Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescent adsorbent.
We studied various parameters affecting the sensitivity of assays that use nucleic acid hybridization in solution followed by capture of the hybrid on a solid phase. Sensitivity is limited not only by nonspecific binding of the detection components but also by reannealing of the target or probe to itself. To perform sensitive assays, the probe concentration must be low enough to reduce high nonspecific binding. Under these conditions, however, the strand displacement reaction or the reannealing of the target to itself drastically decreases the hybridization yield, particularly when the target and the probes are different sizes. To improve DNA detection, we propose a sandwich method based on hybridization of oligonucleotides with a single-strand DNA obtained by polymerase chain reaction under asymmetric conditions. The assay can be performed in one step using a bioluminescent detection procedure which does not require any separation step. The specificity of the method is sufficient to perform a rapid detection and quantification of papillomavirus in biological samples.
Balaguer
P
P
INSERM Unité 58, Montpellier, France.
Térouanne
B
B
Boussioux
A M
AM
Nicolas
J C
JC
D000327:Adsorption; D001426:Bacterial Proteins; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry; D006801:Humans; D008163:Luminescent Measurements; D008863:Microspheres; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D027383:Papillomaviridae / Q000235:genetics; D016133:Polymerase Chain Reaction; D012685:Sepharose; D012996:Solutions; D019809:Streptavidin
D001426:Bacterial Proteins; D004279:DNA, Viral; D009841:Oligonucleotides; D012996:Solutions; D004247:DNA; D012685:Sepharose; D019809:Streptavidin; D005954:Glucosephosphate Dehydrogenase