Ultrafast Reversible Photo-Cross-Linking Reaction: Toward in Situ DNA Manipulation

“…Key to this approach is efficient anchoring of the complementary strands to the surface strands on solid surface. A previously reported 3-cyanovinylcarbazole nucleoside (CNVK) (16,17) can be used as an ultrafast reversible DNA interstrand photo-crosslinking reagent via irradiation at 366 nm for 1 s. Thus, we synthesized an oligonucleotide containing CNVK reagent and dU, which, when attached to the 5500 flowchip surface, acts as a surface primer (Fig. 3A).…”

Section: Significancementioning

confidence: 99%

Isothermal amplification method for next-generation sequencing

Lee

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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We report an approach for generating immobilized monoclonal templates for next-generation sequencing applications. Our isothermal amplification method is based on a template walking mechanism using a pair of low-melting temperature (Tm) solidsurface homopolymer primers and a low-Tm solution phase primer. The method can generate more than one billion submicrometersized colonies in a single lane of a next-generation sequencing flowchip. An alternative paired-end sequencing method using interstrand DNA photo cross-linking to covalently link the complementary strands of the original templates to the solid surface is also demonstrated.genome | in situ PCR | forming colonies | DNA breathing | polony T he introduction of massively parallel next-generation sequencing (NGS) has revolutionized the biomedical research of human disease and health. The general workflow for NGS involves library generation, colony formation, sequencing, and analysis (1, 2). All NGS technologies, except for single-molecule sequencing platforms, require library amplification to generate many copies of each single library fragment before sequencing. The polymerase colony (polony) technology developed by Church's group was the first reported approach to generating monoclonal colonies by performing PCR on a thin polyacrylamide gel on a microscope slide support, and then covalently attaching the amplified DNA to the polyacrylamide matrix using a 5′ acrydite-modified PCR primer (3). Polony technology was used in digital genotyping, haplotyping (4), and later in sequencing (5).Commercial NGS platforms use two major amplification methods: emulsion PCR (emPCR) with microbeads, which is used by Roche's 454 (6) and Life Technologies' SOLiD (7) and Ion Torrent (8) platforms, and bridge PCR (bPCR) amplification (9-12) on a glass surface, which is used by Illumina's HiSeq/ MiSeq sequencing platforms. Dressman et al. (13) first demonstrated an emPCR approach with superparamagnetic beads in 2003. emPCR requires performing PCR on billions of microbeads, each isolated in its own emulsion droplet, followed by emulsion breakup, template enrichment, and bead deposition before sequencing. bPCR generates monoclonal clusters directly on a solid surface with a minimum of hands-on time. A disadvantage of bPCR is that each of the multiple amplification cycles requires the addition of fresh mixture containing polymerase, followed by a chemical denaturation step, making it cost much more than regular PCR. bPCR also requires a specialized pumping system, such as cBot or a dedicated module built inside the HiSeq/MiSeq system. Another library amplification method developed by Complete Genomics uses rolling-circle replication to amplify small fragments of genomic DNA into DNA nanoballs (14). This approach requires multiple PCR steps to construct a complete circular template before the final amplification step to generate nanoballs.We report here a solid-phase, in situ isothermal PCR approach that generates more than one billion monoclonal colonies in a single lane of a 5500 fl...

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“…40 They also achieved an ultrafast reversible DNA interstrand photoinduced cross-linking between a modified ODN containing a 3-cyanovinylcarbazolenucleoside ( CNV K) and an adjacent pyrimidine base upon UV irradiation. 41 Recently, the reversible photoinduced cross-linking reaction was used to select a target RNA sequence (Figure 4). 42 Mesmaeker and co-workers reported that ODNs labeled with ruthenium(II) complexes (RuODNs) ( Figure 5) photo-crosslinked to guanine (G) through their complementary strand under visible irradiation ( Figure 6).…”

Section: Cross-linking Reaction By Reactive Oligonucleotidesmentioning

confidence: 99%

Synthesis of Reactive Oligonucleotides for Gene Targeting and Their Application to Gene Expression Regulation

Nagatsugi

Sasaki

2010

Bulletin of the Chemical Society of Japan

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Many genetic disorders have recently been identified as a major cause of diseases and the regulation of gene expression has been proposed as an attractive therapeutic strategy. Synthetic oligodeoxynucleotides (ODNs) are valuable tools that interfere with gene expression by specifically binding to target genes in a sequence-specific manner. In particular, reactive ODNs are expected to be more efficient because they covalently bind to the target genes. This review summarizes the synthesis of reactive ODNs with inducible reactivity, in particular, their applications to efficient crosslinking reactions, have been developed by the author's group. We also describe applications of the cross-linking reactions to the antisense method in cells and their impact on mutagenesis in model systems.

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Ultrafast Reversible Photo-Cross-Linking Reaction: Toward in Situ DNA Manipulation

Cited by 171 publications

References 25 publications

Molecular Robotics: A New Paradigm for Artifacts

Molecular Robotics: A New Paradigm for Artifacts

Isothermal amplification method for next-generation sequencing

Synthesis of Reactive Oligonucleotides for Gene Targeting and Their Application to Gene Expression Regulation

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