The
PALB2 genomic sequence was obtained from the National Center for Biotechnology Information (reference sequence number NG_007406.1). Primers (Geneworks, Hindmarsh, South Australia, Australia) (Additional file
1) were designed by using Primer3 software (Whitehead Institute and Howard Hughes Medical Institute, Cambridge, MA, USA). For optimal performance of the HRM curve analysis, primers were designed to amplify DNA products between 100 and 310 bp. A total of 35 fragments were designed to cover the coding and flanking intronic regions of
PALB2. The primer sequences and annealing temperatures are listed in Additional file
2. Initially, 96 DNA were Sanger-sequenced as described in Tischkowitz
et al. [
22]. These data and the corresponding DNA were then used to establish the optimal conditions for HRM curve analysis. DNA extracted from peripheral blood samples provided by 1,473 case probands (695 from women diagnosed with breast cancer under the age of 40 years participating in the ABCFR and 778 from kConFab) were screened for germline
PALB2 mutations using HRM curve analysis [
23,
24]. DNA was then systematically screened using this established method. HRM reactions were carried out in 15-μL volumes and included 1.5 μL of 10 × polymerase chain reaction (PCR) buffer (Applied Biosystems, Victoria, Australia), a 3 mM final concentration of MgCl
2 (Applied Biosystems), a 100 μM final concentration of deoxyribonucleotide triphosphate (dNTP) (Bioline, Alexandria, New South Wales, Australia), a 200 nM final concentration of each primer (Geneworks) (Additonal File
2), a 2.3 μM final concentration of Syto9 (Invitrogen, Victoria, Australia), 0.25 U of AmpliTaq Gold (Applied Biosystems) and 3 μL of Q solution (Qiagen, Victoria, Australia). Each reaction underwent a hold of 10 minutes at 95°C and 40 cycles of amplification of 30 s at 95°C and 1 minute at annealing temperature followed by melting to dissociate double-stranded DNA. The temperature range for melting was set at ± 10°C of the melting temperature of each amplicon with a rise in temperature of 0.05°C/s. HRM analysis was performed using Rotor-Gene 6000 Series Software 1.7 (Qiagen). Fragments displaying aberrant melt curves were sequenced to determine potential underlying genetic variations. For sequencing reactions, we utilized larger amplicons than those generated during HRM curve analysis (Additional file
1). Sequencing was carried out in 10-μL reactions, which included 1 μL of 10 × PCR buffer (Applied Biosystems), a 3 mM final concentration of MgCl
2 (Applied Biosystems), a 100 μM final concentration of dNTP (Bioline), a 200 nM concentration of each primer (Geneworks), 0.25 U of AmpliTaq Gold (Applied Biosystems) and 3 μL of Q solution (Qiagen). PCR products were purified and analyzed on a 3130xl Genetic Analyser (Applied Biosystems) and the results were viewed using Chromas 1.45 (Technelusium, Tewantin, Queensland, Australia).