Primers with 5' flaps improve real-time PCR.
Primers that contain portions noncomplementary to the target region are usually used to add to the PCR product a utility sequence such as a restriction site or a universal probe binding site. We have demonstrated that primers with short 5'AT-rich overhangs increase real-time PCR fluorescent signal. The improvement is particularly significant for difficult to amplify templates, such as highly variable viral sequences or bisulfite-treated DNA.
Afonina
Irina
I
Nanogen Inc., Bothell, WA 98021, USA. iafonina@nanogen.com
Ankoudinova
Irina
I
Mills
Alan
A
Lokhov
Sergey
S
Huynh
Phan
P
Mahoney
Walt
W
D024506:5' Flanking Region / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000379:methods / Q000592:standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D013447:Sulfites
D017931:DNA Primers; D004279:DNA, Viral; D005456:Fluorescent Dyes; D013447:Sulfites; C042345:hydrogen sulfite