Figures 31-32 show examples of genotyping by using primer oligo base extension and MALDI/TOF/MS (41). Several known mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis, and four of them involve a short segment of the gene (Figure 31). These mutations can be identified and distinguished from the wild-type allele by using PCR primers complementary to the region of the gene adjacent to the mutation site and then performing PCR with mixtures of dXTP that permit chain extension and ddYTP that causes chain termination.
In the example in Figure 31a, using the primer and ddTTP as the chain terminator results in production of an oligonucleotide of m/z 8841.8 from the wild-type allele and one of m/z 7889.8 from the DF508 mutant allele. In contrast, Figure 31b illustrates that when ddCTP is used as chain terminator, the wild-type allele produces an oligonucleotide of m/z 11,604.5, and the DF508 mutant allele produces a fragment of m/z 10,652.0. Figure 32 illustrates MALDI/TOF/MS analyses of products of these primer oligo base extension assays for the genotypes homozygous wild-type (WT/WT, Figure 32a-b), homozygous DF508 mutant (DF508/DF508 mutant, Figure 32e-f), and heterozygous WT/DF508 (Figure 32c-d). Note that these analyses were performed before delayed extraction and reflectrons were developed for MALDI/TOF/MS, and the resolution and mass accuracy of the spectra are rather poor, although still adequate to achieve the objective of genotyping.