Genetic analysis using polymerase chain reaction-amplified DNA and immobilized oligonucleotide probes: reverse...

The reverse dot-blot method is a simple and rapid diagnostic procedure that allows screening of sample for a variety of mutations/polymorphisms in a single hybridization reaction. Several methods of immobilizing the oligonucleotide probes are discussed. The reverse dot-blot method has several unique properties that are valuable in a diagnostic setting: (1) the typing results from a single sample can be located on a single strip. This facilitates scanning and interpretation of the probe reactivity patterns and minimizes the potential for user error. (2) The test can utilize premade typing strips. This minimizes user labor as well as error potential and allows the use of standardized reagents. (3) Unlike dot-blot/oligonucleotide typing, only the PCR product is labeled, eliminating the potential problem of probes labeled to different specific activities. This method has already been used in the areas of forensic genetic typing (the HLA-DQ alpha Amplitype test), tissue typing for transplantation (the HLA-DR beta) test, cystic fibrosis screening, as well as in a variety of research applications.