Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides

Abstract

; 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 solutionphase polymerase reaction and the fluorophore could be cleaved using laser irradiation (Ϸ355 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 azidolabeled 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. 2-nitrobenzyl linkerNA sequencing is a fundamental tool for biological science. The completion of the Human Genome Project has set the stage for screening genetic mutations to identify disease genes on a genome-wide scale (1). Accurate high-throughput DNA sequencing methods are needed to explore the complete human genome sequence for applications in clinical medicine and health care. Recent studies have indicated that an important route for identifying functional elements in the human genome involves sequencing the genomes of many species representing a wide sampling of the evolutionary tree (2). To overcome the limitations of the current electrophoresis-based sequencing technology (3-5), a variety of new DNA sequencing methods have been investigated. Such approaches include sequencing by hybridization (6), MS-based sequencing (7-9), and sequence-specific detection of single-stranded DNA by using engineered nanopores (10). More recently, DNA sequencing by synthesis (SBS) approaches such as pyrosequencing (11), sequencing of single DNA molecules (12), and polymerase colonies (13) have been widely explored.The concept of DNA SBS was revealed in 1988 (14). This approach involves detection of the identity of each nucleotide immediately after its incorporation into a growing strand of DNA in a polymerase reaction. Thus far, no complete success has been reported in using such a system to sequence DNA unambiguously. We proposed an SBS approach using photocleavable fluorescent nucleotide analogues on a surface in 2000 (15). In this approach, modified nucle...

Design and synthesis of a 3′- O -allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis

“…successful, we irradiated it with near-UV light at 355 nm for 10 sec to cleave the fluorophore from the DNA, generating product 12. In an SBS system, this step would ensure that there would be no carryover of the fluorescence signal into the next incorporation cycle so as to prevent the generation of ambiguous data at each step, as shown in the companion article (15). The photocleavage product 12 was then incubated with a Na 2 PdCl 4 ͞ TPPTS catalyst system at 70°C for 90 sec to perform deallylation.…”

“…The 10-l extension reaction mixture consisted of 45 pmol of the deallylated DNA product as a primer, 100 pmol of a single-stranded synthetic 60-mer DNA template (sequence shown in ref. 15) corresponding to a portion of exon 7 of the p53 gene, 100 pmol of biotin-11-2Ј,3Ј-dideoxyguanosine-5Ј-triphosphate (Biotin-11-ddGTP) terminator (PerkinElmer), 1ϫ Thermo Sequenase reaction buffer, and 4 units of Thermo Sequenase DNA polymerase. The extension reaction consisted of 15 cycles at 94°C for 20 sec, 48°C for 30 sec, and 60°C for 60 sec.…”

“…The above deallylated DNA product 13 was used as a primer in a single-base extension reaction. The 10 l reaction mixture consisted of 50 pmol of the above deallylated product 13, 100 pmol of the 60-mer template (15), 125 pmol of dGTP-PC-Bodipy-FL-510 (14), 4 units of Thermo Sequenase DNA polymerase, and 1ϫ reaction buffer. The extension reaction consisted of 15 cycles of 94°C for 20 sec, 48°C for 30 sec, and 60°C for 60 sec.…”

“…After the fluorescent signal is detected and the nucleotide identified, the 3Ј-OH needs to be regenerated to continue incorporating the next nucleotide. In the accompanying report, we have demonstrated that four photocleavable fluorescent nucleotides can be efficiently incorporated by DNA polymerase into a growing DNA strand base specifically in a polymerase extension reaction, and that the fluorophores can be completely removed by photocleavage under near-UV irradiation (Ϸ355 nm) with high efficiency (15). Using this system in a four-color sequencing assay, we were able to accurately identify multiple bases in a self-priming DNA template covalently attached to a glass surface.…”

Simcluster: clustering enumeration gene expression data on the simplex space

“…However, recent improvements in sequencing technology, powered by the "$1000 genome" effort [29], promises to transform the transcript enumeration approach into a fast and accessible alternative [24,28,13] paving the way for a systems-level absolute digital description of individualized samples [22].…”