Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification.
BACKGROUND
A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies.


METHODS
The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by "sandwich" assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background.


RESULTS
Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis.


CONCLUSIONS
We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.
Edman
C F
CF
Nanogen, Inc., San Diego, CA 92121, USA. cedman@nanogen.com
Mehta
P
P
Press
R
R
Spargo
C A
CA
Walker
G T
GT
Nerenberg
M
M
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004582:Electronics, Medical; D005165:Factor V / Q000235:genetics; D005784:Gene Amplification; D005798:Genes, Bacterial; D005820:Genetic Testing; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C095381:factor V Leiden; D005165:Factor V