BRCA2 exon 11 Mutation Number of samples Wild type 188del11 185delAG 185insA 13 1 4 2 Wild type 5396+1G>A 5382insC 5331G>A 16 4 6 5 Wild type 6174delT 6293C>G 6024delTA 26 3 2 2 Page 3 of 7 (page number not for citation purposes)