Real-time monitoring of rolling-circle amplification using a modified molecular beacon design.
We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2'-O-Me-RNA to prevent 3' exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.
Nilsson
Mats
M
Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. mats.nilsson@genpat.uu.se
Gullberg
Mats
M
Dahl
Fredrik
F
Szuhai
Karoly
K
Raap
Anton K
AK
D002851:Chromatography, High Pressure Liquid; D004270:DNA, Circular / Q000235:genetics*; D005092:Exonucleases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004270:DNA, Circular; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D005092:Exonucleases