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 quantify the hybridization parameters of a 35-mer oligonucleotide probe with target DNAs immobilized on transfer membranes. The amount of the immobilized target DNA remaining accessible to hybridization (Rt) was shown to be about 4% of the spotted DNA. The time course of the hybridization of a target DNA reacting with an excess of full-match probe exhibited a first-order kinetics, in which rate constant k was the highest for the hybridization temperature close to the calculated Tm. The effect of temperature on the hybridization kinetics of the probe sharing 37 to 100% identity with the immobilized target DNA was assessed: A significant fall of both the rate constant k and Rt values at the plateau was observed when the identity shared by the target DNA and the probe decreased from 100 to 71%. The highest k and Rt values were also obtained for temperatures closest to the calculated Tm. A good estimate of the degree of sequence identity may be calculated from the corresponding hybridization signals. Washing procedure did not improve the discrimination between related sequences, except for closely similar sequences. Practical conclusions for the detection of sequences belonging to gene families are presented.
Vernier
P
P
Laboratoire de Génétique des Maladies Neurodégénératives et du Vieillissement, CNRS, Gif-sur-Yvette, France.
Mastrippolito
R
R
Helin
C
C
Bendali
M
M
Mallet
J
J
Tricoire
H
H
D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics*; D006358:Hot Temperature; D007700:Kinetics; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012588:Scintillation Counting / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid
D008567:Membranes, Artificial; D015345:Oligonucleotide Probes; D004247:DNA