title: Isothermal DNA amplification coupled with DNA nanosphere-based colorimetric detection.
abstract: We present a simple, rapid method for detecting short DNA sequences that combines a novel isothermal amplification method (EXPAR) with visual, colorimetric readout based on aggregation of DNA-functionalized gold nanospheres. The reaction is initiated by a trigger oligonucleotide, synthetic in nature for this proof-of-principle study, which is exponentially amplified at 55 degrees C and converted to a universal reporter oligonucleotide capable of bridging two sets of DNA-functionalized gold nanospheres. This reaction provides >10(6)-fold amplification/conversion in under 5 min. When combined with a solution containing DNA nanospheres, the bridging reporter causes nanosphere aggregation. The resulting color change from red to dark purple or blue is enhanced through spotting the solution onto a C18 reversed-phase thin-layer chromatography plate. The reaction can easily be adapted for detection of different trigger oligonucleotides using the same set of DNA nanospheres. It permits detection of as low as 100 fM trigger oligonucleotide in under 10 min total assay time, with minimal reagent consumption and requirement for instrumentation. We expect that combining this simple, versatile assay with trigger generation from a genomic target DNA sequence of interest will be a powerful tool in the development of rapid and simple point-of-care molecular diagnostic applications.
authors.affiliation: Keck Graduate Institute of Applied Life Sciences, and Harvey Mudd College, Claremont, California 91711, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Tan
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wong
authors.forename: Doris
authors.initials: D
authors.lastname: Nguyen
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Zhang
authors.forename: Barbara
authors.initials: B
authors.lastname: Erwin
authors.forename: Lori K
authors.initials: LK
authors.lastname: Van Ness
authors.forename: Shenda M
authors.initials: SM
authors.lastname: Baker
authors.forename: David J
authors.initials: DJ
authors.lastname: Galas
authors.forename: Angelika
authors.initials: A
authors.lastname: Niemz
mesh_terms: D001483:Base Sequence; D002855:Chromatography, Thin Layer / Q000379:methods; D003124:Colorimetry / Q000379:methods; D004247:DNA / Q000032:analysis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D043942:Nanotubes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D009838:Oligodeoxyribonucleotides; D004247:DNA