Miniaturized multi-chamber thermocycler

Details Miniaturized multi-chamber thermocycler Miniaturized multi-chamber thermocycler

1996-12-26

1999-08-17

Sisson, Bradley L.

Chemistry: molecular biology and microbiology

Apparatus

Including measuring or testing

435 911, 422 50, 422 63, C12M 100, C12P 1934, G01N 2100

Patent

active

059393122

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

The present invention relates to a miniaturized multi-chamber thermocycler particularly applicable in polymerase chain reaction methods in which desired DNA sequences are amplified, as well as for carrying out other thermally controlled biochemical and biological molecular processes.
Thermally controlled biochemical and biological molecular processes very often involve procedural steps conducted at different temperatures. Such exposure to varying temperatures is particularly applicable to the polymerase chain reaction.
The polymerase chain reaction (PCR) has been recently developed to amplify definite DNA sequences, and its essential features have been outlined, for example, in "Molekulare Zellbiologie", Walter de Gruyter, Berlin-New York 1994, pg. 256/257' by Darnell, J.; Lodish, H.; Baltimore, D. As noted, PCR requires thermal cycling of mixtures of DNA sequences. To this end, stationary sample treatment devices containing reaction chambers are employed into which the respective samples are introduced and then subjected to periodical heating and cooling, the respectively desired DNA sequences being amplified in accordance with the specifically preselected primers contained in the samples.
Presently, PCR is preferably carried out on a plurality of samples in one-way plastic vessels (microtubes) or in standardized micro-titre plates. The sample volumes used therein range between about 10 and 100 .mu.l (A. Rolfs et al, Clinical Diagnostics and Research, Springer Laboratory, Berlin/Heidelberg, 1992). Recently, C. C. Oste et al., The Polymerase Chain Reaction, Birkhauser, Boston/Basel/Berlin (1993), page 165, reports the use of smaller sample volumes ranging from about 1 to 5 .mu.l.
The above referred microtubes are subjected to a temperature regime of conventional heating and cooling units (Marktubersicht Gentechnologie III, Nachr. Chem. Tech. Lab. 41, 1993, M1). Due to the bulky nature of such typical heating and cooling units, parasitic heat capacities of transmitter, and heating and cooling elements physically limit a reduction in the cycle times, in particular with reduced sample volumes. As much as 20 to 30 seconds is required for the temperature of the samples in the microtubes to reach desired equilibrium. Moreover, in practice, overheating and subcooling cannot be entirely avoided. In addition, one of the greatest problems with a PCR carried out in microtubes is that the temperature gradients within the samples may lead to differences in temperatures up to 10.degree. K. To overcome this drawback, heatable covers have been employed with some effectiveness, however resulting in increased cost of the apparatus.
For purposes of automation of PCR, micro-titre plates predominantly made of heat-proof polycarbonate are used for charging and sample analysis. These behave thermally in a manner similar to the microtubes mentioned hereinbefore, however, they are more advantageous when used in manual or automatic sample charging. Overall, the devices used for these applications are bulky and not easy handle.
The effectiveness of the prior sample chambers is subject to a variety of drawbacks. Therefore, a miniaturized sample chamber has recently been proposed (Northrup et al, DNA Amplification with microfabricated reaction chamber, 7th International Conference on Solid State Sensors and Actuators, Proc. Transducers 1993, pg. 924-26) which permits a four times faster amplification of desired DNA-sequences than prior known arrangements. The sample chamber, taking up to 50 .mu.l sample liquid, is made of a structurized silicon cell with a longitudinal extension in an order of size of 10 mm which, in one sample injection direction, is sealed by a thin diaphragm via which the respective temperature exposure is executed by miniaturized heating elements. Also, with this device, the DNA sequence to be amplified is inserted via micro-channels into the cell, subjected to a polymerase chain reaction and subsequently drawn off. Notwithstanding the advantages obtained with said device, the reaction

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Clinical Chemistry, vol. 40, No. 9, Sep. 1, 1994, pp. 1815-1818, XP000444699 Wilding P et al.: "PRC in a Silicon Microstructure" see p. 1815, right-hand column, paragraph 4 -p. 1817, right-hand column, paragraph 1.
"Molekulare Zellbiologie", Walter de Gruyter, Berlin-New York 1994, pp. 256-257 by Darnell, J.; Lodish, H.; Baltimore, D.
A. Rolfs et al., "PCR: Clinical Diagnostics and Research", p. 29-31, Springer Laboratory, Berlin/Heidelberg, 1992.
C.C. Oste et al. "The Polymerase Chain Reaction", Birkhauser, Boston/Basel/Berlin (1993), p. 165.
Marktubersicht Gentechnologie III, Nachr. Chem. Tech. Lab. 41, 1993, M2, M4, M5 and M6.
Northrup et al. "DNA Amplification With A Microfabricated Reaction Chamber", The 7th International Conference on Solid State Sensors and Actuators, Proc. Transducers 1993, pp. 924-926.

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