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/cm2 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.

Second-generation non-invasive high-throughput DNA sequencing technology in the screening of Down's syndrome in advanced maternal age women

“…Illumina (Solexa, San Diego, CA, USA) sequencing was adopted in the non-invasive DNA second-generation high-throughput sequencing using the sequence by synthesis method (6)(7)(8). Briefly, maternal blood (8 ml) was collected in EDTA and anticoagulant tubes.…”

The next-generation sequencing technology and application

“…The adapters on the surface also act as primers for the following PCR amplification. Adding mixtures containing the PCR amplification reagents to the flow cell surface, the DNA fragments are amplified by "bridge PCR" (Adessi, 2000;Fedurco et al, 2006). After several PCR cycles, about 1000 copies of single-stranded DNA fragments are created on the surface, forming a surface-bound colony (the cluster).…”

PCR Advances Towards the Identification of Individual and Mixed Populations of Biotechnology Microbes

“…Bridge PCR (Adessi et al, 2000;Fedurco et al, 2006) involves the use of spatially distributed oligonucleotides that are covalently attached to a support (shown in Fig. 1 C,D).…”