Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups.
Nucleoside triphosphates having a 3'-ONH₂ blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH₂ group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH₂ blocking group in "next generation sequencing."
Foundation for Applied Molecular Evolution, Gainesville, FL 32604, USA. sbenner@ffame.org
Daniel
D
Hutter
Myong-Jung
MJ
Kim
Nilesh
N
Karalkar
Nicole A
NA
Leal
Fei
F
Chen
Evan
E
Guggenheim
Visa
V
Visalakshi
Jerzy
J
Olejnik
Steven
S
Gordon
Steven A
SA
Benner
D017931:DNA Primers / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure; D009705:Nucleosides / Q000737:chemistry*; D011122:Polyphosphates / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D005456:Fluorescent Dyes; D009705:Nucleosides; D011122:Polyphosphates; D004259:DNA-Directed DNA Polymerase