Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing.
A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.
Finn
Patrick J
PJ
Amersham Biosciences, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
Bull
Matthew G
MG
Xiao
Haiguang
H
Phillips
Paula D
PD
Nelson
John R
JR
Grossmann
Greg
G
Nampalli
Satyam
S
McArdle
Bernard F
BF
Mamone
J Anthony
JA
Flick
Parke K
PK
Fuller
Carl W
CW
Kumar
Shiv
S
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004396:Coloring Agents / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015224:Dideoxynucleosides / Q000737:chemistry*; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D004396:Coloring Agents; D015224:Dideoxynucleosides; D009711:Nucleotides; C003712:trimethyllysine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D008239:Lysine