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

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.

Figure 1

(A) Simplified diagram of steps required for glass functionalization with amino (ATS) and carboxyl (BTA) groups. Staining of derivatized glass surfaces using NBD reagents of (B) aminated (ATS) and (C) carboxylated (BTA) glass slides.

Figure 2

Characterization of BTA-derivatized slides (A) Slide staining with NBD. Example of staining of aminosilanized (ATS) slides with (A) 4-fluoro-7-nitrobenzofurazan (NBD-F) and of BTA slides with (7-nitrobenzo-2-oxa-1,3-diazol-4-yl)ethylenediamine (NBD-NH₂), and corresponding negative controls. Fluorescence of NBD-modified slides was measured in air with 5 s acquisition time, using the optical filter xf22 from Omega. (B) Consecutive hybridizations using a complementary TR-labeled probe. Four successive treatments of hybridization-denaturation (1 to 4) of the Texas Red-labeled reverse-P1 primer (500 nM) shown for 5′-amino-10T-P1 primer (34mer) grafted at 1 µM for 60 min. Hybridization conditions: 500 nM Texas Red-labeled oligonucleotide in TMN buffer. Denaturation conditions: three times soaking in 50% formamide (v/v in H₂O) for 5 min at 80°C.

Figure 3

Probe concentration and time dependence for the attachment of aminated oligonucleotides to BTA-derivatized slides monitored by hybridization using a TR-labeled oligonucleotide. (A) Concentration dependence for the grafting of 5′-amino-10T-P₂ primer (34mer) on BTA glass. Grafting conditions: 10 mM EDC/10 mM 1-Methyl-imidazole (50°C)/1 h. Hybridization: 500 nM reverse-P₂-Texas Red in TMN buffer. The solid line is to guide the eye. Background fluorescence is subtracted. (B) Time dependence for the grafting of 5′amino-10T-SS-P₂ primer on BTA glass. Grafting conditions: 1 µM primer, 10 mM EDC/10 mM 1-Methyl-Imidazole (50°C at different incubation times. Hybridization (circles), 500 nM reverse-P₂-Texas Red in TMN buffer. Denaturation (triangle): three times, 5 min at 80°C in 50% formamide (v/v in H₂O). Experimental data points are background subtracted.

Figure 4

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.

Figure 5

Yield of DNA attachment using different cross-linkers between aminated slides (ATS) and modified oligonucleotides. Experiment is performed in one glass channel chip. The channels differ by the cross-linker used: First 3 channels are treated with s-MBS. Channels 4 to 6 are treated with TMA and 7–8 with BTA. The surfaces are then grafted and hybridized with the appropriate DNA material (see text for grafting and hybridization conditions).

Figure 6

Characterization of DNA colonies generated using BTA chemistry by Sybr-Green I staining. Histograms of colonies average intensity (A) Before BbvI digestion colonies are double stranded and their length is 347 bp. (B) After BbvI digestion, colonies are still double stranded and their length is reduced to 43 bp, showing consequently a lower average intensity.

Figure 7

In situ restriction digestion of dsDNA colonies. Analysis of products resulting from in situ enzymatic digestions of solid-phase-amplified dsDNA. Numbers on the right indicate approximate base lengths of BbvI cleavage products harvested from the surface.

Figure 8

Characterization of in situ restriction digestion of dsDNA colonies. (A) Autoradiograms obtained from surface-released products labeled at their 3′ ends with [α-³³P]ddNTP. Digestions were performed for 1 h at various concentrations (indicated) of BbvI. The gel images show DNA cleavage as a function of enzyme concentration (left) and near-quantitative cleavage of the 347-bases substrate to the expected products of 300–304 and 47 bases (right). (B) Electropherograms obtained from surface-released products at different enzymatic reaction times for 0.25 µ/µl BbvI followed by 3′ end labeling with ddNTP-Cy5.