Device for processing biological specimens for analysis of nucleic acids

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This application is the national filing of PCT application No. PCT/US90/06768 filed Nov. 16,1990, claiming priority of U.S. patent application Ser. No. 07/438,592 filed Nov. 17, 1989, now U.S. Pat. No. 5,188,963.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method and apparatus, including component devices, for use in automating the detection of target nucleic acid sequences in biological-containing samples. The method involves a sequence of physical and chemical reactions, and more particularly to a system for the exposure of, amplification of, and labelled-probe coupling to, a specific, known nucleic acid sequence. The process of the invention consists of the following stages: 1) matrix dispensing, sample mixing and DNA immobilization; 2) preparing DNA; 3) amplifying DNA target sequences; 4) hybridizing a labeled probe to the target; and 5) scanning the matrices for signal produced by bound label.

The invention is especially suited to the automated detection of single, specific genetic sequences present at random in multiple samples containing biological material without labor-intensive DNA extraction and purification procedures being performed separately on each sample. The ability to detect single copies of a specific nucleic acid in biological or environmental samples makes this process revolutionary.

2. Description of the Related Art

There is no laboratory apparatus or equipment currently on the market that automates DNA preparation, modification and detection in one, unattended operation. The apparatus and devices described herein embody an automated process, including a fluid-delivery system and a thermal reaction chamber.

Devices for receiving biological specimens for diagnostic purposes are varied and adapted to the methods of detection. The devices may take the form of tubes for liquid specimens, flat surfaces such as glass slides suitable for microscopy, microtiter dishes, Petri dishes and cubes containing growth medium, or filters made of various materials to which cell and viral components will adhere.

These specimen samples are then treated in such a way as to indicate either the presence or absence, or quantity, of a specific biological entity. Test reagents may either be preapplied to the device or added in series after the specimen is present. Test results are read manually by a technical person or automatically with instrumentation specifically designed for that assay. In some instances the specimen is diluted with a diluent, or an aliquot of the specimen is removed from the original collecting device and transferred to another vessel at some point in the assay. In some cases physical and chemical means are used to amplify the signal of the assay for greater sensitivity. Some assays require extraction or separation to isolate a specific component from other parts.

In DNA-based diagnostics the sequence specificity of base-pairing or enzymatic or other types of cleavage is exploited. The linear sequence of nucleotides in double-stranded DNA molecules forms the basis of replication of the genetic code. Hybridization is the binding of two single-stranded DNA strands whose base-pairing sequences are complementary. Temperature and salt concentration affect the stringency of these base-pairing matches. A change from high stringency to low stringency can cause the same DNA probe to be either exquisitely specific to detect a particular target or less specific and detect a group of related targets.

In some instances the sizes of DNA fragments, produced by restriction endonuclease digestion or by amplification of a target sequences between primer pairs, are used to make a DNA-print for individual identification or aid in diagnosis of a genetic disease, cancer or infectious disease. For example, electrophoresis may be used to size-fractionate different-sized nucleic acids which have been specifically cleaved or whose native length puts them in a distinguishable size-length class.

In the electrophoresis method, a current is applied to DNA loaded at the cathodal end of a gel matrix, which causes the DNA to migrate towards the anodal end of the matrix. The electrophoretic mobility of DNA is dependent on fragment size and is fairly independent of base composition or sequence. Resolution of one size class from another is better than 0.5% of fragment size (Sealy P. G. and E. M. Southern. 1982. Gel electrophoresis of DNA, p. 39-76. In D. Rickwood and B. D. Hames (EDS.), Gel Electrophoresis of Nucleic Acids. IRL Press, London). This reference and all other publications or patents cited herein are hereby incorporated by reference.

Electrophoresis methods thus require a vessel to hold the matrix material and the biological specimens to be subjected to electrophoresis. Such vessels may mold the gel matrix during its formation and may hold it during processing.

Diffusion of reagents is faster where the ratio of the matrix surface area to matrix volume is greatest as in thin, flat matrices. Likewise, electrophoresis of macromolecules requires less voltage and is faster in ultra-thin matrices or tiny (glass) capillaries. In these aqueous matrices, the vessel is necessary to prevent evaporation and to add strength in handling. Existing vessels that enclose matrices impede rapid diffusion of reagents and molecular probes. Once the existing vessels are taken apart in processing, they cannot be put back together to continue automated processing.

Accordingly, the invention aims to provide a system for automated gene identification of multiple samples, which prepares nucleic acids in the samples for testing, sufficiently amplifies target nucleic acid sequences and accurately detects their presence or absence in the samples.

Another object of the invention is to provide a carrier to contain specimens and be used as the sole vessel for completion of all steps of an assay, including sample preparation, electrophoresis, amplification and hybridization.

Yet another object of the invention is to provide support of the matrix and specimen, molding the matrix and embedding the specimen in it for automated processing.

A further object of the invention is to provide such a system which is adaptable to dispensing different quantities of different reagents for saturating specimens quickly with a series of solutions automatically.

A further object of the invention is to provide such a system wherein airflow and heating regulate and monitor temperature and humidity in the matrices including drying them.

A further object of the invention is to provide a system which can accommodate partial capacity loads, i. e., fewer matrices per run, or that can accommodate more than one probe per run.

A further object of the invention is to provide an automatic process and apparatus allowing identification of nucleic acid sequences that have been embedded or fractionated in a matrix whether or not prior extraction or purification of DNA has been performed in the invention.

A further object of the invention is to carry the specimen in transport from the point of collection to the processing point.

A further object of the invention is to provide a convenient way to make the particles containing target nucleic acids of a specimen in a matrix available and sufficiently spread for signal detection in a two-dimensional array.

A further object of the invention is to concentrate specimen nucleic acids, or amplified products thereof, for detection of their presence.

A further object of the invention is provide a barrier to evaporation of solutions during processing.

A further object of the invention is a mechanism to change configuration of the carriers during processing of the specimen to adapt to processing conditions.

A further objective of the invention is to provide support for reading the test results.

A still further object of the invention is to permanently store the nucleic acids present in the specimen for possible retesting and serve as a permanent record of the test, if an archival record is desired.

Other objects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.

SUMMARY OF THE INVENTION

The process and invention in this patent utilizes the fundamental methodology from several state-of-the-art techniques to automate nucleic acid detection directly from biological material. The direct detection is automated by immobilizing the nucleic acids of each sample in a semi-solid matrix for DNA preparation, amplification and hybridization. The frequency of the target sequence in the sample can be determined by measuring hybridization of the label to the single gene targets in situ.

In addition, the apparatus of the invention may be used to process gels of other known techniques in a new way and to automate these techniques or increase their sensitivity.

In a broad aspect, the component device of the invention comprises:

a top piece and a bottom piece, said bottom piece having a matrix holding area, said top piece having a closed position;

said top piece and said bottom piece hinged together along a first side of said bottom piece, said top piece having a first area that extends beyond said first side, whereby force on either piece causes the top piece to hingedly move away from the bottom piece and upward from the closed position to an open position; and

said bottom piece having an overlap area on a second side of said bottom piece, said overlap area extending beyond said top piece, said overlap area having a fluid receiving depression, whereby fluid added to said fluid receiving depression may diffuse into and over matrix material placed in said matrix holding area.

The bottom piece and top piece are preferably parallel to each other except where the matrix is not of a uniform thickness.

In more detailed aspects of the device of the invention, the "first side" of the bottom piece may be an end or a long side of the preferably elongated bottom piece. Thus, in a first embodiment of the carrier device of the invention the top piece is hinged to the bottom piece along a short edge of said bottom piece and towards the short edge of the top piece, and said first side is opposite and parallel to said second side.

In a second embodiment of the device invention, the top piece is hinged to the bottom piece along a side edge of said bottom piece and said top piece, and said first side is perpendicular to said second side.

In the device of the invention, multiple carriers are housed in a reaction chamber through which reagents, solutions, enzymes and nucleotide primers and probes required for identification in this system are circulated. The carriers are stacked, preferably in a horizontal plane, and remain relatively stationary. Fluids move through the matrices and the spaces between them and the carrier covers. The liquid buffers and washes are delivered into the reaction chamber, and gravity flow through the thin matrices and dehydration/rehydration of the matrices facilitate and control diffusion. This approach contrasts to the way that dried agarose gels or solid-support systems such as filters are agitated in hybridization solutions.

The method of the invention utilizes the component device of the invention. The DNA present in the sample, that has been introduced into an individual matrix in a carrier, remains anchored in the corresponding matrix and is separated from the other cellular particles or sample debris by lysing solutions and thorough washing. Several volumes of wash buffer are diffused through the matrix to clear away biological molecules (except nucleic acids, which are immobilized because of the nature of their structure) and also matrix contaminants (for example, sulfonated groups found in agarose) that might interfere with subsequent enzymatic activity. The wash solution also normalizes pH. The matrices are dehydrated by a drying cycle. The sample to be analyzed for the presence of a particular DNA component (or RNA or polypeptide moiety) is thus suspended in matrix material placed in the matrix holding area of the device.

Any one or more of the following steps may then be performed on the matrix and suspended sample, depending on the sample and the results desired: (a) removal of undesired components, e.g. cell wall material, proteins, etc., (b) denaturing the DNA in situ, (c) amplifying a desired nucleic acid component in the matrix material; (d) applying an electric current to the matrix material; and (e) hybridizing a labeled probe to a desired component. Subsequent steps known in the art may be used to detect the particular component in the matrix, or the component as amplified and/or labeled in the matrix.

The device of this invention facilitates automation of DNA-based diagnostics and genetic surveillance and detection. Although the discussion and examples herein are directed primarily to DNA analysis, it is clear that the device of the invention may be used with RNA with equal facility. The device of the invention serves as the specimen container. It can also serve as a mold for embedding a specimen in its matrix. It serves as a specimen holder for manual and mechanical handling and transport. The device serves as an individual archival record for each sample specimen. The sample nucleic acids are preserved in such a way that they may be tested more than once, or the sample may be analyzed for the presence of other nucleic acid targets.

Its parts are configured to open and close via a hinge connection. The closing mechanism (not shown), which is incorporated into the automated instrument, may open and close the hinged parts. The invention may also be opened and closed manually.

One way the invention is different from other diagnostics is that in the invention nucleic acids in specimens may be dispersed randomly in the matrix, and detected as individual targets in the specimen. The significance of this two-dimensional format is that target nucleic acids in spread or dispersed cells or viral particles are enumerated in order to quantify the number of cells or viral particles containing the suspected target DNA. A given degree of amplification of target DNA in a matrix will distinguish locations that represent a few copies of original target from many copies of target. The difference in amplitude of these signals, and construction of a total signal by summing individual signals, reflects a more accurate quantitative answer for each specimen as opposed to measuring a single amplitude for total signal of each specimen. In addition to improving measurement of signals over background noise, the method is useful to distinguish individual particles/cells having a few copies of a target DNA from those with many copies. This information can be predictive (1) in cancer when in vivo gene amplification means a more aggressive malignancy or (2) in viral infections to distinguish latent from active infection.

DNA sequences are excellent molecular probes because of the complementarity of primer and probe sequences to target DNA for the purpose of amplification and hybridization. Similarly the recognition sites of restriction endonucleases are DNA-sequence specific. Restriction fragment length polymorphisms (RFLP's) are the result of restriction endonuclease cleavage and require electrophoretic size fractionation. Detecting a particular sequence variation may indicate individual identity, disease susceptibility or disease state.

To perform amplification and/or hybridization, the gel matrices are dehydrated by the introduction of heated, moving air while the gel matrices remain stationary in the reaction chamber. The matrices are then rehydrated with the solution containing primer, nucleotide and polymerase molecules. The DNA is amplified by rounds of primer extension of target DNA. A short time is allowed for annealing of one or more primer pairs (a pair is defined as two primers that border opposite ends of a linear target DNA and are complementary to the opposite DNA strands) at an appropriate temperature. The number and choice of primer pairs and the number of replication cycles will vary according to the target nucleic acid. The sequence of a target nucleic acid must be known to determine a system to be used for detection. As more sequence information becomes available, the choice of primers for any one system may be changed to reflect a conserved genetic region and improve the specificity of detection. New technology may improve fidelity of primer annealing and DNA polymerization to allow accurate detection by incorporating labeled nucleotides in the amplification step, thus eliminating the need for a separate hybridization step in the detection process.

The gel matrices are dehydrated after the gene amplification reaches the level needed for detection by the hybridizing probe. The hybridizing probe consists of single-stranded DNA complementary to, but shorter than, the DNA target sequence and has one or more label molecules attached. The choice of nucleotide sequences for the hybridization probe reflects the same considerations stated for primer sequences. The hybridizing solutions are pulse-sprayed into the reaction chamber. Shorter DNA probes diffuse and bind to the amplified copies within the matrices, while diffusion conditions retard leaching-out of the longer, amplified segments or the carrier surface may be used to trap small amplification products.

An alternate procedure involves primer pairs back to back along a target sequence in order to extend longer targets efficiently. The number of primer pairs in a linear or nested series may vary to accommodate the size-length of DNA required to immobilize the amplified segments during treatment. This alternative requires a ligase to incorporate each primer covalently to the linear molecule at its 5-prime end and the ligase needs to be thermo-resistant. In a particular system, such an enzyme would need to be isolated from nature, if it has not been already isolated.

Another alternate procedure involves adding the hybridization probes during the amplification phase. When single-stranded, labeled probe molecules are incorporated into the growing chains, they become part of the amplified DNA and sequential hybridization is not necessary. Since the process time is dramatically reduced in simultaneously amplifying and labeling the DNA, this step is desired. An enzyme for joining single strand nicks as described in the preceding paragraph is also necessary in order to insure the target sequence was labeled unambiguously over a background of randomly-primed, amplified DNA.

Each kind of labeled probe that hybridizes to the target DNA is detected according to the nature of its label molecule. The number of aggregates of detection signals corresponds to the number of original target sequences directly. In the case of higher density of targets or remelted agarose, the number can be interpolated.

Either specific restriction endonucleases, ribozymes (non-protein RNA molecules that cut and resplice RNA into genetic messages) or polymerases may be introduced into the gel matrix to act upon the nucleic acids, which are selectively embedded. "Selectively embedded" means that the nucleic acids of specimens are trapped and other specimen components and excess or unbound reagent molecules are washed away. Experiments have shown DNA sequences of a few hundred nucleotides or more remain essentially immobilized during amplification and hybridization conditions in given matrix materials while allowing short oligonucleotides, mononucleotides or enzymes to diffuse as necessary. The composition and concentration of the matrix may be altered to selectively immobilize a specific size class of nucleic acids. The endonucleases break linear DNA into restriction fragment polymorphisms. Polymerase molecules, together with DNA or RNA primers, are used to expand a selected DNA or RNA fragment population. With addition of electrical current, the fragments move through the gel matrix toward the anode, according to their size. Subsequent staining or hybridization within the matrix and carrier enables the identification of specific band patterns. Amplification products may be identified by electrophoretic separation and non-specific DNA staining; but in some cases hybridization probes are necessary to distinguish them from spurious amplification products which cause ambiguities.

The purpose of the electrical current in electrophoresis within the device of the invention is to fractionate and concentrate the macromolecules by size. Electrophoretic mobility of specific DNA restriction fragments, RNA messages or amplified nucleic segments are then compared with those similarly treated from another specimen. For example, specimens from two or more individuals may be compared for paternity identification. Forensic specimens may be compared to specimens from suspects. Family groupings may be compared for markers of genetic disease. Tumor specimens may be compared to standards for classification.

The electrophoretic character of this device is different from other electrophoresis equipment in that the macromolecules in the matrix are automatically processed before, after or in between electrophoretic phases. Different fluid treatments are applied automatically in series to the matrix carrier. The ability to automatically change the solution saturating the matrix heretofore was not possible. The instrument provides processor-controlled fluid delivery to individual matrices. An equivalent electrical current is supplied to each matrix carrier in each rack by design of the circuits.

Previously, unrelated specimens were grouped together in the same non-standardized gels. In this invention, related specimens contained in each matrix are processed and compared with both a standard built into each matrix and a standard matrix processed with each instrument operation. The carriers and matrices are preferably manufactured according to specifications that will standardize them and make their electrical resistance equivalent, when saturated with buffer, whereby the interpretation of the test results determined from them may be standardized and variability originating from individual gel preparations eliminated.

In standard electrophoresis the prepared sample is manually loaded in the gel for electrophoresis, and the gel, or the nucleic acids in it, are manually handled for hybridization and detection. The feature of the matrix carrier of the invention is that the physical and chemical handling of it is automated within the instrument. Other kinds of prepackaged, prepared gels for electrophoresis are opened before or after electrophoresis and the gel is removed for running, staining or other processing. This carrier is unique in that it can be opened and closed mechanically by the instrument in coordination with the fluid and air flow systems in the thermal chamber of the instrument. This feature allows the genetic specimen to undergo further treatments without transfer to another vessel.

Furthermore, the automated system represents versatility in applications. A unique matrix carrier is intended for each specific diagnostic or DNA identification test. Matrix size and composition will be adapted to perform a particular kind of assay. The two-dimensional format allows spatial enumeration of signal identification positions of target sequences during repetitive probing.

Racks are designed to hold matrices of the same design. The same basic instrument design will hold any rack configuration and accommodate processing for any of the tests. It is also clear that instead of or in addition to using the carrier for electrophoretic separation of DNA or RNA, the carrier may be used for analysis of sample proteins using standard electrophoretic techniques or in situ immunohistochemistry.

Sequence-specific nucleic acid identification depends upon one or more of three fundamental methods: amplification, hybridization and electrophoresis, all of which may be performed using a matrix carrier according to an embodiment of the invention. The automated system for DNA-based diagnostics herein incorporates one or more of these methods in a given order depending upon the nature of the specimen and the quantity of nucleic acid in a particular type of specimen. Microprocessor-controlled processing starts with a sample preparation phase. Lysing and deproteinizing treatments are performed automatically to prepare the sample specimen after it is incorporated into the matrix carrier and loaded into the instrument. The application of treatments that follow are programmed to perform methods appropriate and prearranged for a batch of similar matrix carriers.

As illustrated in the schematic of FIG. 17, the automated system has great flexibility in the inclusion and ordering of methods used. After sample preparation any one of the three fundamental methods are performed first: amplification, hybridization or electrophoresis. Detection of the sequence-specific nucleic acid target may occur after treatments for any one of the methods. A particular test can involve one, two or all three methods before detection, in any order.

Furthermore, there are advantages to performing multiple steps or methods in one vessel. They are: (1) standardization of accurate assay results (2) less technician skill and less technician preparation and handling time required and thus lower test cost (3) more convenient sample collection and (4) less human error in switching samples or labels. Current methods require a technician to prepare a sample and transfer it to another container or a gel together with other specimens. A specimen may go through several container changes during processing, and each container change is a possible source of error in identifying a patient specimen or sample source. The matrix carrier in this invention contains the patient specimen or sample throughout the entire processing. The uniqueness of this technology is the stabilization of nucleic acids in a matrix without extensive preparation of the biological sample, and the subsequent treatment of this matrix to prepare and identify the target genetic sequences automatically.

The invention includes any possible coating of the carrier surfaces with selected biomolecules, natural or synthetically-manufactured, by chemically attaching them to the carrier material. For carriers made of glass, a known standard method of binding biomolecules to surfactants is with sulfonyl chlorides (Nilsson et al., In W. B. Jakoby (Ed.) Methods in Enzymology, Vol. 104, 1984, Academic Press, Inc., Orlando Fla.). For carriers made of polypropylene or polystyrene, chemical attachment may be by hydrophobic binding to their phenyl groups. The purpose of adhering molecules to the carrier is to facilitate the processing of genetic detection.

For a fuller understanding of the nature and objects of the invention reference should be made to the following detailed description taken in connection with the accompanying drawing figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a first embodiment of the carrier of the invention in an open position.

FIG. 2 is a side view of the first embodiment of the carrier of the invention in a closed position.

FIG. 3 is a side view of the first embodiment of the carrier of the invention with carrier edge removed showing the matrix space and the channel.

FIG. 4 is a perspective view of a hinge of the first embodiment.

FIG. 5 is a perspective view of a second embodiment of the carrier in a closed position.

FIG. 6 is a perspective view of the second embodiment of the carrier of the invention in an open position showing a side hinge and subsections of a matrix.

FIG. 7 is a back perspective view of the second embodiment of the invention showing the hinge.

FIG. 8 is a perspective drawing of a third embodiment of the carrier showing a base that snaps onto standard microscope slides.

FIG. 9 is perspective drawing of a cover for use with the snap-on base of FIG. 8.

FIG. 10 is a cross-section view of the snap-on base and cover of the third embodiment as it would be placed on a standard slide in the rack.

FIG. 11 is a lengthwise section drawing of the snap-on base and cover of the third embodiment to show liquid flow and cover positions.

FIG. 12 is a perspective view of a tray rack to hold the matrix and carriers.

FIG. 13 is a schematic drawing of the automated gene identification apparatus of the invention.

FIG. 14 is a perspective drawing of the overall gene identification apparatus embodying the invention.

FIG. 15 represents temperature profiles in which thermal cycling such as that used for PCR is attained in the matrix.

FIG. 16 is a schematic drawing of an electrical circuit closed by a matrix.

FIG. 17 is a schematic diagram showing some of the various analyses and methods for which the invention may be used.

FIG. 18 illustrates a microscopic view of cells in the matrix after amplification and detection of a specific sequence in the cells while embedded in the matrix of a carrier and processed in the invention.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The invention broadly comprises a fluid flow system comprising an apparatus and component carriers 10, each of which carrier contains biological specimens in a matrix 12. The system, shown in FIGS. 1-14 & 16, is capable of fluid flow through the matrices and collection of fluids drained from the reactor before being either discarded or recycled. The system also includes blowers and heating elements to control the air or fluid temperature in the chamber. In the preferred embodiment of the invention, the programing of the number and time intervals of treatments, the endpoints of each treatment, valve-control and electrical switching are computerized into the microprocessor.

The carrier 10 is composed of an upper rectangular piece 14 and a lower rectangular piece 16 hinged together; which, when folded, encase the matrix 12 and, when unfolded, expose one surface of the matrix 12.

Three embodiments of the carrier device are depicted in the figures. The first two embodiments may have electrical contacts for electrophoresis and all may have multiple matrix sections and subsections, but for ease of depiction, these variations are not shown in all embodiments.

In a first embodiment (FIGS. 1-4) the hinges 18 are along a shorter side of the lower rectangular piece 16, while in the second embodiment (FIGS. 5-7), the hinges