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Surface DNA content analysis by TdT labeling of 3′-OH using Cy5-ddNTP Visual Genetics sequencer trace recorded for disulfide-T2 template (80mer) grafted on BTA glass, labeled with 500 nM ddNTP-Cy5.0 at 3′ end (TdT) and cleaved from the surface in 50 mM DTT/Tris solution pH 8.5 (1 h). Grafting conditions: 130 nM 5′-amino-SS-T2 template, 10 mM EDC/10 mM 1-Methyl-Imidazole (50°C)/1 h. Terminal Deoxynucleotidyl Transferase (TdT), 20 µ/ml, NEB buffer4, CoCl2 (250 mM). Sequencer (Visible Genetics, VG90008), SureFill 6% Sequencing Gel (Visible Genetics, Ref. #VG40006), Stop Loading Dye (Amersham). The upper traces represent DNA size markers labeled with Cy5.5. In the lower trace, the Cy5 labelled oligonucleotide extracted from the surface migrates as a single peak with an approximate apparent size of 87 bases.
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fig4: Surface DNA content analysis by TdT labeling of 3′-OH using Cy5-ddNTP Visual Genetics sequencer trace recorded for disulfide-T2 template (80mer) grafted on BTA glass, labeled with 500 nM ddNTP-Cy5.0 at 3′ end (TdT) and cleaved from the surface in 50 mM DTT/Tris solution pH 8.5 (1 h). Grafting conditions: 130 nM 5′-amino-SS-T2 template, 10 mM EDC/10 mM 1-Methyl-Imidazole (50°C)/1 h. Terminal Deoxynucleotidyl Transferase (TdT), 20 µ/ml, NEB buffer4, CoCl2 (250 mM). Sequencer (Visible Genetics, VG90008), SureFill 6% Sequencing Gel (Visible Genetics, Ref. #VG40006), Stop Loading Dye (Amersham). The upper traces represent DNA size markers labeled with Cy5.5. In the lower trace, the Cy5 labelled oligonucleotide extracted from the surface migrates as a single peak with an approximate apparent size of 87 bases.

Mentions: Another advantage of this direct-labeling approach over PCR-based QC methods is that no artifacts are introduced during the QC from the thermally surface-released DNA and its eventual extension or amplification in the solution phase. TdT labeling can be quite useful and informative for the determination of ratios of grafted template and primers provided, both contain groups that are cleavable under mild conditions, such as disulfides, vicinal diols, RNA linkers or protease motifs. As this method is 10-fold less sensitive than the asymmetric PCR approach (10-fold amplification expected using 10 cycles of linear amplification), it may be used to perform QC on cleavable DNA colonies (Figure 8) or in cases where the surface concentration of DNA template is relatively high. While we were unable to detect a disulfide linked template (462mer), or disulfide 80mer synthetic oligonucleotide (T2) using TdT labeling approach (both having been grafted onto BTA surfaces from 1.25 nM solutions, it was possible to detect the latter template when grafted onto BTA from a higher solution concentration (130 nm) (Figure 4). It is important to note that the TdT-mediated labeling reaction at 37°C takes place at a relatively low temperature, at which practically no thermally induced release of grafted template from the glass surface has been observed (in contrast to PCR thermocycling, which requires temperatures between 60 and 95°C).

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

Fedurco M, Romieu A, Williams S, Lawrence I, Turcatti G - Nucleic Acids Res. (2006)

Bottom Line: This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation.The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm(2) from the amplification of initial single-template DNA molecules immobilized.DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.

Affiliation: Manteia Predictive Medicine S.A. Zone Industrielle Coinsins, CH-1267, Switzerland.

ABSTRACT
The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5'-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm(2) from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.

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