Detection of Human DNA Mutations using Base Excision Sequence Scanning (BESS) and the LI-COR IR² Automated DNA Sequencer

Human BRCA1 Exon 11 Analysis

Procedure

Human DNA samples form individuals with BRCA1 mutations were obtained from the Coriell Medical Institute (Camden, NJ). The PCR primers were selected for a 271 bp region of BRCA1 exon 11, IRD700-GTGAGGATGAAGAGCTTCCC) and the reverse primer (5'-GCAGTCAAGTCTTCCAATTCA-3'). The PCR and BESS reaction was carried out as described above. A separate PCR was conducted with dTTP and unlabeled primers. The product was sequenced in ddT reactions using Thermo Sequenase^®. Both the Bess and Sequencing samples were analyzed on a 6% gel on a LI-COR DNA Sequencer.

Results

Figure 3 shows the sequencing reactions produced lighter signal intensities. In addion the sequencing samples ran 1 bp slower than the BESS T-scan™ reactions, as is expected based on the strand excision mechanism for Exonuclease VII cleavage. The banding patterns of the ddT and the BESS reactions are identical. A G to T transversion at codon 1250 (lanes 2 and 7) is easily detected as well as a 4 base deletion in codon 1252 that appears as a shift in the banding pattern. Comparison of banding patterns in lane 6 (heterozygous) and lane 8 (homozygous) demonstrate the ability of the method to detect mutations in heterozygotes.