Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.
Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.
Metzker
M L
ML
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030.
Raghavachari
R
R
Richards
S
S
Jacutin
S E
SE
Civitello
A
A
Burgess
K
K
Gibbs
R A
RA
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013698:Templates, Genetic
D003854:Deoxyribonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase