RESUMEN
A microarray method for bacterial species identification based on cpn60 and 16S rDNA hybridization was developed. Specific cpn60 or 16S rDNA oligonucleotides from various Helicobacter or Campylobacter species were printed and immobilized onto a proprietary plastic solid support. Using universal primers, fragments derived from either cpn60 or 16S rDNA genes from single isolates or from a complex human waste sludge DNA sample spiked with Helicobacter pylori were biotinylated and hybridized to the plastic slide. Subsequent querying with a streptavidin-horseradish peroxidase conjugate followed by color development using tetramethylbenzidine resulted in accurate Helicobacter species identification with no cross-hybridization to either the 16S rDNA or the cpn60 sequence of a closely related strain of Campylobacter jejuni. The combination of a nonfluorescence visual detection system with a polymer-based DNA microarray slide has resulted in a molecular tool that should prove useful in numerous applications requiring rapid, low-cost bacterial species identification.
Asunto(s)
Chaperonina 60/genética , ADN Bacteriano/genética , ADN Ribosómico/genética , Helicobacter/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Ribosómico 16S/genética , Campylobacter jejuni/genética , Chaperonina 60/análisis , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Hibridación Genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Ribosómico 16S/análisis , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
Carbohydrate-carbohydrate interactions between Gg3 trisaccharide-carrying polystyrene (PN(Gg3)) and monolayers of several glycosphingolipids (GSLs) were quantitatively investigated by surface plasmon resonance techniques. PN(Gg3) was adsorbed onto a GM3 monolayer strongly and specifically with an apparent affinity constant of K(a) = 2.5 x 10(6) M(-1), and the apparent affinity constants onto GSLs decreased in the following order: GM3 > LacCer > (KDN)GM3 approximately GlcCer > GM2 approximately GD3 approximately GM4 > GM1 approximately 2,6-isoGM3 > ceramide. These results suggest that PN(Gg3) recognizes not only some specified portions of GM3 but also the trisaccharide as a whole. On the other hand, PN(Lac) and PN(Cel) were bound to GSLs less strongly (K(a) approximately 10(4) M(-1)) and less selectively. The kinetic analysis revealed that the selectivity in the adsorption of PN(Gg3) onto the GM3 monolayer is dominated by the faster adsorption rate.
Asunto(s)
Carbohidratos/química , Gangliósidos/química , Poliestirenos/química , Resonancia por Plasmón de Superficie/métodos , Secuencia de Carbohidratos , Datos de Secuencia MolecularRESUMEN
Oligonucleotide-based DNA microarrays are becoming increasingly useful tools for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Here, we present a method that permits the manufacture of microarrays from non-modified oligonucleotides on a poly carbodiimide-coated glass surface by UV-irradiation. The use of UV-irradiation facilitates an increase in the level of signal intensity, but it does not affect signal discrimination by the oligonucleotides immobilized on the surface. The signal intensity obtained for an array fabricated using non-modified oligonucleotides with UV-irradiation is approximately 7-fold greater than that without UV-irradiation. The detection of SNPs was tested to ascertain whether this technique could discriminate specific hybridization signals without causing significant UV-irradiation-induced damage to the immobilized oligonucleotides. We found that this immobilization method provides greater hybridization signals and a better match/mismatch ratio of SNPs than do the established aminosilane techniques. Application of this technology to manufacturing DNA microarrays for sequence analysis is discussed.