Australasian Biotechnology (backfiles) AusBiotech ISSN: 1036-7128 Vol. 6, Num. 5, 1996

Capillary Flow PCR: High Throughput Genetic Analysis

Ken C Reed

Director, Queensland Agricultural Biotechnology Centre, DPI Gehrmann Laboratories, The University of Queensland 4072

Code Number: AU96012
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Summary

A continuous flow approach to thermal cycling is briefly reviewed. This sets the scene for assessment of capillary flow PCR as a tool for high throughput, automated analysis employing in-line laser fluoremetry for detection.

The emphasis in health care will continue to evolve from repair to prophylaxis. The cost of illness treatment is escalating to the point where it will become unsupportable, the result of increasing sophistication in medical technology and an informed, ageing population. Preventive maintenance is inherently a more desirable mode of health care. We see this reflected in growing emphasis on nutrition and exercise, immunisation and "therametrics" (illustrated by quantification of relevant therapeutic parameters in management of chronic diseases, such as viral load in HIV infections).

To date, prophylaxis has concentrated largely on extrinsic factors. Society has been slow to accept the magnitude and impact of genetic variation in predisposing humans to physical and mental ailments. In truth, the data have been slow in coming since technologies to easily measure genetic variation have only recently arrived with the invention of PCR. However, there is still no platform that can process routine genetic analyses on the scale of blood biochemistry automated analytical technologies.

In designing such a platform we took advantage of relativity. Analysis of components in solution depends on subjecting them to sequential change: either the environment can be changed (as in a thermal cycler) on a fixed solution (microtitre plate), or the solution can be moved through fixed environments. The latter approach lends itself more readily to automation (and illustrates the fundamental difference between optimal human activity and optimal robotics - human actions are constrained by anatomy and are path-dependent; automated pathways are determined solely by the end product).

The basic principle of capillary flow PCR is to pump the assay solution through a flexible capillary that passes repeatedly between two or three different temperature zones to achieve the cycle of repeated denaturation-(annealing)-extension (see Figure). Samples are injected into a stream of oil which serves as a continuous flow substrate and separates successive samples. Thermal equilibration is extremely rapid, allowing rigorous control over dwell times so that each amplification cycle usually takes no more than 45 sec (set by path length).

Sample volumes are small (5-10 ul), with resultant economies, and sample injection is easily automated. The system is completely enclosed and so avoids contamination. Every sample is subjected to identical conditions, ensuring excellent quality control.

The different assay conditions required for different target amplifications are met by purpose-designed cassettes.

In this system, analysis of PCR reaction products is done in-line by laser fluorometry for bimodal assays that give a simple yes/no answer. Further development of energy-transfer adducts should allow in-line analysis to be extended to multiplex reactions and allele discrimination, although length variants (microsatellite alleles, DNA sequencing) will continue to require electrophoretic resolution for the foreseeable future.

Capillary flow PCR provides a platform to enable genetic analysis to become a routine clinical test. The demand for genetic diagnostics is not yet strong but community concerns, combined with the enabling outputs of the Human Genome Project and commercial enterprise, will shortly revolutionise our concepts of wellbeing through individualised preventive care.

Legend to Figure.

Two alternative cassette designs for capillary flow PCR. Samples (5-10 ul) are injected sequentially into a continuously flowing stream of oil that carries them through alternating temperature zones. The time for which a sample remains at a set temperature is determined by the diameter of the capillary tubing, the flow rate and the path length in that temperature zone. Separate cassettes are designed for optimal amplification of each PCR target sequence.

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