Detection of single base substitutions in polynucleotides by capture with immobilized oligonucleotides.
We have improved a sandwich hybridization assay to detect single base substitutions in polymerase chain reaction (PCR) amplified DNA sequences. The target DNA was captured by an immobilized oligonucleotide and revealed using a second oligonucleotide coupled to an enzyme. Short oligonucleotides (13, 15 bases) were used to obtain specific hybridization at 37 degrees C. We developed two different assay formats for rapid identification of PCR products: a microtitration plate format with oligonucleotides bound to polystyrene and a channelling assay using oligonucleotides immobilized on Sepharose, which did not require any separation step. The specificity and advantages of both methods are described.
INSERM Unité 58, Montpellier, France.
P
P
Balaguer
B
B
Térouanne
P
P
Allibert
P
P
Cros
A M
AM
Boussioux
B
B
Mandrand
J C
JC
Nicolas
D000469:Alkaline Phosphatase; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005954:Glucosephosphate Dehydrogenase; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D009195:Peroxidase; D011119:Polynucleotides / Q000235:genetics*; D012680:Sensitivity and Specificity; D012685:Sepharose
D015345:Oligonucleotide Probes; D011119:Polynucleotides; D012685:Sepharose; D005954:Glucosephosphate Dehydrogenase; D009195:Peroxidase; D000469:Alkaline Phosphatase