Strategy and result from high-throughput genotyping on allele-specific primer extension arrays. (A) The design of an array is illustrated schematically. Eighty separate reaction chambers have been formed on each microscope glass slide, using a 384-well formatted silicon rubber grid. Each of these reaction chambers contain an identical array of 72 primers for detection of 36 mutations or SNPs. The vertical columns (A–D) contain pairs of allele-specific primers for each site ordered into nine horizontal rows (1–9). The acronyms corresponding to the diseases and the mutant or variant nucleotides analyzed on the array are given in the lower part of A. INCL = infantile neuronal ceroid lipofuscinosis (Vesa et al. 1995); AGU = aspartylglucosaminuria (Ikonen et al. 1991); FV = Factor VLeiden (Bertina et al. 1994); CCD = congenital chloride diarrhea (Höglund et al. 1996); APECED = autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (the Finnish-German APECED Consortium 1997); HTI = hereditary tyrosinemia type I (St-Louis et al. 1994); HFI = hereditary fructose intolerance (Cross et al. 1990); OAT1 & 2 = gyrate atrophy of the choroid and retina (Mitchell et al.1989); Batten = Batten disease (the International Batten Disease Consortium 1995); vLINCL = variant late infantile neuronal ceroid lipofuscinosis (Savukoski et al. 1998); NKH = nonketotic hyperglycinemia (Kure et al. 1992); CF Δ508/ΔTT394 = cystic fibrosis (Kere et al. 1994); A1AT = α1-antitrypsin deficiency Z-mutation (Cox et al. 1988); HH = hereditary hemochromatosis C282Y (Feder et al. 1996); DFNB=nonsyndromic deafness, connexin 26 35insG (Denoyelle et al. 1997); ODG = hypergonadotropic ovarian dysgenesis (Aittomaki et al. 1995); DTD = diastrophic dysplasia (Hästbacka et al. submitted); CNFmaj/min = congenital nephrosis (Kestilä et al. 1998); PKU R408W = phenylketonuria (Guldberg et al. 1995); MCAD = medium-chain acyl-CoA dehydrogenase deficiency (Matsubara et al. 1990); LCHAD = long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (Ijist et al. 1996); PT = prothrombin G20210A (Poort et al. 1996); MPS = mucopolysaccharidosis type I (Bunge et al. 1994); RS1/2 = X-linked juvenile retinoschisis (Huopaniemi et al. 1999); LPI = lysinuric protein intolerance (Torrents et al. 1999); Salla = Salla disease (Verheijen et al. 1999); SNP1 = WIAF-11062, SNP2 = WIAF-11091, SNP6 = WIAF-10964 (Cargill et al. 1999); SNP7 = HLA-H IVS2 (Beutler et al. 1996); SNP8 = HLA-H 5569 (Jeffrey et al. 1999) ; EPMR = progressive epilepsy with mental retardation (Ranta et al. 1999). (B) A fluorescence image of the array described in A obtained when 40 DNA samples were genotyped in duplicate (>2400 genotypes) by allele-specific primer extension. The genotype assignments for a larger number of samples based on numeric ratios from arrays are shown in Figures 3 and 4. (C) Enlargements of 16 individual arrays (a–p in B). The genotyping results from samples of mutation carriers and patients are indicated by the disease acronym and genotype below the array images. Both allele-specific primers give similar signal intensities in heterozygous samples, while only one of the primers gives a signal for homozygotes.
