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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a device for use in automating the detection of
target nucleic acid sequences in biological-containing samples. The device
described herein is for use with an automated process, including a
fluid-delivery system and a thermal reaction chamber, as is described in
U.S. patent application Ser. No. 07/227,348, filed Aug. 2, 1988 and now
abandoned in favor of a continuing application Ser. No. 07/935,637, filed
Aug. 24, 1992. The disclosure of the co-pending application is hereby
incorporated herein by reference.
The invention in said co-pending application relates to a method and
apparatus for automating the detection of target nucleic acid sequences in
biological-containing samples involving 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 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.
2. Description of the Related Art
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.
Wherever a unique, organism-specific polynucleotide sequence is identified,
it is possible to use a labeled, synthetic molecule of the unique sequence
to determine the presence of the organism by hybridization of the unknown
sample to the labeled sequence. This detection method involves
hybridization between DNA:RNA hybrids or DNA:DNA duplexes. The probe is a
single-stranded nucleic acid molecule complementary to a unique nucleic
acid sequence of the gene being tracked. The probe is labeled with an
identifying molecule and introduced to the test sample. Hybridization has
been an important research tool, but its use is limited to a few clinical
laboratories because of the time, skill and knowledge required of the
technician performing the test. DNA probes are being used as commercial
diagnostics for a few infectious or genetic diseases, but their individual
cost is prohibitive for mass screening.
While the common laboratory procedure for hybridization binds the target
DNA to a solid support, an alternative approach is solution hybridization
or hybridization which requires individual column separation of the
unbound, labeled probe for each sample. This invention uses a gel matrix
as a solid support. It is not necessary to transfer DNA to a membrane
filter after purification and amplification. This approach eliminates any
loss of DNA that occurs during transfer. (Purrello and Balazs, 1983, Anal.
Biochem. 128:393-397).
Presently DNA preparation and amplification require labor-intensive
protocols just as hybridization methods do. The only apparatus which
automates DNA preparation is the Applied Biosystems Nucleic Acid
Extractor, which will process sixteen tissue samples simultaneously in
four hours. The sample must comprise homogenous tissue and already contain
enough copies of the target DNA to be detected, i.e. about a million
copies. The laboratory technician must then either fractionate the
extracted DNA by gel electrophoresis or transfer the DNA to a solid
support for detection by hybridization to a labeled probe. There is no
laboratory apparatus or equipment currently on the market that automates
hybridization so that it may be left unattended.
Suspending cells in agarose beads or cubes is a common laboratory procedure
for preparing unsheared nucleic acid molecules for subsequent enzymatic
modifications. (P. R. Cook, 1984, EMBO 3:1837-1842 and L. Van der Ploeg et
al. 1984. Cell: 37:77-84). After solidification the agarose beads or cubes
are subjected to extensive treatment with detergent, protease and salt. It
is possible to remove all cellular constituents except DNA because the
pores in the agarose matrix are large enough to allow rapid diffusion of
proteins and other small macromolecules while quantitatively retaining
genomic DNA (Smith and Cantor, 1986, Cold Spring Harbor Symposium on
Qualitative Biology 51:115-122).
FMC Bioproducts, Rockland, Me., has a nonradioactive-label for DNA in which
their product information states that the labeling is done directly in
diluted, remelted agarose. This protocol allows electrophoretic
fractionation of DNA in agarose and then quick and easy preparation of
specific probes (Resolutions 1987 Newsletter 3(2):1-2). FMC also markets a
new grade of agarose certified for reliable restriction endonuclease
activity. Many other examples exist where research scientists are
performing enzymatic modifications on DNA in agarose. D. Persons and O.
Finn, (Biotechniques, 1986, 4:398-403) reported primer extension of cDNA
on a poly A+ RNA template using a reverse transcriptase in remelted
agarose. The method and device of this invention also involves primer
extension with polymerase enzymes in agarose.
Immunodiagnostics are commonly used to identify organisms directly by
antigenic determinants or to identify individuals by their antibodies
which are produced by exposure to the antigen. The same problem is
encountered with antigen identification as with DNA probes, i. e. the
organism must be cultured if it is not present in sufficient numbers for
detection. There is no in vitro method to amplify antibody- binding
antigens accurately like there is with primer extension gene
amplification. Low population targets in a mixed background cannot be
identified immunologically. The gene amplification in vitro has given DNA
probes the potential to outperform immunological detection. The accuracy,
sensitivity and quantitation potential of DNA probes will make them the
diagnostic of choice.
An automated system for simultaneously detecting target nucleic acid
sequences from multiple samples must accommodate several different steps
and varying reaction conditions. It must be constructed to change reagents
and solvents automatically for each stage and monitor time, temperature
and pH. If tests are automated and the same apparatus that performs one
test for multiple samples in one run could be used for many different
tests by changing a few selected reagents, the cost of gene detection
would be relatively inexpensive and the system would supersede other
methods because of its speed and preciseness.
In order to have enough gene copies for detection, present methods rely on
selective cultivation of the organism which takes days to weeks depending
upon the organism. A selective DNA amplification technique has been
practiced whereby synthetic primers are annealed to single stranded or
denatured, double-stranded nucleic acid target sequences and polymerase
molecules incorporate nucleotides that replicate a portion of the nucleic
acid extending from the primers. Using excess primers in pairs bordering a
target sequence in a way that each polymerase extension includes sequences
that are complementary to the other primer sequence is a method now termed
polymerase chain reaction (PCR) (see U.S. Pat. Nos. 4,683,195 and
4,683,202). This method continues in repetitive rounds of replication
until the target sequence has been amplified by a factor of more that 10
million. Saiki et al. reported that a thermostable DNA polymerase improves
the specificity, yield, sensitivity and length of products that can be
amplified (Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R.
Higuchi, G. T. Horn, K. B. Mullins, and H. A. Ehrlich, Science, 1988,
239:487-491). A selective gene amplification protocol that can duplicate a
single copy of a nucleic acid target in vitro to a sufficient number of
copies that can be detected over non-specific background binding with a
labeled hybridization probe is the level of sensitivity that will enable
easy screening of multiple samples. The accuracy of a gene detection is
assured by labeling a probe complementary to a polynucleotide sequence
between the two primer sequences for the purpose of hybridization
identification. Thus, even if the primers had amplified non-target
sequences because of duplicity of sequence or mismatch, the label would
only be detected that bound to the target sequence.
The ability to amplify a single target DNA and/or RNA sequence enough to
detect it without the cultivation of cells or organisms will simplify gene
detection and attempts to automate it. Saiki et al. reported that PCR
detects a single copy of target DNA present in one in 1.5 million cells.
There is no reason to doubt that gene amplification by primer extension
will detect a target DNA segment present at one copy per organism in the
starting material. The ability to then quantify how many original copies
or organisms there were per sample before amplification will make mass
sampling and fate-monitoring possible by hybridization detection.
Quantifying methods depend upon diluting the amplified gene so that
individual signals are enumerated or intensity of total signal matches
that of a known standard concentration.
Using the aforementioned gene amplification protocol, the presence of HIV-1
in peripheral blood mononuclear cells (PBMC) was determined by in situ
hybridization to DNA from the PBMC's without prior cultivation of them
(Ou, C., S. Kwok, S. Mitchell, D. Mack, J. Sninsky, J. Krebs, P. Feorino,
D. Warfield, and G. Schochetman, Science, 1988, 239:295-297). This direct
detection method reduces the time to three days from the three to four
weeks required for cell cultivation and virus isolation. The polymerase
chain extension technique started with DNA isolated from PBMC's,
repetitively amplified the target DNA in solution, and analyzed bands on
an autoradiogram produced by gel electrophoresis of restriction enzyme
digests of the target DNA bound to end-labeled radioactive probe
molecules.
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.
Accordingly, the invention aims to provide a vessel for the individual
specimens to be contained.
Yet another object of the invention is to mold matrix material which is to
contain the specimen.
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 support of the specimen,
embedding it in a matrix for automated processing.
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 for optimal signal detection.
A further object of the invention is to allow for saturating specimens
quickly with a series of solutions or for drying them automatically.
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 to provide such a system wherein
airflow and heating regulate temperature and humidity.
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 its
configuration 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 automation of this invention makes direct detection available for
innumerable clinical diagnoses and for environmental gene-tracking.
Current practice for tracking microbes released into the environment is
done by selecting marker genes on the same episome or genome as the
engineered rDNA. The system of this invention provides direct,
simultaneous monitoring of the rDNA from many samples without the time and
expense required to cultivate the microbe. 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. The target DNA in each sample is
immobilized, exposed, amplified and located in a series of treatments to
the matrix block in which the sample has been introduced.
The DNA present in the sample that has been introduced into an individual
matrix remains anchored in the corresponding matrix and is separated from
the other cellular particles or sample debris by lysing solutions and
thorough washing. After washing, the sample is exposed to another solution
to denature the DNA in situ. The denaturing solution is followed with a
neutralizing treatment.
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 temperature is changed to
the optimal temperature for polymerase activity for a time period long
enough to extend the DNA segment past the sequence to which its primer
pair partner binds. The temperature is raised to a denaturation
temperature for the DNA during a simultaneous partial dehydrating period.
A new round is initiated by rehydrating with pulses of fluid to rehydrate
the gel and the temperature is lowered to the annealing temperature. Each
amplifying round theoretically increases logarithmically the copies of DNA
target segments; the actual increase depends upon the efficiency of the
polymerase. Approximately twenty to twenty-five rounds of amplification
increase one DNA copy to two million copies, which is more than the number
of copies needed for detection by current labeled probes. 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. The shorter DNA probes
diffuse and bind to the amplified copies within the matrices, but the
diffusion conditions retard the leaching-out of the longer, amplified
segments.
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.
In a broad aspect, the 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 downward pressure on said first area 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 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.
The method of the invention utilizes the device of the invention. The
sample to be analyzed for the presence of a particular DNA component (or
RNA or polypeptide moiety) is 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) amplifying a desired nucleic acid
component in the matrix material; (c) applying an electric current to the
matrix material; and (d) 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 may be incorporated into the automated instrument (for
automatic gene processing and detection according application Ser. No.
07/227,348, abandoned in favor of continuation application Ser. No.
071,935,637), which has been abandoned in favor of file wrapper
continuation application Ser. No. 07/935,637filed on Aug. 24, 1992 which
opens and closes the hinged parts. Said application is incorporated herein
by reference. 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 format is that target nucleic acids in the 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.
The purpose of the electrical current in electrophoresis within the device
of the invention is to fractionate and concentrate the macromolecules by
size. In the case of nucleic acids, 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 cell components 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 endonucleases break linear DNA into
restriction fragment polymorphisms. Polymerase molecules, together with
DNA 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.
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 in my
co-pending patent application 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, multiple specimens were grouped in the same matrix for
simultaneous electrophoretic comparison. In this invention, 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. A matrix carrier manufactured with quality control
standards is another advantage of the automated system. The electrical
resistance of each matrix will be reproducible when it is saturated with
standard buffer. Advantages of the automated handling and separating of
specimens into multiple matrices 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.
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 gel matrices molded in a
carrier are removed from the carrier for staining or further 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. 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 histochemistry.
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 as discussed in application Ser.
No. 7/227,348, abandoned in favor of continuation application Ser. No.
07/935,637. 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. 9, the automated system has great
flexibility. 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.
The invention includes any possible coating of the carrier surfaces with
selected biomolecules, natural or synthetically-manufactured, by
chemically attaching them to surfactants which normally adhere to the
carrier material. For carriers made of glass, a known (standard method of
binding biomolecules is with sulfonyl chlorides (Nilsson et at., 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 preadhering 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 invention 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 schematic drawing of an electrical circuit closed by a matrix.
FIG. 9 is a schematic diagram showing some of the various analyses and
methods for which the invention may be used.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF
The invention broadly comprises a carrier 10 and a matrix 12 capable of
containing biological specimens. 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.
Two embodiments of the invention are depicted in the figures. Both
embodiments may have the electrophoresis electrical contacts, multiple
matrix sections and subsections, but for ease of depiction, these
variations are not shown in both 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 18 are along a portion of a longer edge of the lower
rectangular piece 16. Having the hinge 18 along a longer side portion of
the lower piece 16 (second embodiment) is valuable for electrophoresis,
where a longer matrix is preferred. Such a side hinge arrangement allows a
longer, narrower matrix while requiring less overhead space for opening
the carrier than an elongated cover for a carrier that was hinged at an
end would require.
The edges of the two pieces 14 and 16 are juxtapositioned to overlap each
other so that in the first embodiment (FIG. 1) the upper piece 14 overlaps
and extends beyond the lower piece 16 at the end having the hinge 18, and
the lower piece 16 extends beyond the upper piece 14 at the end of the
carrier 10 that is not hinged. In the second embodiment (FIG. 5), the
upper piece 14 extends beyond the lower piece 16 at the hinged side of the
lower piece 16, and the lower piece 14 extends beyond the upper piece 14
at an edge perpendicular to the edge having the hinge 18.
The lower overlap 22 on the lower piece 16 functions to receive fluids
which may diffuse into the matrix 12 whether the invention is in the
closed or open position. The upper overlap 24 of the upper piece 14
functions as a lever end to open the invention. Mechanical pressure
applied to the upper overlap 24 lifts the upper piece 14 away from the
matrix 12, which rests on to the inner surface of the lower piece 16.
Each hinge 18 (FIG. 4) in the first embodiment preferably comprises an
upper portion 26 grippingly engaging the upper piece 14 of the carrier and
a lower portion 28 attached to the lower piece 16. The means of attachment
may be g | | |
