GENOTYPING OF HUMAN DEOXYRIBONUCLEASE-I POLYMORPHISM BY THE POLYMERASE CHAIN-REACTION

We recently completely elucidated the molecular basis of genetic polym orphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE11, *2, *3 and *4. In this paper we des cribe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the th ree nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitution s neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE11 (or *3) a nd the DNASE12 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE 13. A 100% correlation was found between the results of this genotypi ng method and those obtained by phenotyping using conventional isoelec tric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.