BMC Cancer 2008, 8:59 http://www.biomedcentral.com/1471-2407/8/59 Background BRCA1 and BRCA2 are the two most frequently mutated genes underlying inherited breast and ovarian cancer. As both are large multi-exon genes, with inactivating muta- tions occurring across the entire coding region, the conse- quent cost of BRCA1/2 mutation screening has limited mutation testing to those who are most likely to have a mutation based on their family history of cancer. Conse- quently, a significant proportion of women carrying germline mutations are missing out on the opportunity to have mutation screening and thereby to modify their risk of breast cancer, particularly those with a small pedigree structure or where the inheritance is through the paternal lineage. The costs of full sequencing are reducing, but it will be some years before they drop significantly enough to have a serious impact on the numbers of genetic tests provided by scarce health dollars. In the meantime, an alternative approach is to use a mutation scanning tech- nique to highlight variations in genomic sequences which are then characterised by sequencing. High resolution melting (HRM) is a new methodology for mutation scanning in which the mutation scanning is car- ried out in the same tube or well in which the sequence is amplified (reviewed in [1]). The PCR reactions and HRM can all be performed in a single run of less than 2 hours resulting in extremely rapid screening. Up to 384 scans (generally 192 samples in duplicate) can be done at the same time enabling very high throughput. We selected several regions of the BRCA1 and BRCA2 genes known to carry a number of founder mutations in order to investigate the ability of HRM for reliable detec- tion of both known founder mutations and other muta- tions occurring in close proximity to those founder mutations. Founder mutations are specific mutations found at high frequency in a particular population as a result of geographic, cultural or ethnic isolation. Individ- uals of Ashkenazi Jewish ancestry have a particularly high carrier rate for three mutations predisposing to the hered- itary breast and ovarian cancer syndrome: the 185delAG and the 5382insC in BRCA1 and the 6174delT in BRCA2 with a population prevalence of approximately 2.5% [2- 4]. The 5382insC in exon 20 of BRCA1 is also one of the most common recurrent mutations in Central and Eastern Europeans [5-9]. This exon also contains the 5331G>A BRCA1 mutation recently shown to be a founder muta- tion in Greek Europeans [10]. Methods Samples Samples from 29 carriers of known mutations in one of the regions of the BRCA1 and BRCA2 genes that contain the Ashkenazi founder mutations were obtained from existing samples in the Diagnostic Molecular Pathology laboratory at the Peter MacCallum Cancer Centre. Up to five biological replicate samples (i.e separate individuals) were chosen for each mutation. The study was performed under guidelines approved by the Peter MacCallum Ethics of Human Research Committee (approval 03/90). HRM Assay Conditions Effective primer design is an important component of HRM analysis. Primers were designed to flank the coding regions of BRCA1 exons 2 and 20 and to amplify a 534 bp region surrounding the 6174delT mutation on BRCA2 exon 11. All amplicons were chosen so that they con- tained a single melting domain using the Poland program [11]. It was also necessary to ensure that the primers did not overlay known single nucleotide polymorphisms (SNPs) that may lead to single alleles not being amplified. An example of this was previously reported for the 6174delT mutation where the wild type allele was not amplified[12]. In other cases, this would lead to lack of amplification of the mutant allele. The primers were designed to be annealed at 60°C using Primer Express software (Applied Biosystems, Foster City, CA) to calcu- late the "Tm". Primer sequences are listed in Table 1. PCR and HRM Assay Conditions The PCR and HRM were performed in a single run on a LightCycler 480 (Roche Diagnostics, Penzberg, Germany) in a reaction mix containing 2.5 µl (25 ng) of genomic DNA, either 400 nM (exon 2 and exon 11) or 200 nM (exon 20) of each primer and 3 mM MgCl2 in the LightCy- cler® 480 High Resolution Melting Master containing Table 1: List of primers used to amplify BRCA1 exon 2, BRCA1 exon 20 and part of BRCA2 exon11. Gene BRCA1 exon 2 BRCA1 exon 20 BRCA2 exon 11 Forward primer Reverse primer Forward primer Reverse primer Forward primer Reverse primer Sequence 5'-AAAAGATATAGATGTATGTTTTGCTAATGTGT-3' 5'-TCCCAAATTAATACACTCTTGTGCTGA-3' 5'-GAGTGGTGGGGTGAGATTTTTGTC-3' 5'-CCTGATGGGTTGTGTTTGGTTTCT-3' 5'-CGAAAATTATGGCAGGTTGTTACG-3' 5'-GCTTTCCACTTGCTGTACTAAATCCA-3' Page 2 of 7 (page number not for citation purposes)