Heat activatable 3'-modified dNTPs: synthesis and application for hot start PCR.
Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95 degrees C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.
Koukhareva
Inna
I
Trilink BioTechnologies, Inc., 9955 Mesa Rim Road, San Diego, CA 92121, USA.
Haoqiang
Huang
H
Yee
Joyclyn
J
Shum
Jonathan
J
Paul
Natasha
N
Hogrefe
Richard I
RI
Lebedev
Alexandre V
AV
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D016133:Polymerase Chain Reaction
D004279:DNA, Viral; D003854:Deoxyribonucleotides; D004259:DNA-Directed DNA Polymerase