Enhancement of deoxyribonucleic acid microarray performance using post-hybridization signal amplification.
Microarray performance depends upon the ability to screen samples against a vast array of probes with the appropriate sensitivity and selectivity. While these factors are significantly influenced by probe design, they are also subject to the particular detection methodology and reagents employed. Herein we describe the incorporation of super avidin-biotin system (SABS) and secondary enzymatic enhancement (SEE) as post-hybridization signal amplification techniques to improve the sensitivity of oligonucleotide microarrays. To these ends, we tested these methods on electrochemically interrogated arrays using both purified influenza A PCR products and randomly amplified genomic Francisella tularensis DNA as targets. While SABS treatment did not improve sensitivity for CombiMatrix ElectraSense(®) arrays using purified influenza A cDNA, chip sensitivity was improved 10-fold for randomly amplified targets. SEE improved performance to a greater degree and was able to lower the detection limits 10-fold for influenza A and 100-fold for F. tularensis DNA. These results indicate the promising capability of post-hybridization amplification techniques for enhancing microarray performance.
Wojciechowski
Jason
J
Center for BioMolecular Science and Engineering, Bld. 30, U.S. Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20376, USA. jason.wojciechowski@nrl.navy.mil
Chase-Baldwin
Kitty
K
Wasieloski
Leonard P
LP
Padilla
Susana
S
Vora
Gary J
GJ
Taitt
Chris Rowe
CR
D001360:Avidin / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D005604:Francisella tularensis / Q000235:genetics*; D006735:Horseradish Peroxidase / Q000737:chemistry; D009980:Influenza A virus / Q000235:genetics*; D008839:Microelectrodes; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D015345:Oligonucleotide Probes; C058865:Strep-avidin conjugated horseradish peroxidase; D001360:Avidin; D001710:Biotin; D006735:Horseradish Peroxidase