Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing.

The discovery of RFLPs and their utilization as genetic markers has revolutionized research in human molecular genetics. However, only a fraction of the DNA sequence polymorphisms in the human genome affect the length of a restriction fragment and hence result in an RFLP. Polymorphisms that are not detected as RFLPs are typically passed over in the screening process though they represent a potentially important source of informative genetic markers. We have used a rapid method for the detection of naturally occurring DNA sequence variations that is based on enzymatic amplification and direct sequencing of genomic DNA. This approach can detect essentially all useful sequence variations within the region screened. We demonstrate the feasibility of the technique by applying it to the human retinoblastoma susceptibility locus. We screened 3,712 bp of genomic DNA from each of nine individuals and found four DNA sequence polymorphisms. At least one of these DNA sequence polymorphisms was informative in each of three families with hereditary retinoblastoma that were not informative with any of the known RFLPs at this locus. We believe that direct sequencing is a reasonable alternative to other methods of screening for DNA sequence polymorphisms and that it represents a step forward for obtaining informative markers at well-characterized loci that have been minimally informative in the past.