Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5'-triphosphates to 'direct-load' DNA sequencing.
A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.
Finn
Patrick J
PJ
Amersham Biosciences, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
Sun
Lei
L
Nampalli
Satyam
S
Xiao
Haiguang
H
Nelson
John R
JR
Mamone
J Anthony
JA
Grossmann
Greg
G
Flick
Parke K
PK
Fuller
Carl W
CW
Kumar
Shiv
S
D004396:Coloring Agents / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry*; D004586:Electrophoresis / Q000379:methods; D009154:Mutation; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism
D004396:Coloring Agents; D003854:Deoxyribonucleotides; D004247:DNA; D019914:Taq Polymerase