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Alpha -antitrypsinCo AT)deficiency ia an autoaoaal recessive diaeaee with a frequency (Pi ZZ and Pi SZ) of More than l/looo in northern Europe. By amplifying the DMA segaent (1), that contains the Z Mutation, we have developed a sensitive, reliable and rapid Method for detection of the a AT Z allele. The two primers used sere both 2o-»er oligonucleotides, I: 5' aaATAQACATQQQTATQaCC, II: 5" QQCCTaaaATCAaCCTTACA. They were constructed to anneal to target sequences, 135 bases apart in genoMic DNA, Baking it possible to aaplify the intervening segaent including the site of the Z Mutation, which is a transposition froM Q to A in ezon V. DNA froM individuals with a AT types MM, HZ and ZZ were prepared froM peripheral blood leucocytes. 1 ug genoMic DNA in buffer (67 ••ol/l Tris-HCl (pH 8.8), 6.7 ••ol/l MgCl , 16.6 ••ol/l ( NH ) SO , 10 ••ol/l BHE, 6.7 unol/1 EDTA, 10X DMSO) was Mixed with 0.3 ug priMer I, 0. 3 ug priner II and 33 u«ol/l of each dNTP in a total voluMe of 50 Ml. The solution was denatured (95 C, 8 «in), the priners were annealed 122 C, 5 Min) and Genotypes MM HZ ZZ MM and 1 Unit of tlenow DNA polyMerase ( New Eng. , Bio Labs) was added. The reM probe ^h gMt ^k action proceeded at 53 C for 5 Min. ^^ ^^ In subsequent cycles denaturation was for 5 Min, annealing for 1 "in and polynerization for 6 Min. After 25 cycles 1 probe A ^fe 1 ul of the solution was adjusted to 0. 4 MOI/1 NaOH and 0. 025 MOI/1 EDTA, spotted onto Zeta •eibranes (Bio-Rad) Amplified DHA from individuals with and baked for 1 hr at 80°C. The spots the a AT genotypes MM, HZ and ZZ were prehybridixed in 5 x SSPE (1 x hybridized with P-labelled M or SSPE = 10 ••ol/l NaH PO , (pH 7.4), Z oligonucleotide probes. 0.15 MOI/1 HaCl, 1 MMOI/1 EDTA), IX SDS and 100 ug/al herring sperM DNA at 52 C for 30 Min. Hybridization was performed at 52 for 60 Bin in the aa«e buffer, using 21-ner allele specific oligonucleotide probes (2). The sequence of the M probe was 5' ACCATCQACrjAQAAAQaQACT and the Z probe was 5' ACCATCaACA>QAAAaaOACT. 10 pnol oligonucleotides were labeled at the 5'end with P. The probe (1x10 cpn/ug) was used without purification. The spots were washed in 2xSSPE, 0. 1X SDS at 22°C for 2x3o «in, followed by 5iSSPE, 0.1X SDS at 52°C for 15 nin. The final wash was for 15 Min in 5zSSPE, 0. IX SDS at 60°C for the spots hybridized with the Z probe and at 63 C for the spots hybridized with the M probe. The spots were autoradiographed for 3 hrs (fig). The whole procedure, including the isolation of DNA, can be performed within 3 days.

Detection of alpha1-antitrypsin genotypes by analysis of amplified DNA sequences

Detection of alpha₁-antitrypsin genotypes by analysis of amplified DNA sequences