Hydrolink gels: a rapid and simple approach to the detection of DNA mutations in thromboembolic disease.

摘要

AIMS: To develop a simple and rapid technique for detecting DNA mutations based on the polymerase chain reaction, followed by electrophoresis, in a novel polymer--Hydrolink D5000--specifically designed to separate double stranded DNA fragments. METHODS: Eleven subjects with previously characterised mutations within the antithrombin gene (including single base pair mutations and insertions) and three normal controls were studied. DNA was amplified and one sixth of the PCR product electrophoresed in a 20 cm x 20 cm x 1 mm 100% Hydrolink D5000 gel for two to six hours, followed by staining in ethidium bromide for 20 minutes. The gel was then visualised under ultraviolet light. RESULTS: After amplification and electrophoresis a single additional band was observed in five out of nine variants in which the mutations involved a single base pair substitution, while two additional bands were seen in four out of nine mutants which arose as a result of a single base pair insertion. No abnormality was detected in two known variants. CONCLUSION: This method provides a simple and rapid approach to the screening and detection of mutations at the DNA level which does not involve the use of either toxic reagents or radioisotopes. It may also provide evidence about the type of mutation.