RADIOIMAGER QUANTIFICATION OF OLIGONUCLEOTIDE HYBRIDIZATION WITH DNA IMMOBILIZED ON TRANSFER MEMBRANE - APPLICATION TO THE IDENTIFICATION OF RELATED SEQUENCES

The radioimager scintillating optical fiber imager was used to quantif y the hybridization parameters of a 35-mer oligonucleotide probe with target DNAs immobilized on transfer membranes. The amount of the immob ilized target DNA remaining accessible to hybridization (R(t)) was sho wn to be about 4% of the spotted DNA. The time course of the hybridiza tion of a target DNA reacting with an excess of full-match probe exhib ited a first-order kinetics, in which rate constant k was the highest for the hybridization temperature close to the calculated T-m. The eff ect of temperature on the hybridization kinetics of the probe sharing 37 to 100% identity with the immobilized target DNA was assessed: A si gnificant fall of both the rate constant k and R(t) values at the plat eau was observed when the identity shared by the target DNA and the pr obe decreased from 100 to 71%. The highest k and R(t) values were also obtained for temperatures closest to the calculated T-m. A good estim ate of the degree of sequence identity may be calculated from the corr esponding hybridization signals. Washing procedure did not improve the discrimination between related sequences, except for closely similar sequences. Practical conclusions for the detection of sequences belong ing to gene families are presented. (C) 1996 Academic Press, Inc.