Methods of detection of single base substitutions in clinical genetic practice

The ability to diagnose human diseases at the DNA level has become possible because of a rapid development in DNA technology, particularly in the area of detection of single base substitutions. Mutations in the genomic DNA of a particular gene may be inferred indirectly using linkage analysis and restriction fragment length polymorphisms. However, direct detection of the mutation is the more favourable approach. The advent of the polymerase chain reaction to amplify specific regions of genomic DNA or mRNA has enhanced the speed and sensitivity of many of the screening and diagnostic procedures. Screening methods have been developed that will detect at least 70% and, with some methods, close to 100% of all mutations. The methods include ribonuclease A cleavage, denaturing gradient gel electrophoresis, chemical cleavage of mismatch and direct sequencing. Choice of method is based on a number of factors and will depend on the structure of the gene to be analysed. Following identification of a mutation using one of the screening procedures, prenatal diagnosis and carrier testing can be offered. The overall aim is to develop a method that has the potential to determine the mutation present in an index case of a previously untested family in a few days, thus allowing any other relevant family member to be tested.