Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators.
DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner.
Ju
Jingyue
J
Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. ju@c2b2.columbia.edu
Kim
Dae Hyun
DH
Bi
Lanrong
L
Meng
Qinglin
Q
Bai
Xiaopeng
X
Li
Zengmin
Z
Li
Xiaoxu
X
Marma
Mong Sano
MS
Shi
Shundi
S
Wu
Jian
J
Edwards
John R
JR
Romu
Aireen
A
Turro
Nicholas J
NJ
D001483:Base Sequence; D003116:Color; D004247:DNA / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*; D015394:Molecular Structure; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005456:Fluorescent Dyes; D009711:Nucleotides; D004247:DNA