TC genotypes identified by solid-phase minisequencing.

(A) TCII-259C homozygotes (PP); (B) TCII-259C/G heterozygotes (PR); (C) TCII-259G homozygotes (RR). Genomic DNA was amplified by PCR using the sense primer biotin-5′-GTGCGAGAGGAGATCTTGAA-3′ and the antisense primer 5′-GTAGGTCTTGTGGTTCAGAA-3′. Biotinylated products were bound to streptavidine-coated microtiter plates (Wallac, Turku, Finland) and denatured with NaOH. Thermo Sequenase DNA polymerase (Thermo Sequenase Dye Terminator Kit, Amersham, Buckinghamshire, United Kingdom), fluorescent ddNTP, and the antisense detection primer 5′-CTGTTCCCAGTTCTGCCCCA-3′ were added to generate an allele-specific pattern. After the minisequence reaction, the plates were washed and the extended sequence primers were released by incubation with formamide. These were separated and analyzed by capillary electrophoresis and laser-induced fluorescence in an ABI 310 genetic analyzer (Perkin-Elmer, Cambridge, England).