DENATURATION FINGERPRINTING - 2 RELATED MUTATION DETECTION METHODS ESPECIALLY ADVANTAGEOUS FOR HIGH G+C REGIONS

Two versions of denaturation fingerprinting (dnF(2R) and dnF(1R)) are described for detecting mutations. DnF2R is a sensitive screening meth od in which fingerprints are generated by performing denaturing gel el ectrophoresis on bidirectional ''cycle-sequencing'' reactions with eac h of two dideoxy terminators, e.g., ddATP and ddCTP. When the fingerpr ints generated by ddATP and ddCTP are combined, all sequence changes a re expected to result in one extra and one absent segment. DnF(2R) was performed on 246- and 318-bp segments of the human factor IX gene, an d the products were electrophoresed through a 6% Long Ranger(TM) gel w ith 7 M urea. All 32 single-base mutations were detected in hemizygous males with hemophilia B. DnF(2R) has been applied to detect a total o f seven heterozygous sequence changes in large-scale screening and was found to be especially suitable for high G+C regions. In a blinded an alysis, all of twenty-four additional single-base mutations were detec ted, but 7 of 31 heteroxygous mutations were missed (23%). To reduce t he effort of dnF(2R) by almost twofold while retaining the ability to detect all types of single-base changes, one fingerprint (dnF(1R)) was generated by performing a single reaction with ddATP and a second che mically modified terminator (e.g., ROX-conjugated ddCTP), which retard s the mobility of the same termination products. The sensitivity and s pecificity of dnF(1R) equaled that of dnF(2R), with the exception that the blinded analysis of heterozygotes in the 318-bp segment, which re vealed the presence of an additional mutation. DnF under partially den aturing conditions may have optimal sensitivity for the detection of h eterozygotes.