Detection of oligonucleotide hybridization at femtomolar level and sequence-specific gene analysis of the Arabidopsis thaliana leaf extract with an ultrasensitive surface plasmon resonance spectrometer.
A flow-injection (FI) device is combined, through the use of a low-volume (4 microl) flow cell, with an ultrasensitive surface plasmon resonance (SPR) spectrometer equipped with a bi-cell photodiode detector. The application of this novel FI-SPR device for sequence-specific ultratrace analysis of oligodeoxynucleotides (ODNs) and polydeoxynucleotides was demonstrated. Self-assembled monolayers of ODN probes are tethered onto Au films with a mercaptohexyl group at the 3' ends. The FI-SPR provides a detection level (< or =54 fM) 2-3 orders of magnitude lower than other SPR devices and compares well with several ultrasensitive detection methods for labeled DNA targets (e.g. fluorophore-tagged and radiolabeled DNA samples). The technique is also highly selective, since a 47mer ODN target with a single-base mismatch yielded a much smaller SPR signal, and a specific interaction was detected when the complementary target was present at 0.001% of the total DNA. The FI-SPR was extended to the measurement of two individual genes in a cDNA mixture transcribed from an Arabidopsis thaliana leaf mRNA pool. The greatly enhanced sensitivity not only obviates the necessity of DNA labeling, but also significantly reduces sample consumption, allowing direct quantification of low abundance mRNAs in cellular samples without amplification.
Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032, USA.
Fayi
F
Song
Feimeng
F
Zhou
Jun
J
Wang
Nongjian
N
Tao
Jianqiao
J
Lin
Robert L
RL
Vellanoweth
Yvonne
Y
Morquecho
Janel
J
Wheeler-Laidman
D017360:Arabidopsis / Q000235:genetics*; D060387:Ascorbate Peroxidases; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics; D010544:Peroxidases / Q000235:genetics; D010936:Plant Extracts / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics*; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
D015342:DNA Probes; D018076:DNA, Complementary; D009841:Oligonucleotides; D010936:Plant Extracts; D010544:Peroxidases; D060387:Ascorbate Peroxidases