Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides.
We report four-color DNA sequencing by synthesis (SBS) on a chip, using four photocleavable fluorescent nucleotide analogues (dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, and dCTP-PC-Bodipy-650) (PC, photocleavable; Bodipy, 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene; ROX, 6-carboxy-X-rhodamine; R6G, 6-carboxyrhodamine-6G). Each nucleotide analogue consists of a different fluorophore attached to the 5 position of the pyrimidines and the 7 position of the purines through a photocleavable 2-nitrobenzyl linker. After verifying that these nucleotides could be successfully incorporated into a growing DNA strand in a solution-phase polymerase reaction and the fluorophore could be cleaved using laser irradiation ( approximately 355 nm) in 10 sec, we then performed an SBS reaction on a chip that contains a self-priming DNA template covalently immobilized by using 1,3-dipolar azide-alkyne cycloaddition. The DNA template was produced by PCR, using an azido-labeled primer, and the self-priming moiety was attached to the immobilized DNA template by enzymatic ligation. Each cycle of SBS consists of the incorporation of the photocleavable fluorescent nucleotide into the DNA, detection of the fluorescent signal, and photocleavage of the fluorophore. The entire process was repeated to identify 12 continuous bases in the DNA template. These results demonstrate that photocleavable fluorescent nucleotide analogues can be incorporated accurately into a growing DNA strand during a polymerase reaction in solution and on a chip. Moreover, all four fluorophores can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing continuous identification of the DNA template sequence. Optimization of the steps involved in this SBS approach will further increase the read-length.
Seo
Tae Seok
TS
Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Bai
Xiaopeng
X
Kim
Dae Hyun
DH
Meng
Qinglin
Q
Shi
Shundi
S
Ruparel
Hameer
H
Li
Zengmin
Z
Turro
Nicholas J
NJ
Ju
Jingyue
J
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D010777:Photochemistry; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D005456:Fluorescent Dyes; D004247:DNA