Use of glucose-6-phosphate dehydrogenase as a new label for nucleic acid hybridization reactions.
We describe here a sensitive new procedure for detecting DNA hybridization by dot blots. The method utilizes DNA or oligonucleotide probes labeled with biotin, sulfone, or haptens that can be detected by glucose-6-phosphate dehydrogenase (G6PDH) conjugates. Biotin labeling of DNA gave the best sensitivity. G6PDH activity was revealed by staining or by bioluminescence using an FMN oxidoreductase and a luciferase from Beneckea harveyi. Bioluminescent detection offered better sensitivity and faster revelation than the colorimetric assay and was found to be very useful in visualizing single mutations in human DNA after hybridization with an allele-specific biotinylated oligonucleotide probe. Revelation can be performed using a luminometer, photographic films, or a very sensitive video camera. The detection is limited by the nonspecific binding of the labeled reagent (streptavidin or antibodies). This limit is similar to that obtained with other nonisotopic labeling procedures, but our method is faster and several hybridization reactions can be performed on the same support.
Balaguer
P
P
INSERM Unité 58, Montpellier, France.
Térouanne
B
B
Eliaou
J F
JF
Humbert
M
M
Boussioux
A M
AM
Nicolas
J C
JC
D010582:Bacteriophage lambda; D001710:Biotin; D003124:Colorimetry; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D038181:FMN Reductase; D005784:Gene Amplification; D005954:Glucosephosphate Dehydrogenase; D015151:Immunoblotting; D008156:Luciferases; D008163:Luminescent Measurements; D009247:NADH, NADPH Oxidoreductases; D009693:Nucleic Acid Hybridization; D011827:Radiation; D019914:Taq Polymerase
D004279:DNA, Viral; D001710:Biotin; D005954:Glucosephosphate Dehydrogenase; D008156:Luciferases; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase