Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-04-04
2002-08-27
Sisson, B. L. (Department: 1655)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S091100, C435S287200, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06440724
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to a device for preparing samples for detecting a nucleotide sequence by means of polymerase chain reaction.
2. Description of Related Art
U.S. Pat. No. 5,455,175 discloses a so-called thermocycler with which a plurality of liquid biological samples can be exposed repeatedly to a pre-set temperature profile in order to carry out the PCR. To shorten the time required for the temperature treatment, a small volume of each of the biological samples is taken up in a thin-walled glass capillary. To this end, each of the individual samples must be filled into the capillary and then sealed in. This is time-consuming.
DE 33 36 738 A1 discloses a titer plate, a lid of which can only be removed from the support with the expenditure of considerable force. A sample taken up in the known device is difficult to heat externally; the device is unsuitable for carrying out the PCR.
SUMMARY OF THE INVENTION
It is an object of the present invention to overcome the disadvantages of the prior art. In particular, it is intended to provide a method and a device with which the time required for preparing the samples for PCR is reduced. A further aim of the invention is a simplified and improved, in particular real-time, detection and an increase in sensitivity.
In accordance with the invention, there is provided a method for preparing samples for detecting a nucleotide sequence by means of polymerase chain reaction, where
a) an analysis solution is filled into at least one cavity provided on a support,
b) a lid configured complementary to the shape of the cavity is placed onto the support in such a way that at least some of the analysis solution is displaced into a gap formed between the cavity and the lid and
c) the gap is sealed by means of at least one seal provided near an opening of the cavity.
The proposed process considerably shortens the time for sample preparation. The procedure of sealing the sample solution into a capillary is dispensed with.
The analysis solution can have added to it a first and/or a second primer. It is regarded as particularly advantageous to have a third primer, preferably with its 5′-terminal end, bound to the internal face of the lid which extends into the cavity. In this manner, it is possible to bind, to the third primer, a nucleotide sequence which may be present in a sample. This can be achieved in a particularly simple fashion by immersing the internal face of the lid into the sample. After the amplification cycles have been concluded, it is furthermore possible to accumulate the amplified nucleic acid on the internal face by binding it to the third primer. The accumulation is expediently carried out by applying an electrical field. The nucleotide sequence is shifted toward the third primer under the influence of an electrical field.
To detect the presence of the nucleotide sequence of interest, it is expedient to examine the analysis solution and/or one of the primers for their fluorescence properties. Upon binding of the nucleotide sequence to be detected to one of the primers, a change in the fluorogenic properties of the substances present in the analysis solution may take place. Upon binding of the nucleotide sequence to be detected to one of the primers, a spatial relationship between two fluorophoric groups is preferably alterable in such a way that a fluorescence reaction can be generated, altered or quenched.
In a further embodiment, the analysis solution is heated and cooled cyclically. A typical temperature cycle consists of a first heating of the analysis solution to 90 to 92° C., a cooling to 50 to 55° C. and a second heating to 72 to 75° C. During first the fit heating, denaturation takes place, during the cooling, renaturation, and during the second heating the synthesis of the nucleotide sequence. The aforementioned cycle is repeated approximately 30 times.
Heating can be effected by means of light, preferably infra-red radiation, resistance heating or by passing of gas or a fluid around the cavity. Rapid cooling is effected expediently by passing a gas, for example air, or a fluid around the cavity, or by means of a Peltier element.
Furthermore provided in accordance with the invention is a device for detecting a nucleotide sequence which may be present in a sample by means of polymerase chain reaction, where the cavity has a surrounding lateral wall which widends conically toward the opening and the gap has a width of not more than 1 mm.
The proposed device allows time-saving sample preparation. By using it, the time required for carrying out a PCR can be reduced considerably. The lid can be lifted without great expenditure of force after the PCR has been carried out.
It is advantageous to provide a facility for cyclically heating and cooling the analysis solution. Moreover, a facility for examining the fluorescence properties of the analysis solution and/or one of the primers may be provided.
The support can be made of a translucent material, preferably of glass or plastic. The cavity is expediently designed to have planar sections; preferably, it has a flat bottom. On the support and/or on the internal face of the lid, a further seal may be provided at sections located between the cavities or on projections arranged on the internal face of the lid. Like the seal, the further seal can be made of, for example, rubber, silicone, Teflon or other suitable materials.
It is considered to be especially advantageous for the support to have 96 cavities and for the lid 96 projections which are complementary to the shape of the cavities. Thus, for example the support can have approximately the dimension of a conventional 96-well microtiter plate. Naturally, the support may also have a fraction or a multiple of the abovementioned number of cavities.
In accordance with a further embodiment, the lid can be made of an electricroconductive material, preferably a plastic. The support can exhibit an electrode, preferably an electrode made of platinum, so that an electrical field can be applied between the lid and the support, by which nucleotide sequence present in the analysis solution can be shifted to the internal face and accumulated by field-inversion cycles.
The plastic can comprise a polycarbonate, a trimenthylthiopnene, triaminobenzene and/or a polycarbene, and at least sections of the internal face of the lid can be provided with a substance which binds biomolecules. Binding of the nucleotide sequence to the plastic can be mediated here by streptavidin or avidin.
The analysis solution advantageously has a first and/or second primer added to it. It is considered as especially advantageous for a third primer to be bound to the internal face of the lid facing the cavity, preferably with a 5′-terminal end. This allows the amplified nucleotide sequence to be removed from file analysis solution.
In accordance with a further embodiment, a means for exciting fluorescence between the bottom and the internal face of the lid is provided. It is possible that the radiation originating from the excitation means can be focused toward the internal face of the lid. This is advantageous hit particular when the nucleotide sequence is bound to the internal face via the third primer. The means for exciting fluorescence is expediently generated by a laser diode. Thus, it takes the form in this instance of laser light. Excitation of the support bottom can also be achieved by a so-called gally-mode laser (Science 1998, 280, p 1501, 1544 ff.) in a pre-set manner, either simultaneously or successively.
There may furthermore be provided a facility for detecting the fluorescence, a facility for evaluating the fluorescence observed, and a facility for shifting the support relative to the means for exciting the fluorescence and/or to the detection facility. Moreover, there may be provided a facet-eye-like means for separately exciting and/or detecting the fluorescence between each bottom and the internal face of the corresponding lid. This saves yet more analysis time.
Expedient
November Aktiengesellschaft Gesellschaft fur Molekulare Medizin
Rankin, Hill Porter & Clark LLP
Sisson B. L.
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