This application is the national phase under 35 U.S.C. xc2xa7371 of PCT International Application No. PCT/SE98/01017 which has an International filing date of May 28, 1998 which designated the United States of America.
1. Field of the Invention
The present invention relates to a method to perform biochemical reactions and a new reaction vessel for use in said method. The invention is applicable for all small volume biochemical reactions in which the reagents are dispensed from reagent capillaries or so called reagent cartridges through centrifugation. The method and reaction vessel according to the present invention is particularly suitable for use in the PCR (Polymerase Chain Reaction) technique.
2. Prior Art
Biochemical reactions are routinely carried out in so-called microtiter platesxe2x80x94sheets of moulded plastic that typically contain eight rows and 12 columns of tiny wells, each of which capable of holding a few milliliters or less of the reaction mixture. Alternatively, 96 separate, detachable reaction vessels, e.g. micro centrifuge tubes or so called Eppendorf(copyright)xe2x80x94tubes or similar reaction vessels are arranged in the same manner. The 96-well format is widely used and the microtiter plates as such offers many benefits. Apparatuses are developed, which allow automatic and simultaneous handling of several microtiter plates, e.g. the automatic dispensing of reagents using pipetting robots and the simultaneous centrifugation in plate centrifuges.
An alternative to both manual dispensing and automatic dispensing using robots are dispensing devices, e.g. the reagent capillaries described in EP 530 283. In these, the reagents are predispensed in capillaries, separated from each other and from the ambient air, thus preventing untimely mixing and reactions and thus extending storage life. These capillaries are then brought in orientation with the reaction vessels or wells on a microtiter plate and emptied through centrifugation together with the reaction vessels. The commercially available Capilette(copyright) system (from Alphahelix AB, Sweden) exemplifies this application.
One widely used technique is the PCR (Polymerase Chain Reaction)-technique, which has found utility in a number of important diagnostic sectors. The PCR-method mainly comprises the following stages:
1) preparation of the reaction mixtures, i.e. preparation of the samples to be tested;
2) the actual amplification, i.e. the chain reaction in which the DNA molecules are replicated exponentially; and
3) the detection of positive samples by means of electrophoresis or hybridisation.
Using the PCR-technology, there are, however, several disadvantages with the presently available microtiter plates and reaction vessels. The PCR-method remains timeconsuming and work-intensive. The amplification steps, involving repetitive heating and cooling cycles, are carried out in capillaries. Capillaries are chosen to allow for rapid heating and cooling during the amplification cycles. The surface area in relation to the volume favours rapid heat transfer.
Normally such capillaries hold from about 5 xcexcl to about 20 xcexcl. The transfer of the reagent to the capillaries, followed by the necessary sealing of the capillaries, is a time-consuming step, additionally a step prone to contamination and errors. The capillaries are filled with the reaction mixture using the capillary force, i.e. one end of an open capillary is immersed in the reaction mixture. Thereafter one end of the capillary is sealed by melting one end in a gas flame. The capillary, filled with the reaction mixture and sealed at one end, is consequently subjected to the heating and cooling cycles to perform the amplification. For the handling of these capillaries, specific laboratory racks and apparatuses have been developed
The object of the present invention is to simplify the handling of small volume reaction mixtures and to remove at least one source of errors and contamination. The present invention aims in particular to simplify the PCR-procedure. The present invention further aims to achieve the previous goals within the physical limits of the presently used 96 well format.
The present invention now discloses a new reaction vessel and a method to perform biochemical reactions according to the attached claims.
USPTO Backgrounds
USPTO Backgrounds