Mode of action and application of Scorpion primers to mutation detection.
Scorpion primers can be used to detect PCR products in homogeneous solution. Their structure promotes a unimolecular probing mechanism. We compare their performance with that of the same probe sequence forced to act in a bimolecular manner. The data suggest that Scorpions indeed probe by a unimolecular mechanism which is faster and more efficient than the bimolecular mechanism. This mechanism is not dependent on enzymatic cleavage of the probe. A direct comparison between Scorpions, TaqMan and Molecular Beacons on a Roche LightCycler indicates that Scorpions perform better, particularly under fast cycling conditions. Development of a cystic fibrosis mutation detection assay shows that Scorpion primers are selective enough to detect single base mutations and give good sensitivity in all cases. Simultaneous detection of both normal and mutant alleles in a single reaction is possible by combining two Scorpions in a multiplex reaction. Such favourable properties of Scorpion primers should make the technology ideal in numerous applications.
Thelwell
N
N
Oswel Research Products Ltd, University of Southampton, Southampton SO16 7PX, UK.
Millington
S
S
Solinas
A
A
Booth
J
J
Brown
T
T
D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004735:Energy Transfer; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics; D012680:Sensitivity and Specificity; D012996:Solutions; D019914:Taq Polymerase / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
C505032:CFTR protein, human; D017931:DNA Primers; D015342:DNA Probes; D015335:Molecular Probes; D015345:Oligonucleotide Probes; D012996:Solutions; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D019914:Taq Polymerase; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I