Biomedical subjects

Thomas M H Lee

Publications and source records attributed to Thomas M H Lee.

4 recordsLinked to original sources

PubMed2006

Electrochemical real-time polymerase chain reaction.

In this work, we report the first electrochemistry-based real-time polymerase chain reaction technique for sequence-specific nucleic acid detection. This new technique builds upon the advantages of the well-established fluorescence-based counterpart, such as short assay time (simultaneous target DNA amplification and detection). In addition, this electrochemical approach could employ simple and miniaturizable instrumentation compared to the bulky and expensive optics required in the fluorescence-based schemes. We have demonstrated a proof-of-concept experiment showing that the utilization of solid-phase extension of the electrode surface-immobilized capture probe with Fc-dUTP during PCR resulted in the accumulation of the redox marker on the transducer surface. This new technique can be applied to a microfabricated PCR electrochemical device for point-of-care diagnostics as well as on-site environmental monitoring and biowarfare agent detection.

DNA↗

PubMed2005

Nanocrystal-based bioelectronic coding of single nucleotide polymorphisms.

A bioelectronic method for coding unknown single nucleotide polymorphisms (SNPs) based on the use of different encoding nanocrystals is described. Four such nanocrystals, ZnS, CdS, PbS, and CuS, linked to the adenosine, cytidine, guanosine and thymidine mononucleotides, respectively, are sequentially introduced to the DNA hybrid-coated magnetic-bead solution. Each mutation captures via base pairing different nanocrystal-mononucleotide conjugates, and yields a characteristic multipotential voltammogram, whose peak potentials reflect the identity of the mismatch. The mismatch recognition events are being amplified by the metal accumulation feature of the stripping voltammetric transduction mode. Each of the eight possible one-base mismatches can thus be identified in a single voltammetric run. The use of nanocrystal tracers for detecting two known mutations in a single DNA target is also illustrated in connection to nanocrystals linked to two nucleotides along with a single voltammetric run. The protocol presented should facilitate the rapid, simple, low-cost, and high throughput screening for SNPs.

Base Pair Mismatch↗

PubMed2002

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.

DNA Probes↗

PubMed2002

Genotyping on a complementary metal oxide semiconductor silicon polymerase chain reaction chip with integrated DNA microarray.

A novel method for the fast identification of genetic material utilizing a micro-DNA amplification and analysis device (micro-DAAD) consisting of multiple PCR microreactors with integrated DNA microarrays was developed. The device was fabricated in Si-technology and used for the genotyping of Chinese medicinal plants on the basis of differences in the noncoding region of the 5S-rRNA gene. Successful amplification of the genetic material and the consecutive analysis of the fluorescent-labeled amplicons in the micro-DAAD by the integrated oligonucleotide probes were demonstrated. Parallel analysis was performed by loading the four PCR reactors of the micro-DAAD with different samples of 3-microL volume. Temperature sensors and heating elements of the micro-DAAD enable precise temperature control and fast cycling, allowing the rapid completion of a combined amplification and analysis (hybridization) experiment.

Genotype↗