TY - JOUR
T1 - Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
AU - Ju, Jingyue
AU - Kim, Dae Hyun
AU - Bi, Lanrong
AU - Meng, Qinglin
AU - Bai, Xiaopeng
AU - Li, Zengmin
AU - Li, Xiaoxu
AU - Marma, Mong Sano
AU - Shi, Shundi
AU - Wu, Jian
AU - Edwards, John R.
AU - Romu, Aireen
AU - Turro, Nicholas J.
PY - 2006/12/26
Y1 - 2006/12/26
N2 - 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.
AB - 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.
KW - DNA chip
UR - http://www.scopus.com/inward/record.url?scp=33845940688&partnerID=8YFLogxK
U2 - 10.1073/pnas.0609513103
DO - 10.1073/pnas.0609513103
M3 - Article
C2 - 17170132
AN - SCOPUS:33845940688
SN - 0027-8424
VL - 103
SP - 19635
EP - 19640
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 52
ER -