Multiwell-plates are used for analyzing samples typically with a nucleic acid amplification technique. The purpose of the analysis is the detection (presence or absence of an analyte) and/or the quantification of the concentration of an analyte in samples. In the current invention the analyte is a nucleic acid: RNA or DNA or derivatives there off. The derivatives (Nucleic Acids, NA) mentioned include molecules which are accessible directly or indirectly (e.g. after chemical modification) to a NA amplification method (e.g. DNA-polymerase, Transcriptase, Reverse-Transcriptase, etc.). The target analytes can be e.g. genetic material with biological origin e.g. for genetic testing, in case of infectious diseases the analyte can be nucleic acid material from a virus or bacteria, in case of gene-expression the analytes can be m-RNAs, the analyte can also be methylated DNA.
A variety of tools and techniques have been developed to detect and investigate the structure and function of individual genes and the proteins they express. Such tools include polynucleotide probes, which comprise relatively short, defined sequences of nucleic acids, typically labeled with a radioactive or fluorescent moiety to facilitate detection. Probes may be used in a variety of ways to detect the presence of a polynucleotide sequence, to which the probe binds, in a mixture of genetic material. Nucleic acid sequence analysis is also an important tool in investigating the function of individual genes. Several methods for replicating, or “amplifying,” polynucleic acids are known in the art, notably including polymerase chain reaction (PCR). Indeed, PCR has become a major research tool, with applications including cloning, analysis of genetic expression, DNA sequencing, and genetic mapping.
There are many circumstances in which multiple batch reactions need to be performed such as Genotyping applications. DNA amplifications by means of polymerase chain reaction (PCR) or primer extension is a method routinely used in genotyping, such as SNP (single nucleotide polymorphism) analysis. SNP specific targets are observed via reaction plate from either top or bottom (after a PCR amplification, primer extension or hybridization step) or sample/reagent removed and interpreted via spectroscopy, mass spectroscopy, sequencing or hybridization. These batch reactions can be performed on reaction plates. These reaction plates, in many such applications, are often referred to as microtitre plates. These reaction plates have generally supplied as injection molded, one piece reaction plates having multiple wells formed therein in the form of miniature test tubes.
In general, the purpose of a polymerase chain reaction is to manufacture a large volume of DNA that is identical to an initially supplied small volume of “target” or “seed” DNA. The reaction involves copying the strands of the DNA and then using the copies to generate other copies in subsequent cycles. Each cycle will double the amount of DNA present thereby resulting in an exponential progression in the volume of copies of the target DNA strands present in the reaction mixture. In general, the purpose of PCR is to manufacture a large quantity of DNA which is identical to an initially supplied small quantity of target or seed DNA. The reaction involves copying the strands of the DNA and then using the copies to generate other copies in subsequent cycles.
A typical PCR temperature cycle requires that the reaction mixture be held accurately at each incubation temperature for a prescribed time and that the identical cycle or a similar cycle be repeated many times. For example, a PCR program may start at a sample temperature of 94° C. held for 30 seconds to denature the reaction mixture. Then, the temperature of the reaction mixture is lowered to 37° C. and held for one minute to permit primer hybridization. Next, the temperature of the reaction mixture is raised to a temperature in the range from 50° C. to 72° C. where it is held for two minutes to promote the synthesis of extension products. This completes one cycle. The next PCR cycle then starts by raising the temperature of the reaction mixture to 94° C. again for strand separation of the extension products formed in the previous cycle (denaturation). Typically, the cycle may be repeated 20 to 30 times.
During a typical PCR process, a small quantity of the sample and a solution of reactants, including the target, are deposited in the wells of a microtiter plate. The plate is placed in a thermocycler which operates to cycle the temperature of the contents within the wells, as described above. In particular, the microtiter plate is placed on a metal heating fixture that is shaped to closely conform to the underside of the plate and wells. A heated top plate of the thermocycler then tightly clamps the plate onto the metal heating fixture during the heating and cooling cycles.
For real time polymerase chain reaction (“PCR”) measurements, wells containing assay/sample mixtures need to be tightly sealed to prevent water evaporation during thermocycling. The thermal cycling process may include temperatures that are above the vapor point of the solutions used in the process. This creates vapor that is trapped in the wells of the microtiter plate. The presence of this vapor may cause inaccurate fluorescence or other spectrometric measurements. The trapped vapor may also contain needed reactants, thus causing incomplete reactions during the thermal cycling and may cause inaccurate measurements. Furthermore, vapor pressure may create stresses within the sample wells, causing leaking of the cover. Such leaking can lead to loss of sample and cross contamination between sample wells. Further the vapor can condense on the cover placed over the wells, thus causing both incomplete reactions due to reagents missing in the well and measurement errors in the case of optical detection. In extreme cases even the sample in a well can exsiccate.
The polymerase chain reaction (PCR) technology is a major research tool throughout molecular biology, both academically and in the pharmaceutical industry. The limitations of use of such reactions have historically been the high costs resulting from the cost of reagents (particularly the enzyme) and the relatively high volumes of reagent needed to be used in the injection molded microtitre plates; typical well volumes in prior art devices could be as large as 200 microliters. However, it could be possible to obtain effective results from plates that have smaller well volumes. To date, however, effective reaction plates of well volume down to two microliters and lower have not been readily achieved.
Another problem with the relatively large volume in the prior art devices is that the excess air gap in the wells of such reaction plates causes evaporation and condensation problems that can reduce the efficiency of the reactions. Sometimes, mineral oil will be used on top of the reaction to prevent/stop evaporation/condensation problems (oil capping). However this may give rise to problems of getting rid of the oil after the reaction has gone to completion. In the prior art it therefore has been considered to be desirable to minimize the size of the excessive air gap in the multi-well reaction plates to minimize evaporation or to avoid the need for oil capping.
Another problem of same plates according to the prior art is that the base of the prior art is complex. This makes it difficult to mate to a thermal transfer plate. Therefore, each well will not transfer externally applied heat into the wells of the reaction plate efficiently, thereby making heat dependant reactions less reliable. This results in variations in the heat transferred to the various wells in the reaction plate. It would therefore be desirable to provide a reaction plate that allows heat easily to be transferred into the wells and which transfer is uniform. Use of injection molding would appear not to allow thin enough bases to be reliably formed for such transfer to occur.
Multi-well reaction plates should have a high density of wells, i.e. a large number of wells per surface area. In conventional prior art multiwell-plates, arrays of, for example, 8 by 12 wells and 16 by 24 wells have been provided. This limits each reaction plate to 96 and 384 reactions at a time, respectively. The contemporary standard is 96 or 384 wells per plate. Also there also known reaction plates having more wells, e.g. 1,536 or 3,072, but these plates are presently not yet used in PCR because they do not fulfill the requirements described above. It would be desirable therefore to increase the number of wells at a much reduced reagent volume to allow an increased reaction turnover at reduced costs.
A further use for such reaction plates is in genotyping. Genotyping is a vast, commercial industry. Most genotyping methods require a DNA amplification process. This is also where the majority of process costs occur. By reliably and routinely working with low volumes of reagent and with high throughputs, the cost per reaction could be substantially reduced. However, prior art devices have not achieved this reliably. For this reason, costs of approximately $0.50 per reaction are frequently incurred.
The well known TaqMan™ (Applied Biosystems) biotyping systems, is a government approved systems for GMO (Genetic Modified Organisms), as well as for most large SNP clinical diagnostic markers. The existing TaqMan 7700™ system uses 8 by 12 (96) well reaction plate technology. Each well is at least approximately 200 μl in volume. By using the reaction plates of the present invention, this could be reduced to 2 μl, and less. The current TaqMan 7700™ 96 well plate will not work at these lower sample/reagent volumes due to the high internal volume problems.
The dimensions of multi-well plates are standardized, see e.g. ANSI American National Standards Institute and SBS Society for Bimolecular Sciences, Standard ANSI/SBS 2-2004. The pitch, i.e. the well-to-well step size, is usually 9 mm for plates with 96 wells, 4.5 mm for plates with 384 wells and 2.25 mm for plates with 1,536 wells.
Multi-well plates according to the prior art are described in GB 2369086 A and WO 2005/028109 A2. Further, similar reaction plates are known from the manufactures KBioscience and KBiosystems. These known plates have the common disadvantage that the bottom base of the plates is formed by a rigid plate for which a material has to be used which has a low thermal conductivity. In addition, the cover has to be heated, which is in particular a difficult task upon optical detection of the samples. However, the heating of the cover is required in order to avoid condensation of the liquid sample on the cover.
It should be noted that despite these incentives, no suitable reaction plate device, until now, had been devised.