Optimization of the performance of the polymerase chain reaction in silicon-based microstructures.

We have demonstrated the ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures. Optimal design/ processing result in equivalent performance (yield and specificity) for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes. Amplifications in volumes as small as 0.5 microl and thermal cycling times reduced as much as 5-fold from that of conventional systems have been demonstrated for the microstructures.

Quantitation of metallothionein mRNA by RT-PCR and chemiluminescence.

A general procedure has been developed for the determination of mRNA expression by reverse transcription polymerase chain reaction (RT-PCR), over a wide concentration range, with quick quantitation of amplified products by luminescence. The discriminating power of this approach is the specific hybridization of PCR product to ruthenium-labeled oligonucleotide probe(s). This method is sensitive enough to detect increases in the formation of PCR product by the 6th cycle. The accumulation of PCR product was successfully modeled with a recursive relationship. This procedure was capable of accurately determining starting template copies over a 9-log dynamic range, with a sensitivity limit of 10(2) copies. Inclusion of an mRNA internal standard (identical to amplified template except for a 6-bp deletion) corrected variabilities in the reverse transcriptase as well as PCR, allowing for the expression of data as mRNA copy number/micrograms total tissue RNA. This procedure was used to detect changes in levels of winter flounder (Pleuronectes americanus) liver metallothionein mRNA. Liver metallothionein mRNA levels ranged from 1.0 x 10(6) copies/micrograms total tissue RNA in control samples to 1.0 x 10(9) copies/micrograms total tissue RNA in samples treated with Cd (a known metallothionein mRNA inducer).

A high-sensitivity electrochemiluminescence-based detection system for automated PCR product quantitation.

A high-sensitivity nonisotopic system has been developed for post-PCR product detection. The probe-based detection system exploits a chemiluminescent reaction that takes place on the electrode surface in an electrochemical cell. The detection system incorporates a biotin-streptavidin capture reaction onto a solid support that permits fast post-PCR product detection at the attomole level. The system precision is within 5% relative standard deviation over a linear dynamic range of greater than three orders of magnitude. In this paper, the principles and features of the electrochemiluminescent-based detection system, together with its application to PCR product quantitation, are described in detail.

A high-performance system for automation of the polymerase chain reaction.

A high-performance PCR system has been developed which reduces the time required for PCR, increases the throughput, reduces reagent consumption and ensures reproducibility of amplification. Integration of sophisticated temperature control with optimally designed vessels has resulted in an amplification system which produces unique benefits. These include rapid amplification, the elimination of the need for oil, even for small volumes, and a microplate format which provides liquid handling automation benefits.

Time-resolved fluorimetric detection of terbium-labelled deoxyribonucleic acid separated by gel electrophoresis.

Deoxyribonucleic acid (DNA) was reacted with a strong chelating agent and labelled with terbium, yielding a highly fluorescent conjugate with a lifetime of 1.5 ms. When a pulsed source and gated detection electronics were employed, the long-lived decay allowed effective discrimination against background fluorescence and scattered excitation. Detection limits should therefore be significantly improved in comparison with covalent labels with fluorescence lifetimes in the nanosecond regime or stains such as ethidium bromide. The conjugate is very stable, remaining fluorescent on dilution and in an electric field at elevated temperatures (60 degrees C), conditions typically encountered during polyacrylamide gel electrophoresis. As an enhancement solution is not required to develop the fluorescence, this system could be utilised in situations where on-line detection is desirable. Although only DNA was labelled, the method is equally applicable to ribonucleic acid.