Dna Analysis of Gametes and Embryos: Analysis of Delta-F508 Cystic Fibrosis Mutation in Single Cells

Abstract

The delta-F508 mutation is the most common mutation causing cystic fibrosis (CF) (Kerems et al, 1990). A method to consistently identify the presence of the delta-F508 mutation in single cells has been developed using decontamination of buffers and reagents by restriction digestion with Msel followed by heat inactivation before Polymerase Chain Reaction (PCR) (Strom et al, 1990; Verlinsky et al, 1990). After PCR, the amplified products are allocated to three tubes. PCR products from a homozygous normal individual are added to one tube, and products from a CF patient homozygous for delta-F508 are added to the second tube. These tubes are heated to 95°C for 10 min. and allowed to reanneal at 68°C for 10 min. The procedure permits heteroduplexes to form between delta-F508 and normal sequences (Rommens et al, 1990). The third tube is untreated. The DNA from the three tubes is then analyzed by Polyacrylamide gel electrophoresis (equipment and reagents required are presented in annexes 1 and 2).

In previous publications Svetlana Milayeva