Visible Genetics Inc.

Robert Shipman

James M Dunn

James Leushner

Jean-Michel Lacroix

Marina T Larson

May Hui

Samples are tested for mutations in the BRCA1 gene using a hierarchical approach. First, each sample is amplified in one or more multiplex PCR amplification reactions. Each multiplex PCR reaction produces a mixture of amplified fragments. The sizes and amounts of these fragments are evaluated and compared to standard values reflecting the sizes and amounts of fragments produced when the same multiplex amplification is performed on the wild-type BRCA1 gene. Differences between the observed fragment sizes and/or amounts and those for the wild-type gene are indicative of a mutation with the BRCA1 gene of the sample. Next, one or more of the exons of the BRCA1 gene are sequenced, preferably only for those samples where no mutation was detected by analysis of the multiplex PCR fragments. The sequencing procedure can be performed by amplification and sequencing of the multiplex amplification mixture.

Method and reagents for testing for mutations in the BRCA1 gene

A method is provided for simultaneously determining the positions of a selected nucleotide base in a target region of both strands of a denatured duplex nucleic acid polymer. The nucleic acid polymer is combined with a reactant mixture comprising first and second oligonucleotide primers, said primers binding to the sense and antisense strands, respectively, of the nucleic acid polymer at a location flanking the target region; a thermostable DNA polymerase; a chain-terminating nucleotide triphosphate complementary to the selected nucleotide base; and other reagents for synthesis of chain extension products to form a reaction mixture. This mixture is processed through a plurality of thermal cycles, each including at least a chain extension phase and a denaturation phase to produce chain extension products. These chain extension products are evaluated to determine the positions of the selected bases. The method of the invention differs from the prior art, because the first and second oligonucleotide primers are each labeled with different, spectroscopically-distinguishable fluorescent labels. The method therefore obtains information about both DNA strands simultaneously while providing improved sensitivity as a result of the non-linear increase in the amount of DNA which results from the production of additional templates molecules from unterminated fragments.

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Mississauga, Canada

Robert Shipman