Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry.
DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to site-specifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence.
Seo
Tae Seok
TS
Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Bai
Xiaopeng
X
Ruparel
Hameer
H
Li
Zengmin
Z
Turro
Nicholas J
NJ
Ju
Jingyue
J
D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D001483:Base Sequence; D003500:Cyclization; D004247:DNA / Q000032:analysis / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D010782:Photolysis; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D000480:Alkynes; D001386:Azides; D003845:Deoxycytosine Nucleotides; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D004247:DNA; D004259:DNA-Directed DNA Polymerase