We have developed a method for anchored amplification on a microchip array that allows amplification and detection of multiple targets in an open format. Electronic anchoring of sets of amplification primers in distinct areas on the microchip permitted primer-primer interactions to be reduced and distinct zones of amplification created, thereby increasing the efficiency of the multiplex amplification reactions. We found strand displacement amplification (SDA) to be ideal for use in our microelectronic chip system because of the isothermal nature of the assay, which provides a rapid amplification system readily compatible with simple instrumentation. Anchored SDA supported multiplex DNA or RNA amplification without decreases in amplification efficiency. This microelectronic chip-based amplification system allows multiplexed amplification and detection to be performed on the same platform, streamlining development of any nucleic acid-based assay.

Species-specific bacterial identification of clinical specimens is often limited to a few species due to the difficulty of performing multiplex reactions. In addition, discrimination of amplicons is time-consuming and laborious, consisting of gel electrophoresis, probe hybridization, or sequencing technology. In order to simplify the process of bacterial identification, we combined anchored in situ amplification on a microelectronic chip array with discrimination and detection on the same platform. Here, we describe the simultaneous amplification and discrimination of six gene sequences which are representative of different bacterial identification assays: Escherichia coli gyrA, Salmonella gyrA, Campylobacter gyrA, E. coli parC, Staphylococcus mecA, and Chlamydia cryptic plasmid. The assay can detect both plasmid and transposon genes and can also discriminate strains carrying antibiotic resistance single-nucleotide polymorphism mutations. Finally, the assay is similarly capable of discriminating between bacterial species through reporter-specific discrimination and allele-specific amplification. Anchored strand displacement amplification allows multiplex amplification and complex genotype discrimination on the same platform. This assay simplifies the bacterial identification process greatly, allowing molecular biology techniques to be performed with minimal processing of samples and practical experience.

Oligonucleotides which form triple helical complexes on double-stranded DNA have been previously reported to selectively inhibit transcription both in vitro and in vivo by physically blocking RNA polymerase or transcription factor access to the DNA template. Here we show that a 16mer oligonucleotide, which forms triple helix DNA by binding to a 16 bp homopurine segment, alters the formation of histone-DNA contacts during in vitro nucleosome reconstitution. This effect was DNA sequence-specific and required the oligonucleotide to be present during in vitro nucleosome reconstitution. Binding of the triple helix oligonucleotide on a 199 bp mouse mammary tumour virus promoter DNA fragment with a centrally located triplex DNA resulted in interruption of histone-DNA contacts flanking the triplex DNA segment. When nucleosome reconstitution is carried out on a longer, 279 bp DNA fragment with an asymmetrically located triplex site, nucleosome formation occurred at the border of the triple helical DNA. In this case the triplex DNA functioned as a nucleosome barrier. We conclude that triplex DNA cannot be accommodated within a nucleosome context and thus may be used to site-specifically manipulate nucleosome organization.