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Description  |
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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
co-pending U.S. patent application Ser. No. 07/227,348. The disclosure of
the co-pending application is hereby incorporated herein by reference.
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.
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 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 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
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 co-pending application
Ser. No. 07/227,348), 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 co-pending application Ser. No.
07/227,348. 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 glue or other known means of attachment. A flexible bend area 30
located between the upper portion 26 and the lower portion 28 enables the
hinge movement and the opening and closing of the carrier 10. The hinge 18
may be made of flexible plastic, or may be made of a rigid material such
as a plastic or metallic alloy, except in the flexible bend area 30.
Preferably such a hinge is attached at each side of the carrier 10.
Although the carrier is described as comprising separate pieces, it is
equally possible that the carrier may be molded as one piece with a
"living" plastic hinge connecting the portions or that two or more
components of the carrier may be molded together.
Treatment solutions from fluid lines 32 in the device of the co-pending
application flow or drip into channel(s) 34 on the lower piece 16 and
diffuse into the matrix 12. The fluid system as described in my co-pending
patent application delivers measured volumes from one of multiple
reservoirs through a common line either in a continuous or pulse mode at a
selected flow rate. Fluid application is also important in opening and
closing the carrier halves 14 and 16. Application of a fluid volume at the
time of opening releases surface tension between the upper piece 14 and
the matrix 12. This action reduces the mechanical force required to
separate the upper piece 14 from the matrix without disturbing the matrix
12. Application of fluid to the matrix 12 prior to closing the carrier
pieces 14 and 16 leaves a liquid film between carrier piece 14 and the
matrix 12 upon closing. Closure of the carrier 10 at the hinged joint 18
brings the surfaces that are closest to the hinge 18 together first and
gradually those farther away from the hinge 18 make contact. The wave-like
closing action smoothes out the bubbles whose presence may cause aberrant
test results.
The fluids from channel 34 saturate the matrix 12 and fill the space
between matrix 12 and the upper half of the carrier 14, and excess liquids
may exit at the opening(s) 36 between the hinged pieces 18. Collecting
troughs on the racks and the shelf below the racks within the instrument
provide for fluid disposal (not shown). Any opening(s) along the edge are
plugged during addition of the matrix material to the carrier 10 when the
carrier is being prepared for use until after the matrix 12 has been
formed. Taping the opening(s) may be used to close them, but other means
of temporarily covering the opening are possible. The tape or other
fastener is removed when the carriers 10 are loaded in a rack, or a sealed
barrier over the opening(s) may be broken by the opening or closing action
of the carrier pieces 14 and 16.
Opening 36 between the hinges 18 or an open side or end of the matrix 12
also allow electrical contact with the matrix material. The electrical
contacts 38 and 40, in each embodiment and shown for the second embodiment
in FIGS. 5 and 6, permit a constant or deliberately variable electrical
current to flow through individual matrices in order to optimally resolve
different size classes of macromolecules. A coating with negatively
charged groups such as Nafion.TM. (DuPont Co., Wilmington, Del.) on the
lower surface of the upper carrier half 14 and/or the upper surface of the
lower carrier half facing the matrix 12 may be used to help reduce
electroendosmosis, in which cations in aqueous fluids and hydrogels tend
to flow toward the cathode.
The carrier pieces 14 and 16 may be made of glass or plastic or
combinations thereof, sheets of polymer (such as polyetherimide,
Ultem.RTM., General Electric, Pittsfield, Mass.) or metallic alloys.
Carriers used for assays involving electrophoresis are made of
non-conducting materials in order that current flows through the matrix
and not the carrier. Parts of the carrier may be made of optically clear
material for scanning the matrix.
The matrix 12 is preferably a semi-solid material made with agarose or
acrylamide or similar polymer, or mixture thereof, that incorporates
several times its weight of an aqueous solution (hydrogel). A liquid
specimen or specimen mixed with a liquid diluent may be added to the
carrier at channel 34, or into subsections 48 directly, from where it
either combines with a liquid matrix 12 or diffuses into a pre-formed
dehydrated matrix or a subsection thereof. Application of heat or a
polymerizing agent incorporates the specimen into the matrix 12 or
subsection thereof, forming a gel matrix with embedded specimen. The gel
matrix may be hydrated or not before loading its carrier in the
instrument. If dehydrated in storage or transport, the gel matrix material
is rehydrated with fluid treatments from the fluid lines 32 of the
instrument. A rehydrated matrix, preferably ultra-thin (less than 500
micrometers thick), facilitates diffusion of small molecules and retention
of larger ones, quicker electrophoretic resolution and better detection of
signal.
The carrier 10 may be molded to have edges 42 or the carrier halves 14 and
16 may have edge pieces fastened to them. The edges 42 are formed in order
to enable molding of the matrix material in a space between the upper and
lower carrier surfaces 14 and 16. The space between carrier surfaces 14
and 16 may diverge from one end to the other by placing wedge-shaped edges
42 along the sides in order to form the matrix material thicker at one end
(not shown). Such a wedge-shaped matrix may be made by pouring molten
matrix material into a wedge-shaped enclosure formed between the upper and
lower carrier surfaces and bounded by edges 42. The purpose of the wedge
configuration is for increasing the electrophoretic separation of a wider
range of nucleic acid fragment-size classes over less linear space.
Another aid to better resolution of fragment populations is variation of
the concentration of the matrix material over the linear path of
electrophoresis, i.e., making a gradient gel matrix. When matrix material
is preformed on carrier half 14 or 16, it may be applied in a manner to
form a concentration gradient and/or wedge across one dimension for better
electrophoretic resolution.
Edges 42, and extensions 44 which may overlap the surface of the lower
carrier section 16, are molded or fastened to one or both of the carrier
halves. Edges 42 and extensions 44 may form molds for matrix materials,
with and without added specimen material. They may be designed to mold
either matrices 12 of uniform thickness in the space between the upper 14
and lower carrier 16 surfaces or mold subdivisions of the matrices 12 that
contain different matrix materials, volumes or concentrations thereof
(FIGS. 5 and 6). The edge 42 in FIG. 6 is shown cut away where electrical
contact 38 crosses it. The extensions 44 may form molds 46 on the lower
carrier section 16 and subdivide the matrix area into smaller
subdivisions. The subdivision of matrices on one carrier allows the
different matrix sections to include different specimens or standards.
Introducing the specimen into the matrix allows pretreating the specimen
within it to prepare DNA in the sample by a standard method of Smith, Klco
and Cantor (1989, In K. Davies (Ed.), Genome Analysis-A practical
Approach, 1989, pages 41-72, IRL Press, Oxford) or by variations of a
standard method. In a multisection matrix, another section of the matrix
may be pre-formed on the carrier and accept DNA molecules transferred to
it from the initial matrix via electrophoresis. The purpose of varying
materials, or the volume and concentration thereof, in submatrix sections
on the same carrier is to optimize conditions for a specific method For
example, polyacrylamide gel reagents may be introduced, dried and enclosed
in one section of the carrier during manufacture. Later, the sample is
mixed with liquid agarose and added to the matrix carrier 10 filling
subsection spaces 48 and forming subsection 46. After sample preparation
treatments and subsequent drying of the agarose matrix in subsections 46,
the carrier 10 is opened and electrophoresis buffer applied to all matrix
sections. The nucleic acids (or proteins) are electrophoretically
transferred from the agarose to the acrylamide matrix (or an intermediate
matrix in subsections 54) for a processing step for which acrylamide is
better suited than agarose.
The first matrix the specimen encounters may serve to cleanse it. Drying
and rehydrating this matrix reduces total volume and thus concentrates
samples. Electrophoretic movement of macromolecules from the concentrated
first matrix into the second matrix has the effect of loading a more
concentrated sample onto the second gel, i.e., more sample target
molecules per unit matrix. The more concentrated the macromolecules are
when starting electrophoresis, the more easily detected they are after
electrophoretic separation due to a narrower band width. The pr | | |
