Containment cuvette for PCR and method of use

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FIELD OF THE INVENTION

This invention relates to cuvettes in which reactions are undertaken to amplify and detect nucleic acids, using PCR technology, without exposing the environment to amplified nucleic acid.

BACKGROUND OF THE INVENTION

Polymerase chain reaction (PCR) technology is only one of several techniques that permit nucleic acid material, such as DNA, often extracted from as little as a single cell, to be amplified to hundreds of millions of copies. This is important since prior to PCR technology it was virtually impossible to detect a single DNA strand. However, when a single DNA strand, such as the DNA of the human immunodeficiency virus (HIV, otherwise known to cause AIDS), is added to amplifying reagents that will amplify the DNA of choice, hundreds of millions of copies of that DNA can be obtained in a relatively short time. Technology further allows for the detection of the amplified nucleic acid material (DNA for example), using probes that hybridize to the amplified material of choice, such probes in turn either being immobilized or immobilizable to a solid support, such as a filter membrane, and/or being labeled for detection using enzymes or other moieties.

Conventionally, this has been done by amplifying the nucleic acid material in a stoppered plastic container until the desired number of copies have been formed. Thereafter, the container is reopened, such as by unstoppering, and either the amplified copies are withdrawn and transferred to detection apparatus, or detecting reagents can be added to the container used for the amplification, so that detection is done in the same container.

It has been discovered that such a technique is unsatisfactory for convenient and widespread use of, e.g., PCR technology, because aerosols are produced in the act of unstoppering and/or transfer of fluids. Such aerosols contain a few of the amplified nucleic acid material, e.g., DNA. The aerosols then proceed to disperse within the environment. Normally, such few molecules in the environment are not of great concern. However, only one DNA molecule is needed to ruin by contamination other amplifying containers yet to be used for detection. That is, if the errant DNA molecule floats into or is carried, inadvertently, by an operator to another amplifying container yet to be used, that one molecule is all that is needed to provide the DNA needed for the next amplification. Needless to say, if the point of the next test is to see if a particular DNA is present (e.g., for HIV), and it is detected only because of the errant DNA and not that of the patient, the test is ruined. Thus, the very power of DNA amplification becomes the source of potential ruin of the tests. As a matter of fact, an entire lab has been proven to be contaminated by the unstoppering of just a few containers in which the sample has already been amplified. Although such a problem might be avoidable by using highly skilled and trained personnel who painstakingly minimize the aerosols produced, the need for such labor makes the technology impractical for general use.

Thus, it has been a problem prior to this invention to provide apparatus and a method for amplifying and detecting nucleic acid material, without contaminating the surrounding environment.

Yet another problem has been, prior to this invention, to automate the detection steps, that is, minimize the need for operator intervention. The need to transfer amplified nucleic acid material or to add detection reagents makes such automation difficult.

SUMMARY OF THE INVENTION

The above problems are addressed by an apparatus and a method that solve the above-mentioned needs. The invention is based upon the realization that the contamination can be prevented by confining the amplifying reagents and amplified nucleic acid in the cuvette so that it is impossible for any amplified nucleic acid molecules to escape.

More specifically, in accord with one aspect of the invention, there is provided a cuvette for the amplification and detection of DNA, the cuvette including a plurality of compartments including a) means for allowing DNA amplification, the allowing means including a reaction compartment and means adjacent to the reaction compartment for permitting active or passive cycling of the contents of the reaction compartment through a temperature range of from about 30.degree. C. to about 95.degree. C.; b) means for providing liquid interconnection between the compartments by pressurizing the liquid; and c) means for trapping and holding DNA at a detection site for detection, including a detection material capable of generating a detectable signal. The cuvette is improved in that some of the compartments contain the detection material and the reagent in unreacted form in storage, while the cuvette is free of DNA sample, whereby the cuvette need not be reopened between DNA amplification and detection.

In accord with another aspect of the invention, there is provided a closed, disposable cuvette for carrying out amplification and detection of nucleic acid material, comprising: a plurality of compartments including a reaction compartment, means permitting active or passive cycling of the contents of the reaction compartment through a temperature range; at least one detection material being present in at least one of the compartments; and means for fluidly interconnecting the compartments in prescribed order when pressure is applied to the contents of a compartment. The cuvette is improved in that the compartments all are closed to fluid flow to locations outside of the container and said reaction compartment contains nucleic acid material and unreacted amplifying reagents; at least one of the compartments including means at a detection site therein for immobilizing the nucleic acid material for detection after amplification, so that detection of amplified nucleic acid material occurs without contamination of other containers or apparatus by the amplified nucleic acid material. The result is that detection of amplified nucleic acid material occurs without contamination of other containers or apparatus by the amplified nucleic acid material.

In accordance with still another aspect of the invention, there is provided a closed cuvette as described in the previous paragraph, wherein the reagent contents of the reaction compartment comprise polymerase enzyme, primer nucleic acids and deoxyribonucleotides.

In accord with yet another aspect of the invention, there is provided an apparatus for amplifying and detecting DNA, comprising a cuvette containing i) a plurality of compartments and means for interconnecting each of them to at least one other compartment, the compartments including a) at least one reaction compartment for amplifying DNA strands, b) at least one detection compartment for detecting amplified DNA and including a detection site, and c) means for delivering a detection material to amplified DNA strands; ii) means for permitting active or passive cycling of the contents of the reaction compartment through a temperature range; and iii) liquid access means connected only to the at least one reaction compartment for allowing the injection into the reaction compartment of a sample DNA for amplifying; characterized in that the cuvette further includes iv) means sealing the cuvette against passage of DNA after sample DNA is injected; and the apparatus further includes means for moving at least the detection material and a DNA strand into the detection compartment and onto the detection site; so that once a DNA sample is injected into the compartments and the access aperture is closed, the fluid contents of the compartments are contained against contact by the operator and environment during the entire amplification and detection reaction.

In still another aspect of the invention, there is provided a method for amplifying and detecting nucleic acid material in a closed cuvette without allowing aerosols to exit therefrom to contaminate the environment, the method comprising the steps of a) injecting a sample of nucleic acid material into a cuvette comprising a plurality of compartments including a reaction compartment wherein amplifying reagents are present, and a storage compartment for use with a detection material, at least one of the compartments including a detection site, and means for interconnecting the compartments to provide fluid transfer; b) closing off permanently the portions of the cuvette containing the nucleic acid material to lock all nucleic acid into the cuvette; c) amplifying the nucleic acid material by cycling the cuvette through temperature changes preselected to cause the reagents to be effective; d) fluidly transferring amplified nucleic acid material from the reaction chamber to the detection site; e) fluidly transferring detection material to the detection site while keeping the cuvette closed; and f) detecting the amplified nucleic acid material at the detection site with the detection material, all while the nucleic acid material remains confined within the cuvette.

It is an advantageous feature of the invention that a cuvette is provided for amplifying nucleic acids that avoids the risk of contaminating the environment with amplified nucleic acid since it avoids reopening the area of the cuvette containing such nucleic acid.

It is a related advantageous feature of the invention that a cuvette is provided that can be used for such amplification by relatively unskilled labor.

It is another advantageous feature of the invention that such a cuvette is provided that is amenable to automated processing.

Other advantageous features will become apparent upon reference to the detailed description that follows, and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of a cuvette constructed in accordance with the invention;

FIG. 2 is a section view taken generally along the line II--II of FIG. 1;

FIG. 3 is a section view taken generally along the line of III--III of FIG. 1;

FIG. 4 is a fragmentary section view taken along the line of IV--IV of FIG. 1, but without the pipette;

FIG. 5 is an enlarged, fragmentary section view taken along the line V--V of FIG. 1;

FIG. 6 is a fragmentary plan view similar to that of FIG. 1, but illustrating an alternate embodiment;

FIG. 7 is a plan view similar to that of FIG. 1, but illustrating an alternate embodiment;

FIG. 8 is a section view taken along the line VIII--VIII of FIG. 7;

FIG. 9 is a partially sectioned plan view similar to that of FIG. 1, but illustrating an alternate embodiment, the section plane being generally taken along the line IX--IX of FIG. 11;

FIGS. 10, 11, and 12 are section views taken generally along the lines X--X, XI--XI, XII--XII, respectively, of FIG. 9;

FIG. 13 is a partially sectioned plan view similar to that of FIG. 9, but illustrating yet another embodiment;

FIGS. 14 and 15 are fragmentary plan views partially in section, similar to FIG. 9 but illustrating alternate embodiments;

FIG. 16 is a section view taken generally along the line XVI--XVI of FIG. 15;

FIG. 17 is a fragmentary plan view partially in section, similar to FIG. 9 and illustrating still another embodiment;

FIG. 18 is a section view taken generally along the line XVIII--XVIII of FIG. 17; and

FIG. 19 is a section view similar to that of FIG. 5, but illustrating an alternate embodiment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is hereinafter described primarily with respect to the use of PCR technology to amplify and detect DNA, using particular preferred cuvette configurations. In addition, it is useful with any method of nucleic acid amplification, to amplify nucleic acid from any source, in any cuvette, so long as the apparatus and method prevent amplified nucleic acid from exiting the cuvette in any form. The nucleic acid can be obtained, for example, from plasmids or cloned DNA or RNA, or from natural DNA or RNA from any source, including bacteria, yeast, viruses, cells infected by viruses or bacteria, plants or animals. DNA or RNA may be extracted from blood or tissue materials. Another method of amplification called transcription-based amplification and which is different from PCR, that can benefit from the containment cuvette of this invention, is described in Proc. Natl. Acad. Sci. USA, Volume 86, page 1173-1177, February, 1989 (Biochemistry).

PCR TECHNOLOGY

Nucleic acid amplification generally proceeds via a particular protocol. One useful protocol is that set forth in U.S. Pat. No. 4,683,195. Briefly, that protocol features, in the case of DNA amplification, the steps of:

1) Obtaining a sample suspected of containing at least one specific nucleic acid sequence of interest;

2) Denaturing the sample to separate the strands;

3) Contacting the sample with primers, an extension enzyme such as polymerase and other amplification components useful to replicate the nucleic acid;

4) Repeating steps #2 and #3 as many times as necessary; and

5) Detecting the amplified DNA.

A preferred protocol within this class is as follows:

1) A complete DNA double helix is optionally chemically excised, using an appropriate restriction enzyme(s), to isolate the region of interest.

2) A solution of the isolated nucleic acid portion (here, DNA) and nucleotides is heated to and maintained at 92.degree.-95.degree. C. for a length of time, e.g., no more than about 10 minutes, to denature the two nucleic acid strands; i.e., cause them to unwind and separate and form a template.

3) The solution is then cooled through a 30.degree. C.-60.degree. C. zone, to cause a primer to anneal or "attach" to each of the two template strands. To make sure this happens, the solution is held at an appropriate temperature, such as about 55.degree. C. for about 15 seconds, in an "incubation" zone.

4) The solution is then heated to and held at about 70.degree. C., to cause an extension enzyme, preferably a thermostable polymerase enzyme, to extend the primers bound to the template strands by using the deoxyribonucleotides that are present.

5) The completed new pair of strands is heated to 92.degree.-95.degree. C. again, for about 10-15 seconds, to cause this pair to separate.

6) Steps 3)-5) are then repeated a number of times until the appropriate number of strands are obtained. The more repetitions, the greater the number of multiples of the nucleic acid (here, DNA) that is produced. Preferably the desired concentration of nucleic acid is reached in a minimum amount of time, wherein each cycle takes less than one minute. However, as much as five minutes can be used for one cycle.

As used herein, the term "primer" refers to an oligonucleotide, whether naturally occurring or synthetically produced, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced. Such conditions include the presence of nucleotides (such as the four standard deoxyribonucleotide triphosphates) and an agent for polymerization such as a DNA polymerase, and suitable temperature and pH. Generally, each primer used in this invention will have from 15 to 40 nucleotides, and preferably, it has from 20 to 25 nucleotides.

All of this is preferably done in a cuvette, using a cycling of temperature between about 30.degree. C. and about 95.degree. C. The cuvette of the present invention provides a practical approach to allowing PCR technology to be practiced routinely by technicians and those of lesser skills, in an accurate fashion. For a complete understanding of the invention, further details of the PCR technology as it is practiced with this invention will be enumerated first.

Any DNA can be selectively replicated hundreds of millions of times. Selection of the appropriate primer nucleic acid strands insures that, under the best conditions, primarily the DNA of choice will replicate. Preferably, all primers are biotinylated when incorporated into the cuvette, to allow detection to proceed as described hereinafter. Heating of the target DNA now attached to a primer, in the presence of an extension enzyme, produces a double strand that includes a copy of the DNA of choice. The new pair so formed is then separated by very short periods of high temperature denaturing, and the process repeated. This is all done in one reaction compartment by insuring that the primers, deoxyribonucleotides and extension enzymes are present when the sample is added, either as pre-incorporated reagents or reagents that are added with the DNA. If pre-incorporated, the reagents can be applied by spraying and drying, and can include a polymerase, salts, buffers, stabilizers, and the nucleotides needed for replication.

The polymerase enzyme is useful regardless of its sou