Antimicrobial resistance and bacterial identification utilizing a microelectronic chip array.

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.

Rapid, high fidelity analysis of simple sequence repeats on an electronically active DNA microchip.

We describe a method for the discrimination of short tandem repeat (STR) alleles based on active microarray hybridization. An essential factor in this method is electronic hybridization of the target DNA, at high stringency, in <5 min. High stringency is critical to avoid slippage of hybrids along repeat tracts at allele-specific test sites in the array. These conditions are attainable only with hybridization kinetics realized by electronic concentration of DNA. A sandwich hybrid is assembled, in which proper base stacking of juxtaposed terminal nucleotides results in a thermodynamically favored complex. The increased stability of this complex relative to non-stacked termini and/or base pair mismatches is used to determine the identification of STR alleles. This method is capable of simultaneous and precise identification of alleles containing different numbers of repeats, as well as mutations within these repeats. Given the throughput capabilities of microarrays our system has the potential to enhance the use of microsatellites in forensic criminology, diagnostics and genetic mapping.

Character displacement in some Cnemidophorus lizards revisited: a phylogenetic analysis.

Ecological studies have demonstrated the role of competition in structuring communities; however, the importance of competition as a vehicle for evolution by natural selection and speciation remains unresolved. Study systems of insular faunas have provided several well known cases where ecological character displacement, coevolution of competitors leading to increased morphological separation, is thought to have occurred (e.g., anoline lizards and geospizine finches). Whiptail lizards (genus Cnemidophorus) from the islands of the Sea of Cortez and the surrounding mainland demonstrate a biogeographic pattern of morphological variation suggestive of character displacement. Two species of Cnemidophorus occur on the Baja peninsula, one relatively large (Cnemidophorus tigris) and one smaller (Cnemidophorus hyperythrus). Oceanic islands in the Sea of Cortez contain only single species, five of six having sizes intermediate to both species found on the Baja peninsula. On mainland Mexico C. hyperythrus is absent, whereas C. tigris is the smaller species in whiptail guilds. Here we construct a phylogeny using nucleotide sequences of the cytochrome b gene to infer the evolutionary history of body size change and historical patterns of colonization in the Cnemidophorus system. The phylogenetic analysis indicates that (i) oceanic islands have been founded at least five times from mainland sources by relatives of either C. tigris or C. hyperythrus, (ii) there have been two separate instances of character relaxation on oceanic islands for C. tigris, and (iii) there has been colonization of the oceanic island Cerralvo with retention of ancestral size for Cnemidophorus ceralbensis, a relative of C. hyperythrus. Finally, the phylogenetic analysis reveals potential cryptic species within mainland populations of C. tigris.

Variation and evolution of class I Mhc in sexual and parthenogenetic geckos.

We present the first Mhc class I sequences in geckos. We compared Mhc variation in gekkonid species that reproduce sexually (Hemidactylus frenatus, Lepidodactylus aureolineatus, L. moestus, L. sp. Arno, L. sp. Takapoto) to others reproducing parthenogenetically (H. garnotii, L. lugubris). These comparisons include the known maternal (L. moestus) and paternal (L. sp. Arno) ancestors of the asexual L. lugubris. Sequences similar to other vertebrate species were obtained from both nuclear and cDNA templates indicating that these sequences are derived from expressed class I Mhc loci. Southern blot analysis using gecko class I probes, revealed that parthenogenetic clonal lineages of independent evolutionary origin have no within-clone band variation at class I loci and that no detectable recombination between restriction sites had taken place. Variability in the sexual species was similar to mammalian taxa, i.e. class I genes are highly variable in outbreeding sexual populations. Sequence analysis of the alpha-2 domain of class I genes identified point mutations in a clonal lineage of L. lugubris which led to amino acid substitutions. Potential transspecific allelic lineages were also observed. The persistence of asexual lineages with little or no class I diversification over thousands of generations seems to argue against strong selection for Mhc multi-allelism caused by pathogen-Mhc allele specificity. On the other hand, the high level of heterozygosity in the parthenogenetic species (a consequence of their hybrid origin) may provide clonal lineages with adequate antigen presenting diversity to survive and compete with sexual relatives.