Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases.
The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.
Lacenere
Christopher
C
Division of Biology, California Institute of Technology, Pasadena, California, USA.
Garg
Meil K
MK
Stoltz
Brian M
BM
Quake
Stephen R
SR
D002232:Carbocyanines / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015394:Molecular Structure; D013696:Temperature; D014500:Uracil Nucleotides / Q000737:chemistry* / Q000378:metabolism*
D002232:Carbocyanines; D003432:Cross-Linking Reagents; D014500:Uracil Nucleotides; C085321:cyanine dye 5; D004259:DNA-Directed DNA Polymerase