Design and synthesis of a photocleavable fluorescent nucleotide 3'-O-allyl-dGTP-PC-Bodipy-FL-510 as a reversible terminator for DNA sequencing by synthesis.
DNA sequencing by synthesis (SBS) using reversible fluorescent nucleotide terminators is potentially an efficient approach to address the limitations of current DNA sequencing techniques. Here, we report the design and synthesis of a 3'-O-allyl photocleavable fluorescent nucleotide analogue, 3'-O-allyl-dGTP-PC-Bodipy-FL-510, as a reversible terminator for SBS. The nucleotide is efficiently incorporated by DNA polymerase into a growing DNA strand to terminate the polymerase reaction. After that, the fluorophore is photocleaved quantitatively by irradiation at 355 nm, and the allyl group is rapidly and efficiently removed by using a Pd-catalyzed reaction under DNA-compatible conditions to regenerate a free 3'-OH group to reinitiate the polymerase reaction. Two cycles of such steps were successfully demonstrated to sequence a homopolymeric region of a DNA template, facilitating the development of SBS as a viable approach for high-throughput DNA sequencing.
Meng
Qinglin
Q
Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Kim
Dae Hyun
DH
Bai
Xiaopeng
X
Bi
Lanrong
L
Turro
Nicholas J
NJ
Ju
Jingyue
J
D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry
D003848:Deoxyguanine Nucleotides; D005456:Fluorescent Dyes; C029603:deoxyguanosine triphosphate; D004259:DNA-Directed DNA Polymerase