Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2'-deoxyribonucleoside-5'-triphosphates.
Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2'-deoxyribonucleoside-5'-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5'-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In addition, we attached novel fluorophore derivatives. The labeled products were obtained in at least 96% purity after HPLC purification. Enzymatic incorporation into DNA and subsequent extension of the modified DNA chain were studied. Vent(R) exo- DNA polymerase and a defined template-primer system were used to analyze each dye-labeled dNTP derivative. Our data suggest that the incorporation efficiency depends on the selected dye, the nucleobase or a combination of both.
Gnothis SA, PSE-B, EPFL, CH-1015 Lausanne, Switzerland and Römerstrasse 7, D-82398 Polling, Germany.
Gerald
G
Giller
Taurai
T
Tasara
Bernhard
B
Angerer
Klaus
K
Mühlegger
Mario
M
Amacker
Holger
H
Winter
D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure; D009711:Nucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D010078:Oxazines / Q000737:chemistry; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D009711:Nucleotides; D010078:Oxazines; D004247:DNA; C039203:phenoxazine; D004259:DNA-Directed DNA Polymerase