Sequence-specific electrochemical detection of asymmetric PCR amplicons of traditional Chinese medicinal plant DNA.
In this study, an electrochemistry-based approach to detect nucleic acid amplification products of Chinese herbal genes is reported. Using asymmetric polymerase chain reaction and electrochemical techniques, single-stranded target amplicons are produced from trace amounts of DNA sample and sequence-specific electrochemical detection based on the direct hybridization of the crude amplicon mix and immobilized DNA probe can be achieved. Electrochemically active intercalator Hoechst 33258 is bound to the double-stranded duplex formed by the target amplicon hybridized with the 5'-thiol-derivated DNA probe (16-mer) on the gold electrode surface. The electrochemical current signal of the hybridization event is measured by linear sweep voltammetry, the response of which can be used to differentiate the sequence complementarities of the target amplicons. To improve the reproducibility and sensitivity of the current signal, issues such as electrode surface cleaning, probe immobilization, and target hybridization are addressed. Factors affecting hybridization efficiency including the length and binding region of the target amplicon are discussed. Using our approach, differentiation of Chinese herbal species Fritillaria (F. thunbergii and F. cirrhosa) based on the 16-mer unique sequences in the spacer region of the 5S-rRNA is demonstrated. The ability to detect PCR products using a nonoptical electrochemical detection technique is an important step toward the realization of portable biomicrodevices for on-spot bacterial and viral detections.
Lee
Thomas M H
TM
Department of Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon.
Hsing
I-Ming
IM
D015342:DNA Probes; D018744:DNA, Plant / Q000737:chemistry*; D004365:Drugs, Chinese Herbal / Q000032:analysis*; D004563:Electrochemistry; D004586:Electrophoresis; D005292:Ferricyanides / Q000032:analysis; D007202:Indicators and Reagents; D007364:Intercalating Agents; D009841:Oligonucleotides / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015342:DNA Probes; D018744:DNA, Plant; D004365:Drugs, Chinese Herbal; D005292:Ferricyanides; D007202:Indicators and Reagents; D007364:Intercalating Agents; D009841:Oligonucleotides; C007931:hexacyanoferrate III