APC Genotyping in Human Subjects

We applied SOMA to genotyping several variant sites in the human adenomatous polyposis coli (APC) gene. The first of these is a variant (I1307K) present in 6% of the Ashkenazi Jewish population that is associated with an inaeased risk of colorectal cancer of approximately two fold [22]. The polymorphism is an A to T change at codon 1307 and hence represents the most difficult alteration to analyse because there is only a 9Da mass difference between the two variants. PCR primers were designed to yield 15-mer oligonucleotides for MS analysis following 

Samples with wild type sequence only had a signal in the channels monitoring masses representative of both wild type sense (ATA-s) and wild type antisense (ATA-as) alleles. A sample for an individual who is heterozygous at this locus has signals in the sense and antisense channels for both the wild type (ATA) and mutant (AAA) alleles. In a blind analysis of 16 individuals by SOMA and sequencing there was 1(X)% concordance. 

The current test for the I1307K polymorphism uses allele-specific oligonucleotide hybridisation in which labelled probes are used to discriminate between wild type and mutant sequences. Such differential hybridisation requires considerable optimisation and is rarely perfect, resulting in a background signal that 

As a preliminary examination of the potential for multiplex genotyping by SOMA, three common polymorphisms in the APC gene at codons 485, 545 and 1756 were analysed in par lel [10]. The IKIR amplification and digest reactions were performed individually to yield fragments of 8, 9 and 11 bases for codons 485, 545 and 1756, respectively. All the reaction products for one individual were then 

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