Note: Descriptions are shown in the official language in which they were submitted.
~ W091/15768 PCT/US91tO2323
~ 1 '20~001~
PROCESS AND COMPOSITION FOR PERFORMING DNA ASSAYS
BACKGROUND OF THE INVENTION
- 5 This invention relates generally to nucleic acid
hybridization assays and, more particularly, to a fully
automated process for performing nucleic acid
hybridization assays using magnetically responsive -
particles. -
Many current diagnostic and epidemiological
procedures rely on the culture of clinical specimens, -
which can be both time consuming and difficult. ~~
Immunoassays, such as RIA and ELISA, are faster and are
frequently used for viral typing and culture
confirmation. Many viruses, however, do not produce
significant amounts of antigens and therefore cannot be
detected by immunoassays. An alternative procedure for
identifying such pathogens employs nucleic acid
hybridization assays.
.:
Nucleic acids, which are the carriers of genetic
information between generations, are composed of linearly
arranged individual units called nucleotides. Each
nucleotide has a sugar phosphate group to which is
attached one of the pyrimidine or purine bases, adenine
(A), thymidine (T), uracil ~U) guanine (G) or cytosine
tC)- Single stranded nucleic acids form a double helix
through highly specific bonding between bases on two
strands; A will bond only with T or U, G will bond only
with C. Thus, a double stranded, or hybridized, nucleic
acid will form where, and only where, the sequence of the
bases in the two strands is sufficiently complementary as
to allow such hybridization. Depending on the form of
the sugar phosphate group present, the nucleic acid is
termed either deoxyribonucleic acid (DNA) or ribonucleic
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;~ WO91/1S768 PCT/US9l/02323
~. 2~0~19 ~
acid (RNA).
Nucleic acid hybridization assays are based on
this principie of complementarity. Typically, a single
stranded nucleotide sequence complementary to the
sequence of interest is combined with a sample under
conditions allowing hybridizatio~. The former sequence
is termed the "probe;" the latter, the "target." The
presence of double stranded nucleic acid including the
probe indicates that the target sequence is present in
the sample. Such assays have wide applicability,
including the testing of biological samples for the
presence of a pathogen, such as a bacterium, virus or
parasite; the diagnosis of disease associated with a
genetic abnormality; the indication of susceptibility to
certain genetically mediated conditions; paternity or
other relatedness, as for example in forensic analysis;
and the biological contamination of food or other
prod~ct.
Where the amount of target nucleic acid in the
sample is initially small, procedures may be utilized to
amplify the target. Among such amplification procedures
is the polymerase chain reaction (PCR) and ligation-based
amplification procedures. See, for example, United
States Patent Nos. 4,683,195; 4,683,202 and 4,800,159 and
the PCT Publication No. W089/12696.
Various methods are known to those skilled in
the art for determining the presence and extent of
hybridization. Such assays require that the hybridized
probe be distinguishable from non-hybridized probes. The
majority of nucleic acid hybridization assays utîlize
isotopic detection, primarily 32p, and are manual
processes requiring separation steps. For example, the
conventional membrane-based assays involve sample
.
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WO91/15768 PCT/US91/02323
~ 3 r ~ 2 0 ~ ~ ~ 1 9
pretreatment, denaturation, and fixation of nucleic acids
onto solid supports, such as nitrocellulose or nylon
filters. Such procedures are imprecise, labor intensive,
time consuming, and difficult to automate. ~urthermore,
the hybridization probes are usually radiolabeled to high
specific activity in order to obtain the required
sensitlvity. Clinical laboratories are averse to such
probes because they are unstable and present significant
problems of handling and disposal. Consequently, the few
such tes~s on the market have limited practical
application in the clinical laboratory.
Non-isotopic labels have also been used in
nucleic acid hybridization assays. For example, biotin
labPled probes utilizing alkaline phosphatase-avidin
polymeric complexes can be used to detect unique DNA
sequences immobilized on filters through the enzymatic
production of a colored, precipitatable dye, as described
by Ward, et al., U.S. Patent No. 4,711,955. These
nonisotopic, indirect detection systems however are
plagued by intermediate, background-susceptible, binding
and washing steps, and are limited to hybridization on -
membranes.
'
Several membrane-based nonisotopic direct
detection systems have been described utilizing
covalently linked enzyme-DNA. Renz and Kurz (Nucl. Acids
Res. 12:3435 (1984)) describe long probe methodologies
which are limited by lengthy hybridization times and
difficult cloning procedures. Alternatively, well
defined synthetic oligonucleotide probes can be used.
For example, direct labeled enzyme-synthetic
oligonucleotide conjugates are described by Jablonski, et
al., Nucl. Acids Res. 14:6115 (1986); Kerschner, et al.,
Am. Soc. Microb., Abst. 56:309 (1987); McLaughlin, et
al., Lancet 714, March 28 (1987), which are incorporated
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WO91/15768 PCT/US91/02323
2~0~9 4 ~
herein by reference. These conjugates are easily
synthesized for specific analytes, exhibit unaltered
hybridization characteristics with no nonspecific binding
and have exceptional sensitivity and stability. Due to
higher probe concentrations, oligomer hybridizations are
completed more quickly on membrane supports. ~owever,
the full utility of these enzyme conjugates has not been
realized in conventional membrane formats.
An alternative approach to membrane-based assays
is the sandwich assay, as described, for example, United
States Patent No. 4,486,539. The target DNA is removed
from a crude sample by hybridization to a complementary
sequence immobilized on a solid matrix (direct capture) -
or by hybridization with a ligand labeled probe, followed
by capture onto an affinity support (indirect capture)
and removal of unbound probe. Bound target is detected
by a second directly or indirectly labeled probe specific
for a proximal sequence. The addition of this second
hybridization step enhances selectivity for the targct
and reduces the chance of nonspecific interactions.
Althouqh the use of long cloned probes limits the
utility, this is one of the most appropriate formats for
routine diagnostics since it eliminates protracted sample
25 preparation, purification, and nonspecific filter-based ~ -
target immobilization.
Direct capture hybridization supports are
synthesized by attaching nucleic acids, usually cloned
DNA, by chemical modification, adsorption, or enzymatic
processes to a variety of solid phases, including
nitrocellulose, cellulose, nylon, polystyrene, teflon-
polyacrylamide, polypropylene, agarose, sephacryl and
latex. The hybridization efficiencies and capacities of
immobilized DNA have been described in detail. See, for
example, Miller, et al., J. Clin. Microb. July 1988, p.
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WO91/15768 PCT/US91/02323
2.~ 9
1271-1276 and Yehle, et al., Molecular and Cellular
Probes 1:177-193 (1987), which are incorporated herein by -
reference. The supports described thus far are difficult
to make, however, and with the exception of
oligonucleotide resins, have relatively poor loading
capacities. The mixed-phase direct capture
hybridizations are also limited kinetically and may be
inefficient due to inaccessibility of immobilized DNA.
In contrast, hybridization in solution is
extremely efficient and many times faster than
hybridization on solid supports. Solution hybridi2ation
with l25I labeled probes, followed by nonspecific
adsorption onto hydroxyapatite (HAP) is well known in the
art. However, nonspecific background binding to HAP is a
function of probe concentration, and capture is
nonspecific. Therefore, probe inputs are limiting and
hybridization rates are suboptimal.
2Q The DNA assay processes described briefly above
are all manual and have not yet proven to be generally
effective. Current procedures have suffered from the
drawback of not consistently yielding precise and
repeatable results. Variations in the relative amounts
of the various reagents, the timing of their combination
as well as in the needed amount of agitation of the
combined reagents, for example, all contribute to the
somewhat imprqcise and non-repeatable results. In
addition, the possibility of contamination and the
excessive time and labor required by highly-trained
personnèl to carry out the numerous and tedious steps of
manual assays are further disadvantages inherent in the
~ manual DNA assay process.
It should, therefore, be appreciated that there
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W09t/15768 PCT/US9l/02323
2~80~1~ 6 ~
is a need for a fully-automated process and reagents for
performing DNA assays, which yields highly precise and
repeatable results while minimizing the risk of
contamination, and which requires minimal intervention
and monitoring by highly-trained personnel. The present
invention fulfills this need and provides related
advantages as well.
SUMMARY OF THE INVENTION
The present invention resides in an improved
process for performing nucleic acid hybridization assays
that is both highly precise, efficient and repeatable and
that is fully automated, requiring only minimal ~ -
involvement by trained personnel. The process of the
invention includes an initial step of providing a
programmable XYZ pipetter and an associated plurality of
wells, an associated array of thermally-controlled
reaction tube holders, each being adapted to carry a test
tube, ~nd means for effe-ting an alternating or ro-atins
magnetic field about each reaction tube's longitudinal
axis. The XYZ pipetter is used to transfer nucleic acid
samples to separate test tubes carried by the plurality
of reaction tube holders, to transfer successively a
hybridization solution and a suspension of magnetically
responsive particles to the plurality of test tubes at
prescribed times, and to effect position of the reaction
tubes relative to their associated magnetic fields at
prescribed times, such that binding reactions between the
target nucleic acid samples, probe and oligonucleotides
in the hybridization solution, and magnetically
responsive particles can occur and a precise, repeatable
automated nucleic acid hybridization assay can be
provided.
WO91/15768 PCT/US91/02323
~ 2 ~
More particularly, the XYZ pipetter is used by
initially pla~ing the hybridization solution in a first
well, the suspension of magnetically responsive particles
in a second well, a wash buffer solution in a third well,
a substrate buffer in a fourth well and a quench buffer
solution in a fifth well. The separate mlcleic acid
target samples are placed in the plurality of reaction
tubes, each carried in a separate reaction tube holder.
The XYZ pipetter is used initially to transfer the
hybridization solution from the first well to the
plurality of test tubes, in sequence, whereupon
hybridization reactions are allowed to occur.
Thereafter, the XYZ pipetter transfers the suspension of
magnetically responsive particles from the second well to
the plurality of reaction tubes, in sequence, and the
reaction tubes are then selectively rotated or the
magnetic field removed such that the magnetically
responsive particles remain in liquid suspension and are
allowed to undergo a binding reaction with the separate
2n nucleic acid samples. Terminating the step of
selectively rotating allows the magnetically responsive
particles to be moved by the magnetic fields to selected
locations in the reaction tubes, allowing the XYZ
pipetter then to remove the unbound nucleic acid sample
in the liquid phase and hybridization solution from the
plurality of reaction tubes. Alternatively, a
electromagnetic field may be induced around the
stationary tubes to remove the particles from suspension.
The XYZ pipetter then transfers the wash buffer
solution from the third well to the reaction tubes.
After resuspension and separation of the magnetically
responsive particles, the pipetter then removes the wash
buffer solution, to leave behind the magnetically
responsive particles with bound DNA sample and
hybridization molecules. The pipetter then transfers the
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; WO91/1S768 PCT/US91/02323
2~001.~ 8 ~
substrate buffer from the fourth well to the plurality cfreaction tubes whereupon an enzyme probe label catalyzes
a detectable reaction. Alternatively, if the label is a
luminescent or fluorescent moiety, the probe is
dehybridized to allow detection in solution. The
pipetter then transfers the quench buffer solution from
the fifth well to the plurality of reaction tubes, and
then transfers a sample from each of the reaction tubes
to a separate well of the microtiter plate. In this
fashion, the degree of binding reaction between the
hybridization solution and the separate nucleic acid
samples can conveniently be measured.
In another, more detailed feature of the
invention, the XYZ pipetter is further associated with an
optical sensor, and the steps of selectively alternating
and fixing the magnetic field are accomplished by moving
the pipetter's sampling tip to a location where it can be
detected by the optical sensor. By appropriate
programminS, the precise timing cf the steps of
selectively alternating and fixing the magnetic field, as
well as the time delays between the steps of using the
pipetter to transfer the hybridization solution and the
solution of magnetically responsive particles, are
precisely controlled.
In other, more detailed features of the
invention, the hybridization solution includes both
nucleic acid probes havin~ an attached label and nucleic
acid probes having attached biotin or hapten molecules,
and the magnetically responsive particles have attached
to them avidin, streptavidin or antibody molecules. The
process thereby effects a sandwich assay. The label may
be a fluorescent or luminescent moiety, or a labeling
enzyme. The labeling enzyme advantageously can exhibit
fluorescence or luminescence, and the process can further
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WO91/15768 PCT/US91/02323
9 2~8~
include a step of assaying the samples carried by the
microtiter plate using a spectrophotometer, fluorometer,
or luminometer.
In another aspect, the invention utilizes ligand
derivatized magnetically responsive particles to separate
single stranded target from heterologous nucleic acid in
a hybridization assay. The magnetically responsive
particles can have streptavidin stably attached thereto
through a covalent spacer. The length and composition of
the attachment to the particles is critical to their
performance.
Other features and advantages of the present
invention should become apparent from the following
description of the preferred embodiment process, taken in
conjunction with the accompanying drawings, which
illustrate, by way of example, the principles of the
invention.
BRIEF DESCRIPTION OF_THE DRAWINGS
FIG. 1 is a perspective view of an XYZ pipetter
and an associated array of reagent wells and reaction
tube holders, for performing a nucleic acid hybridization
assay in accordance with the preferred process of the
invention.
FIG. 2 is a plan view of the array of reagent
wells and reaction tube holders depicted in FIG. 1.
FIG. 3 is a generalized flowchart depicting the
operational steps performed by the XYZ pipetter of FIG.
1, in performing the nucleic acid hybridization assay of
the invention.
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' WO91/15768 PCT/US91/02323
2~0~ O ~
FIG. 4 is a more detailed flowchart of the
operational steps performed by the X~Z pipetter in mixing
a hybridization buffer with a number of separate nucleic
acid samples.
FIG. 5 is a more detailed flowchart depicting
the operational steps performed by the XYZ pipetter in
admixing a solution of magnetically responsive particles
with the separate nucleic acid samples.
FIG. 6 is a more detailed flowchart depicting
the operational steps performed by the XYZ pipetter in
washing the reacted nucleic acid samples.
FIG. 7 is a more detailed ~lowchart depicting
the operational steps performed by the XYZ pipetter in
transferring the reacted nucleic acid samples to a
microtiter plate, for subsequent reading.
DE~AILED DESCRIPTION OF_T~E INVENTION
Reagents appropriate for use in the process of
the invention and methods of preparing them are herein
described. It will be appreciated that other reagents
and processes, known to those skilled in the art, can
alternatively be utilized.
Oligonucleotide probes can be synthesized using
various standard proced~res and reagents. Preferably,
the probes are chemically synthesized using methods well
known in the art. See, for example, Ruth, PCT
Publication No. W084/032~5, which is incorporated herein
by reference. Capture probes were prepared containing a
biotin moiety attached to either an internal base as
described in W~84/03285, or to the 5'-terminal nucleotide
through a spacer and a C-6 amino modifier. The amino
:
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- :- ~ . . . ~ ~ - .: .. - . . - : .
WO91/15768 PCT/US91/02323
terminated oligomers were synthesized using an automated
DNA synthesizer, such as Applied Biosystems, Inc. Model
380 (Foster City, CA). Prior to the start of synthesis,
spacer phosphoramidite (0-dimethoxytrityl-
diethyleneglycol-0'-(cyanoethyl-N,N-diisopropyl-
phosphoramidite)) and 5'-amino-modifier C~ [3-(4-
monomethoxytrityl- amino)hexyl-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramidite], (Glen Research Corp.,
Herndon, VA) were resuspended in anhydrous acetonitrile.
More specifically, the spacer phosphoramidite -
was synthesized as follows. Diethyleneglycol (10 g, 94.2
mmole) was rendered anhydrous by repeated evaporation of
pyridine under vacuum. 4,4'-dimethoxytritylchloride
(3.19 g, 9.4 mmole) in anhydrous pyridine (20 ml) was
added with stirring for 1 hour. After vigorous stirring
in ice water (100 ml), the reaction mixture was extracted
with 75 mls methylene chloride, 3 times. Three back
extractions with 75 mls water and drying over sodium
sulfate were followed by filtration and rctoevaporatlon
to small volume. The m~xture was applied to a SiO2 column
(2.5 X 30 cm), pre-equilibrated in ethylacetate:
cyclohexane (1:2 v/v) and eluted in the same solvent.
After TLC on SiO2 (ethylacetate:cyclohexane, 2:1 v/v), the
proper fractions were located by W absorbance and color
reaction after HCL vapor exposure. The fractions were
combined and rotoevaporated to dryness. The residue was
dissolved in 20 mls benzene, extracted 3 times with water
and lyophilized (3.16 g, 7.7 mmole 0-dimethoxytrityl-
diethyleneglycol, 82% yield).
The anhydrous residue was redissolved in
tetrahydrofuran (20 ml) and evaporated under vacuum. The
residue was redissolved in tetrahydrofuran (25 ml) and
diisopropylethylamine (3 ml) and cooled in a dry ice-
ethanol bath under argon. 2-cyanoethyl-N,N-
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WO91/15768 PCT/US91/02323
0~ 12
diisopropylchloro- phosphoramidite (2 ml, 9 mmole) was
added with stirring (20 minutes) followed by an
additional 20 minutes stirring at room temperature.
Insoluble salts were vacuum filtered under dry argon and
washed with anhydrous tetrahydrofuran. After vacuum
concentration, ethylacetate (50 ml) was added to the
syrup and extracted 3 times (1 M sodium carbonate, 150
mls each). Drying over sodium sulfate, filtration and
concentration were followed with the addition of
ethylacetate:cyclohexane (1.2, 5 ml). After elution from
a silica column and TLC (as described above), fractions
were combined and rotoevaporated to dryness. The residue
was lyophilized from benzene and gave an oily residue
(2.9 g, 62% yield). The residue was redissolved in
benzene, aliquoted and lyophilized in septum capped vials
under Nz, and stored at -20 C.
: '
The oligomers were purified by reverse phase
HPLC on C-8 silica columns and analyzed by 20%
polyacrylamide gel electrophoresis. The oligonucleotide-
containing reactive amine spacer arm was reacted for 1
hour with a 100 to lO00 fold molar excess of NHS-X-biotin
(N-hydroxysuccinimidyl-aminocaproic-biotin; CalBiochem,
La Jolla, CA) in 0.2 M sodium bicarbonate, pH 8.5.
Biotinylated probe was purified from free probe and
excess biotin by reverse phase ~PLC on C-8 silica columns
eluted with an acetonitrile gradient, then desalted and
ethanol precipitated. Product purity was determined by
analytical gel electrophoresis.
Direct labeled alXaline phosphate-DNA conjugates
can be prepared according to a procedure adapted from the
method of Jablonski, et al. Nucl. Acids Res. 14:6115
(1986), which is incorporated herein by reference.
B~iefly, the linker arm nucleosides are thymidine analogs
modified by replacement of the C-5 methyl with an 11 atom
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.
W091~15768 PCT/~S91tO2323
13 2~;0~19
linker arm which terminates in a primary amine. Such
probes are purified by conventional methodologies and
exhibit essentially unaltered behavior with respect to
physical characteristics as compared to unmodified
probes. More specifically, linker arm nucleoside 3'-
phosphoramidite was prepare by the method of Ruth, et al.
DNA 4:93 (1985), which is incorporated herein by
reference, and incorporated into oligonucleotides using
an automated DNA synthesizer, such as Applied Biosystems,
Inc. Model 380 (Foster City, CA). The oligomers were
purified by reverse phase HPLC on C-8 silica columns and
analyzed by 20% polyacrylamide gel electrophoresis.
Product purity was determined by analytical gel
electrophoresis.
Linker arm oligomers were first derivatized with
disuccinimidylsuberate (DSS). One volume of oligomer in
0.2 M sodium bicarbonate was combined with a 30 fold
excess of DSS in two volumes of DMSO. After 3 minutes at
a~ient temperature, the reaction was applied to FPLC G-
25 gel filtration column and eluted in 1 mM sodium
acetate, p~ 5Ø The fractions were monitored by flow-
through absorptiometry at 260nm, collected and
concentrated by microconcentrator (Centricon lOK; Amicon,
Danvers, MA).
Sodium chloride crystals were added to the
activated probe to a final concentration of 3 M. A two
fold molar excess of calf intestine alkaline phosphatase
(Boehringer Mannheim, Indianapolis, IN) in 3 M NaCl, O.l
M bicarbonate, pH 8.25 was added and maintained for 16
hours at ambient temperature. The conjugate was purified
by FPLC anion exchange and eluted in a NaCl gradient.
Peak fractions were collected by 260/280 nm absorption
ratios. Analytical nondenaturing polyacrylamide gel
electrophoresis confirms product purity.
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WO91/15768 PCT/US91/02323
~ 2~a~9 14 ~'
The invention utilizes ligand derivatized
magnetically responsive particles, including those which
are magnetic or paramagnetic. A variety of materials can
be used, inciuding oxides of iron, chromium and titanium
or other metals. The particles should be small enough to
remain dispersed, having an average settling time of
greater than about three minutes. Preferably, they are
about .l - lO~ in diameter, more preferably about l~.
Various ligands, other than nucleotides or
oligonucleotides can be utilized, including, for example,
avidin or streptavidin, biotin, haptens (including, for
example, dinitrophenol (DNP) or digoxigenin, with a
carrier) lectins, or antibodies (such as those against
biotin, fluorescein or digoxigenin).
The length and composition of the covalent
linkage between the magnetically responsive particles and
the ligands are important to efficient performance.
Conventionally, the protein is crosslinked to the solid
matrix using nonspecific glutaraldehyd2, carbodiimides,
or cyanogen bromide crosslinking. Both methods produce
the protein coupled to the particle. However, activity
is significantly reduced and efficiency and strength of
binding in subsequent particle hybridization assays is
generally unacceptable. For example, Beebe et al., in
PCT Publication #WO 88/02785, use avidin linked to
cellulose beads using N,N'-carbonyldiimidazole, and
require the use of 33 mg of beads per assay to have
adeq~uate binding sites. In PCT Publication #W0 86/073~7,
Snitman et al., coat anti-fluorescein antibody onto l/4
inch polystyrene beads by adsorption, and use one bead
- (equivalent to approximately 120 mg) per assay. In the
method described below, the attachment of avidin or
streptavidin to magnetically responsive particles
produces particles with a binding capacity of
approximately l X lO9 moles (l nanomole) of biotin
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WO91/15768 PCT/US91/02323
2 ~a~
binding sites per mg of particles. This allows the use
of only 15 ~g of beads per assay, or less than l/lOOo the
amount of particles in the prior art methods. The prior
art methods, therefore, cannot provide particles
appropriate for use in the invention since it is
impractical to use milligrams of beads per assay in terms
of cost and performance, and magnetic clearing cannot be
accomplished with such large (milligram) amounts of
beads.
The covalent linkage produced as described below
contains multiple, alternate hydrocarbon and amide
(including urea) residues. The linker contains at least
three total residues and is preferably less than eight
total residues. The method described below produces
proteins attached to magnetically responsive particles
through a relatively long linkage (20-60 atoms) with a
mixed hydrophilic/hydrophobic character. After testing
many lengths and compositions of linkage, this linkage -
has proved to be the most preferred.
Additionally, linkage of proteins to particles
using conventional glutaraldehyde, carbodiimide, or
cyanogen bromide crosslinking also gives a hydrolyzable
linkage which may not be stable to storage conditions in
aqueous solutions or mild acids and bases. The present
invention provides linkages which are chemically stable
to aqueous solutions or mild acids or bases, as, for
example between pH 4 and 8 and is additionally stable to
many amine-containing buffers, such as Tris, which are
often used in biochemical procedures. The resulting
particles can be stored and used much more conveniently.
Streptavidin derivatized magnetically responsive
particles were prepared. Briefly, 10 ml of aqueous
suspension of amine derivatized magnetic particles,
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WO91/1~768 PCT/US91/023t3
~n8~19 16
approximately 1 ~m in diameter (Advanced Magnetics, Inc.,
Cambridge, MA) (50 mg/ml) was centrifuged at 12,000 rpm
for 30 minutes. The supernatant was discarded and the
precipitate was fully resuspended, by vortexing in 20 ml
of H2O and recentrifuged. The particles were further
washed in 20 ml portions of water, twice in methanol, 10
methanol/triethylamine, twice in methanol, and finally
twice in ether. The particles were completely
resuspended each time. The particles were thoroughly
desiccated over P2O5-NaOH in a vacuum.
Precipitated particles were freely suspended in
anhydrous dioxane (10 ml), and then centrifuged off in a
corex tube. After removing the supernatant, the
particles were resuspended in fresh dioxane tlO ml), then
1,6-diisocyanatohexane (2 ml) was quickly mixed in and
the reaction tube placed on a rotator, overnight. Next
day, the magnetic particles were centrifuged, the
supernatant removed, and the particles were carefully
washed ~ith anhydrous dioxane by vortexing and
centrifugation, 3 times. Finally, the precipitate was
resuspended in lo ml of dioxane, to which a solution of l
g 1,6-diamino-hexane in 5 ml of dioxane was added. The
reaction tube was placed on the rotator overnight. Next
day, the particles were centrifuged and then washed three
times with dioxane (20 ml portions). The dioxane-moist
particles were resuspended in a solution of glutaric
anhydxide (1 g), p-dimethylaminopyridine (1 g), anhydrous
acetonitrile (15 ml) and anhydrous pyridine (5 ml). The
reaction mixture was again placed on a rotator overnight.
After centrifugation and removal of the supernatant, the
solution-moist particles were resuspended in a mixture of
anhydrous pyridine (15 ml) and acetic anhydride (3 ml)
and placed on a rotator for 3 hours. The particles were
centrifuged and washed twice in pyridine-water (3:1), 20
ml each, then in acetonitrile, 4 times. Finally, they
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WOgl/15768 PCT/US91/02323
2 ~ 8 ~
17
were resuspended in anhydrous acetonitrile (10 ml) giving
a mixture of approximately 50 mg/ml of carboxy-
derivatized magnetic particles, which can be stored at -
20C for 1 year or longer.
Three ml of the above suspension were
centrifuged. After removing the supernatant, the
particles were suspended in dimethylformamide (1.5 ml).
A solution of N-hydroxysuccinimide (185 mg),
trifluoroacetic anhydride (140 ~1) in dichloromethane
lo (2.5 ml) was made, to which 1-methylimidazole (320 ~1)
was added with cooling and exclusion of moisture. The
mixture was diluted with dimethylformamide t2.5 ml)
before being added to the magnetic particles prepared
above. The mixture was kept in a screw cap vial with
teflon linings and placed on a rotator for 6 to 12 hours.
The particles were centrifuged and thoroughly washed 6
times with dimethylformamide (10 ml each). They were
then transferred into an eppendorf tube and $urther
washed with anhydrous acetonitrile 3 times. They were
finally resuspended in anhydrous acetonitrile (1.5 ml).
A 100 ~1 aliquot of suspension was dried down in a
desiccator in a vacuum to determine weight of particles
per volume suspension.
The activated particles were collected to the
side of the test tube by a magnet and the supernatant was
decanted. A 20 mg/ml solution of streptavidin in 0.1 M
sodium bicarbonate, 0.05~ azide was added to the moist
particles at 0.24 mgs streptavidin per mg particles. The
particles were rotated overnight. The particles were
washed four times in 1 X SSC, 0.1% SDS and then brought
to 50 mg~ml in storage buffer (1 X SSC, 1 mg/ml BSA,
0.05% triton X100 and 0.05% azide). Protein and 14C-
biotin binding assays were performed to determine the
streptavidin loading and biotin binding capacity per mg
,
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WO91/15768 ~CT/US91/02323
~0~ 18
particles, respectively. Other ligands such as
appropriate antibodies, including those to biotin,
fluorescein and digoxigenin, can be attached to magnetic
particles in a similar manner.
The general procedures of nucleic acid
hybridization assays will now be discussed. Preferably,
a sandwich type format is used to detect the presence of
a particular nucleotide sequence in a sample.
Appropriate s~mples include, for example, cell or tissue
extracts, body fluids such as blood or blood products,
urine, saliva, food or other material suspected of~ -
containing nucleic acids or a particular nucleic acid
sequence. Such sample must be treated so that nucleic
acids are in solution and available for hybridization.
Typically, sample preparation includes lysis of the
cells, if present, followed by some separation of
cellular nucleic acids from other cellular material. -
Cells are typically lysed by osmotic pressure, detergent
or heat disruption of the membrane, mechanical shearing,
ultra sound, or a combination of these methods. Nucleic
acids can be purified from other cellular materials using
methods of solvent extraction, for example, phenol
chloroform, ion exchange, or size exclusion, such as
dialysis or gel filtration, centrifugation, or a
combination of these methods. For some samples,
purification of nucleic acids is unnecessary. As one
example, outer cell membranes are lysed by incubation in
buffer containing sucrose and non-ionic detergent.
Cytoplasmic debris is then removed by pelleting of the
nuclei by centrifugation. Addition of detergents and
incubation with proteinase K causes lysis of the nuclear
membranes and releasing chromosomal nucleic acid. These
procedures can be carried out automatically, as described
below.
. , . . , ,, ,, ; . :
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WO91/1~76X PCT~US91/02323
~ 19 ' `'
Where small amounts of nucleic acid 2 ~ ~e
target sequence are present, amplification procedures may
be used to specifically increase the target.
Amplification is typically achieved using standard
procedures such as polymerase chain reaction (PCR), or
ligation amplification or other method of amplification.
Such methods are well known to those skilled in the art.
See, generally PCR TECHNOLOGY, (H. A. Erlich, Ed.
Stockton Press, 1989) and United States Patent Nos.
4,683,1~5; 4,683,202; and 4,800,159, which are
incorporated herein by reference.
In PCR primer nucleotide sequences complementary
to target sequences on opposite strands flanking the
sequence of interest are prepared. In the presence of an
appropriate DNA polymerase and nucleotide precursors, and
under suitable reaction conditions, multiple copies of
the particular target sequences defined by the flanking
primers is exponentially generated. Where appropriate a
label! such as biotin or enzyme, can be covalently
attached to one of the primers, resulting in an amplified
labelled oligonucleotide. Alternatively, primer can
contain long or short linker arms without interference
with PCR procedures.
More specifically, target DNA free of
interfering substances is denatured, as by heating to
about 95~C, and hybridized with primer oligonucleotides.
A polymerase such as ta~ 1 (Cetus Corp., Emeryville, CA)
binds to the hybridized primer sequences and catalyzes
the synthesis of new complementary strands in the
presence of excess nucleotide triphosphates. The newly
formed strands are separated from the template strand by
thermal denaturation. As the temperature is lowered, new
primers bind to the template and the process is repeated.
These repetitive reactions are carried out automatically
, i , , , . ., - : . -
j WO91/15768 PCTtUS91/02323
2~ 19 20 ~
in the thermocycler, as described below.
The present invention includes certain novel
features which are advantageously exploited in a variety
of nucleic acid hybridization formats. The use of
magnetically responsive particles to which the target DNA
can be bound permits the efficiency of solution
hybridization of target and probe while providing a
effective subsequent vehicle for the separation of
hybridized and non-hybridized nucleic acid. Moreover,
the use of magnetically responsive particles facilitates
the automation of the assay system. The direct labeling
of probes permit ligand/ligand, such as biotin/avidin or
hapten/antibody, affinity to be utilized to immobilize
the probe/target complex on the magnetically responsive
particles.
Preferably, a sandwich type for~at is used to
detect the presence of a particular nucleotide sequence
in a sample. For example, biotinylated capture probe,
having a sequence complementary to the target sequence or
a sequence in proximity thereto is combined with a sample
under conditions which permit hybridization between the
capture probe and the target. A detection probe
comprising a labeled sequence complementary to the target
sequence or a sequence in proximity thereto, ? S also
allowed to hybridize with the sample. The affinity of
biotin for avidin or hapten to antibody is utilized to
attach target/capture probe/detector probe complexes to
magnetically responsive particles, where their presence
can be detected or quantified.
Alternative sandwich assay formats can also be
used, as will be appreciated by those skilled in the art.
For example, magnetic or paramagnetic microspheres can be
derivatized with other materials, such as biotin, haptens
, ......... :: . .... . ..... . - . - .. - . . :
-.;: . . : , : , . .. ..
WO91/15768 PCT/US91/02323
~ 21 2~
(including, for example, dinitrophenol (DNP) or
digoxigenin with an appropriate carrier, such as BSA),
avidin lectins, or antibodies (such as to biotin). The
capture probes can, in turn, be derivatized with a non-
oligonucleotide moiety complementary to that on themicrospheres, such as avidin, antibodies, carbohydrates
or haptens. Additionally, various labels can be used on
the detection probe, including radioactive, components of
an enzymatic reaction, chemiluminescent, bioluminescent
and fluorescent moieties.
Preferably the target and the probes are
composed of DNA. However, it will be appreciated that
the target and/or the probes can be composed of RNA such
that the method utilizes DNA-RNA or RNA-RNA
hybridization.
With reference now to the drawings, and
particularly to FIGS. 1 and 2, there is shown a fully-
20 automated apparatus for performing nucleic acid -~
hybridization assays as described above using
magnetically responsive particles and a biotin/avidin
conjugate in performing a sandwich assay. The apparatus
includes an XYZ pipetter 11, a test tube rack 13, a
reaction tube array 15, a plurality of optical sensors 17
associated with the reaction array, a plurality of
reagent wells lg, a thermal cycler 21, and a microtiter
plate 23. The XYZ pipetter includes a conventional
sampling tip Z5 for transporting various DNA samples and
reagents from one location to another, to perform the
assay. Because the apparatus enables the assay to be
performed fully automatically, with substantially no
intervention required by trained personnel, the assay can
be performed to high precision, and with excellent
repeatability.
'. '
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~`~ WO91/15768 PCT/US~1/02323
2~ 9 22
The nucleic acid hybridization performed by the
apparatus of FIG. 1 uses a sandwich technology, in which
a DNA probe, designed to hybridize with the target DNA,
is bound to biotin, while its conjugate, avidin, ls bound
to magnetically responsive particles. The DNA samples to
be tested are first mixed with the biotin-linked DNA
probe and a second proximal DNA probe labeled with a
reporter group and allowed to react. Thereafter, the
mixture is combined with the magnetically-labeled avidin
and allowed to react. The magnetic label is then
separated from the unreacted DNA sample and DNA probes,
and the reporter labeled DNA probe bound to the DNA
sample is then detected, to complete the assay.
: '
The XYZ pipetter 11 is a conventional apparatus
available from several commercial sources. It is -
operated under the control of an associated personal
computer, which allows great flexibility in selecting the -
processing details. The pipetter is equipped with a ~`
positive-flow washing station 27 for use in cleaning the
sampling tip 25 and a liquid level sensor (not shown) on
the tip.
... .
As previously mentioned, the apparatus includes
a reaction tube array 15 that carries reaction tubes 29
in which the binding reactions are made to occur. In
particular, the array includes 48 temperature-controlled
reaction tube wells, in a 6 x 8 arrangement, with
permanent magnets positioned adjacent to each well. Each
well further has associated with it a variable-speed
motor capable of alternating clockwise/counterclockwise
rotation at 400-to-1000 rpm, through 45-to-1080 degrees
of rotation. The reaction tube wells are positioned
slightly off center on the vertical motor shaft. Control
of each bank of six motors is made using the adjacent
WO91/15768 PCT/US91/02323
~. ^. ~
~ 23 2~
photoelectric switch 17, which can be controlled using
the pipetter tip 25. In this fashion, the motors are
controlled by mere movement of the pipetter tip, without
the need for a separate computer interface.
The additional plurality of reagent wells l9 are
provided to accommodate various buffers and reagents, as
will be described below. A particle mixer l9a, which
includes an associated motor (not shown) for providing
vortexing motion, is provided for carrying the
magnetically responsive particle reagent. This particle
mixer has an associated photoelectric switch 31, for
selectively switching the motor on and off, although ~ -
other types of switches can be used.
The magnetic reaction array 15 holds the various
reaction tubes 29 at a selected temperature and functions
to selectively disperse and collect the magnetically
responsive particles. The particles remain in liquid
suspension so long as the tube carrying it is rotated by
its associate~ motor. When the oscillating motion
terminates, however, the particles are quickly collected
to the sides of the reaction tube under the force of the
adjacent permanent magnets, allowing an efficient removal
of excess liquid by the pipetter tip 25. The subsequent
addition of further liquid reagents and resumption of the
oscillating motion causes an immediate uniform
resuspension of the magnetic particles.
The thermal cycler 21 is a temperature-
programmable microfuge test tube holder adapted to carry
out a thermocyclic amplification reaction. Temperature
control is preferably provided from about 25 to 110 C,
with a temperature ramping speed of about 3 seconds per
dçgree. Amplified samples can be obtained from the
thermal cycler for transfer directly into the
. - .: . . - .,, , , :. .
- . -
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WO9l/15768 PCT/US91/02323
~Y; 2~ 19 24 ~
hybridization reaction tubes 29 using the pipetter tip -~
25.
With reference now to the flowchart of FIG. 3,
the preferred process for providing a nucleic acid
hybridization assay will be described. In an initial
step 41, the various nucleic acid samples are manually
placed in separate test tubes of the test tube rack 21, a
hybridization solution containing a hybridization buffer
with oligomers, is placed in a reagent well l9b, and a
solution of magnetically responsive particles is placed
in the particle mixer well 19a. After this has been
done, the XYZ pipetter apparatus is in condition to
initiate the fully-automated portion of the nucleic acid -~
hybridization assay.
Thus, in a subsequent step 43, the
aspirating/dispensing tip 25 of the XYZ pipetter ll
sequentially aspirates the hybridization solution from
the well l9b and the successive nucleic acid samples from
the test tubes of the test tube rack 21 and transfers -
these combined solutions to separate test reaction tubes
of the reaction tube array 15. After a prescribed time
duration, during which time the hybridization solution is
allowed to undergo a binding reaction with the nucleic
acid samples, the XYZ pipetter, in a subsequent step 45,
aspirates the solution of magnetically responsive
particles and transfers it sequentially to the reaction
tubes in the reaction tube array.
.:
Thereafter, after a prescribed time duration in
which the avidin molecules adhered to the magnetically
responsive particles are allowed to undergo binding
reactions with the biotin present in each solution
sample, the liguid portion of each sample is removed at - ~
35 step 47 and the remaining magnetic particles, with bound -
WQ9l/157h8 PCr/US~1/02323
2~ 8~
DNA probe and possible DNA sample, is washed using the
wash buffer solution carried in a well l9c. In a
subsequent step 48, the pipetter tip 25 is used to remove
the wash solution from the reaction tubes and to transfer
to the tubes a substrate buffer solution. After a
prescribed time duration, during which an enzymatic
process is allowed to occur, the pipetter tip 25
transfers a quench buffer solution from the well l9d and
a sample from each reaction tube to a separate well of
the microtiter plate 23 at step 49. The tagged DNA probe
in these samples is thereafter read, for example using a
fluorometer.
FIG. 1 is a flowchart depicting, in greater
detail, the step 43 from FIG. 3 of combining the
hybridization solution with the various nucleic acid
samples in the reaction tubes of the reaction tube array
13. In particular, in an initial step 51, the pipetter's
aspirating/dispensing tip 25 is controllably moved to the
well l9b, where it aspirates a prescribed amount of the
hybridization solution. Thereafter, in step 53, the tip
is moved immediately to the first test tube in the test
tube rack 13, where it aspirates a prescribed amount of
the nucleic acid sample carried in that test tube. The
tip is then moved in step 55 to the first reaction tube
in the reaction tube array 15, where it dispenses the
combined hybridization solution and nucleic acid sample
into the tube. The tip is then washed at the wash
station 27, in step 57.
Thereafter, in step 59, it is determined whether
or not the last of the nucleic acid samples has been
- aspirated from the test tube rack 13. If not, and the
last tube in the current column has not been processed,
the sample number (n) is incremented by one at step 61,
and the pro~ram returns to the initial step 51 of
' ' ~ ~ . :
- . :
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WO9l/15768 PCT/US9l/02323
2~ 9 26 ~
aspirating the hybridization solution from the well l9b.
Eventually, it will be determined at step 60 that
hybridization solution and sample have been dispensed
into the last tube in the current column of tubes. When
that happens, the program advances to step 63 in which
the pipetter tip 25 is moved to the optical sensor 17 for -
the reaction tube column just completed, to activate its
associated column of motors. At step 65 the tip is
washed at the wash station 27. The sample number is then
incremented and the program returns to step 51 where it
aspirates a prescribed amount of hybridization solution.
Eventually, it will be determined at step 59 that the
last of the n nucleic acid samples has been aspirated
from the test tubes-of the test tube rack 13 and
transferred to a separate one of the reaction tubes of
the reaction tube array 15. The program then advances to
a step 63, in which the pipetter tip 25 is moved to the
optical sensor(s) 17 for the affected column(s) of
reaction tubes, to switch on the corresponding motors, to
begin agitating the solution~. Finally, at step 65, the
tip is washed at the wash station 27.
' ' ',
FIG. 5 is a flowchart depicting, in greater
detail, the step 45 in FIG. 3 of adding the magnetically
responsive particle solution to the reaction tubes of the
reaction tube array 15. In particular, in an initial
step 67, the pipetter's aspirating/dispensing tip 25 is
moved to the optical sensor 31, to initiate agitation of
the particle mixer for the particle mixing well l9a
containing the magnetically responsive particle solution.
After a prescribed, relatively short time duration, the
tip, at step 69, is moved to the optical sensor(s) 17 to
inactivate the corresponding column(s) of motors in the
reaction tube array 15. The tip then returns to the
optical sensor 31, at step 71, to terminate agitation of
the particle mixer. At step 73, the tip then aspirates a
.
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W09l/15768 PCT/US91/02323
~` 27 2~
prescribed amount of the magnetically responsive particle
solution and, at step 75, switches the optical sensor 31
to again agitate the particle mixer. At subsequent step
77, the tip dispenses the magnetically responsive
particle solution into the first reaction tube Cl, after
which the tip is washed at the wash station 27, at ~tep
79.
The program then proceeds to step 81, where it
is determined whether or not the magnetically responsive
particle solution has been dispensed into the last of the
reaction tubes carrying nucleic acid samples. If not,
the program proceeds to step 83, where it is determined
whether or not the solution has been dispensed into the
last reaction tube in the current column of tubes. If
not, the reaction tube number is incremented by one, at
step 83, and the program makes a repeated pass through
the subroutine, for the n~xt reaction tube, beginning
with the step 71 of terminating agitation of the particle
mixer. Eventually, it will be determined at step 83 that
the magnetically responsive particle solution has been
dispensed into the last reaction tube in the current
column of tubes. When that happens, the program proceeds
to step 87, where the pipetter tip 25, switches the
optical sensor 17 for the reaction tube column just
completed, to activate its associated column of motors.
The column number is then incremented at step 87, and the
program returns to the step 69 of using the pipetter tip
to inactive the next column of motors in the reaction
tube array 15.
Eventually, it will be determined at step 81
that the magnetically responsive particle solution has
been dispensed into the last of the reaction tubes that
- 35 contains a nucleic acid sample. When this occurs, the
program proceeds to step 91, where the pipetter tip 25
- : ~ - : : . . - . -
-; : .
WO91~1S76X PCT/US91~02323
2 ~ ~a~l9 28
switches the optical sensor 17 for the last reaction tube
column, to activate its associated column of motors in
the reaction tube array 15. The tip is washed at the
wash station 27, at step 93.
FIG. 6 is a flowchart depicting, in greater
detail, the washing step 47 of FIG. 3. In particular, in
an initial step 95, the pipetter's aspirating/dispensing
tip 25 is moved to the first optical sensor 17 to
inactivate the first ~olumn of motors in the reaction
tube array 15. This column is designated by the
referénce variable X. When the motors stop oscillating
the reaction tubes, the magnetically responsive particles
are attracted to the sides of the reaction tubes by the
adjacent permanent magnets. While this is occurring, the
tip 25 is washed at the wash station 27, at step 97.
Thereafter, at step 99, the tip is moved to the next `
optical sensor 17, to inactivate the second, or X + l,
column of motors in the reaction tube array. This allows
the magnetically responsive particles to separate from
the buffer solution for this second column of reaction
tubes while a wash cycle is performed on the first, or X,
column of reaction tubes. Next, at step lOl, the tip 25
aspirates the buffer solution from the middle portion of
the first reaction tube C1 and, at step 103, discards the
aspirated buffer solution and is washed at the wash
station 27. At step lO~ of the program, it is then
determined whether or not the buffer solution has been
aspirated from the last of the six reaction tubes of the
column X in question. If not, the reaction tube number
(n) is incremented by one, at step 107, and the program
returns to the step lOl of aspirating the buffer
solution.
Eventually, it will be determined at step 105 . -
that the buffer solution has been aspirated from the last
.
- : ..
~: . , - : ~ ~ .
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WO9l/15768 PCT/US9l/02323
~ 29 2~80~ ~
of the six reaction tubes of the column X in question,
and the program then will proceed to a step 109, in which
the tip 25 aspirates a wash buffer solution from the
- reagent well 19c, and step 111, where the wash buffer
solution is dispensed into all six reaction tubes. The
tip is then washed at the wash station 27, at step 113,
and the tip then is moved, at step 115, to ~he first
optical sensor 17 to activate the column X of motors in
the reaction tube array 15. This begins agitating the
six reaction tubes of the column, to homogenize the
contained solutions and enhance the washing being
effected.
Thereafter, at step 117, it is determined
whether or not the variable X, identifying the column
number for the six reaction tubes just operated on, is a
maximum. If not, the variable X is incremented by one,
at step 119, and the program returns to the step 99 of
switching the optical sensor 17 ~or the X + 1 column of ~-
reaction tubes, to inactivate the associated column of
motors. By the time this step 99 is again reached, the
motors for the previous column X of reaction tubes will
have been stopped for sufficient time to allow the
magnetically responsive particles and buffer solution to
separate ~rom each other. The program then proceeds
through the subroutine in the same fashion as described
above, for this next column of reaction tubes.
Eventually, it will be determined at step 117
that the final column of reaction tubes has been operated
on. When that happens, the program proceeds to a step
121, where it is determined whether or not a referencP
- variable k has reached the number four. If not, the
variable X is reset to one and the variable k is
incremented by one, at step 123, and the program returns
to the initial step 99 of switching the optical sensor 17
- .: .
WO91/1576X PCT/US9~/02323
2l~0~ 1() 30
to inactivate the first column of motors. The program
then proceeds through the program subroutine in the same
fashion as described above, to effect three wash cycles,
until the variable k has reached the numeral 4. When
this happens, the step 109 is modified such that the tip
aspirates a substrate buffer solution from the well l9d,
rather than the wash buffer solution from the well l9c.
Eventually, it will be determined at the step 121 that
four passes through the program subroutine have been
lo completed, and this program subroutine is then exited. --
FIG. 7 is a flowchart depicting, in greater
detail, the step 49 of quenching the substrate buffer
reaction and the transfer of reaction samples to the
microtiter plate 23. In particular, in an initial step
125, the aspirating/dispensing tip 25 of the XYZ pipetter
11 is moved to the first optical sensor 17 to inactivate
the first associated colum~ of motors in the reaction
tube array 15. This column is designated by the
reference varia~le X. After the motors have stopped
oscillating the reaction tubes, the magnetically
responsive particles will be attracted to the sides of
the reaction tubes by the adjacent permanent magnets.
While this is occurring, the tip 25 is washed at the wash
station 27, at step 125b. The variable X is incremented
by one at step 141. Thereafter, at step 125, the tip is
moved to the ne~t optical sensor 17 to inactivate the
next column in the reaction tube array. This allows the
particles to separate from the buffer for this second
column while the samples in the first column are
transferred to a microtiter plate. The tip is then
washed at the wash station 27, at step 127, and then
moved, at step 129, to the reagent well l9e, where it
aspirates a prescribed amount of the quench buffer
solution. The tip then moves to the first reaction tube
of the column of tubes, at step 131, where it aspirates a
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.
WO91/15768 PCT/US91/02323
~ 31 2~Q~ 9
sample from the tube, which is then dispensed by the tip,
at step 133, into a first well in the microtiter plate
23, at step 117. The program then proceeds to a step
135, where it is determined whether or not a sample has
been aspirated from the last of the six reaction tubes in
the column. If not, the number n is incremented by one,
at step 137, and the program returns to the step 127 of
washing the tip 11 at the wash station 27.
Eventually, it will be determined at step 135
that samples have been aspirated from all n reaction
tubes in the column of tubes. When this occurs, the
program proceeds to step 139, where it is determined
whether or not the last column of reaction tubes has been
operated upon. If not, the column number X is
incremented by one, at step 141, and the program returns
to the initial step 125, where the aspirating/dispensing
tip 25 switches the optica~ sensor 17 for the next
column, i.e., column X, in the reaction tube assay 15.
The program then procee~s through the same subroutine as
described above, this time for the next column of
reaction tubes.
Eventually, it will be determined at step 139
that samples have been aspirated from all of the columns
of reaction tubes in the assay 15. When this occurs, the
fully automated portion of the nucleic acid hybridization
assay process will have been completed. The microtiter
plate 23, which then will carry n reaction samples, is
then transported, in step 143, to a suitable fluorimeter,
to provide an accurate and precise measure of the degree
of reaction for each DNA sample.
The sequential operation of the XYZ pipetter 11,
as described above with re~erence to the flowcharts of
FIGS. 3-7, can be accomplished using a computer program
- : .
. , . . ~.
~j WO91/15768 PCT~US91/02323
2~ 32 ~
appropriately written to interface with the particular
XYZ pipetter being used. An accompanying appendix is a
printout of the source code for one suitable program
written to operate on an IBM PS-2 Model 30-286 personal
computer, or equivalent. The program is written in C,
for use with a conventional Packard XYZ pipetter.
Additional operations can also be performed by
appropriate programming of the XYZ pipetter. As
indicated an amplification procedure can be performed
prior to placing the samples in the test tubes.
Similarly, samples requiring preparation prior to
hybridization, as for example cell lysis, can be placed
directly in the reaction tubes and appropriate solutions
added and removed therefrom.
The following examples are intended to
illustrate, but not the invention. -
20EXAMPLE I ~;
Detection of Cloned HIV Fragment
A model system was constructed in which a human
immunodeficiency virus (HIV) fragment was inserted into a
cloning ~ector. Briefly, a SP6-HIV vector (pBHlO-R3) was
obtained from E.I. Du Pont & De Nemours, Wilmington, DE.
The vector was constructed by cloning a 9 Kb of HIV
genomic insert into the Sst-l restriction site of PSP-64.
The plasmid pMBIl07 was constructed by restriction of
pBHlO-R3 with Bam-~l and then religation which resulted
in the deletion of the 3'-LTR region. Cells were
transformed and selected for ampicillan resistance.
Plasmid was purified by CsCl gradient centrifugation
(Maniatis, et al., Molecular Cloning: A Laboratory
Manual, 2nd Ed., Cold Spring Harbor, New York (1989),
which is incorporated herein by reference).
- -
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,
. . .
WO91/15768 PCT/VS91/02323
2a~ ~al ~
pMBI107 was linearized by restriction with Bam-
Hl. A dilution series of Bam Hl-pMBI107 was made in 10
mM Tris, pH 7A5~ 1 mM EDTA (TE). Ten ~1 of each target
5 dilution was added to 10 ~1 of 0.01 mg/ml human placenta
(HP) DNA in TE. Controls contained equal volumes of TE
and HP DNA. Samples were denatured at 95C for 10
minutes, chilled in an ice water bath for 1 minute and
centrifuged briefly to collect condensate. The samples
were placed in tubes in the magnetic sample rack on the
worktable.
Biotinylated and acid phosphatase labelled
oli~onucleotides were prepared as described above, using
sequences listed in Table I. A processing program for
the XYZ pipetter was iniated and prompted for assay
variables, such as a sample number, replicate number,
etc. The XYZ pipetter automatically combined 102 ~1 of
hybridization buffer (6XSSC, pH 8.0, 10% formamide, 0.1
SDS, 0.1 mq/ml BSA) 3 nM biotin-N231, -N226, -N224,
-N211, -N253 and -N229, and 3 nM AP-N218, -N220, -N227, -
N233 and -N234) and 20 ~1 of sample into reaction tubes
in the magnetic rack. The sampling tip washed itself
between each sample to eliminate the possibility of
carryover. Hybridizations proceed at 37C with rotation
for 30 ~inutes.
The XYZ pipetter then automatically added 15 ~1
of a 1 mg/ml streptavidin-particles (SA-particles),
prepared as descrihed above, in 6XSSC, pH 8.0, 10%
formamide, 0.1% SDS, 0.1 mg/ml ~SA to each reaction tube.
Fifteen ~g of SA-particles posses a biotin binding
capacity of approximately 60 pmoles. Thus, there is a 35
fold molar excess of biotin binding sites over biotin
probe. Capture of the ternary hybrid complex onto the
particles proceeds at 37C with rotation for 30 minutes.
~ . : ~ : , . .
WO91/15768 PCTJUS91/02323
~' 34
2a ~ ~13 TABLE I
Probe Sequences
N178 5' -dATA ATC CAC CTA TCC CAG TAG GAG AAA T
N279 5'-dTTT GGT CCT TGT CTT ATG TCC AGA ATG C
N280 5'-dCAT TCT TAC TAT TTT ATT TAA TCC CA
N231 5'-dCTA GGT GAT ATG GCC TGA TGT
N226 5'-dCCC TAT CA~ TTT TGG TTT CCA T
N224 5'-dTGT TGA CAG GTG TAG GTC CTA
N211 5'-dCTG GCT TTA ATT TTA CTG GTA CA
N253 5'-dTGC CAT TTG TAC TGC TGT CTT
N229 5'-dTGC CAC ACA ATC ATC ACC TGC
N218 5'-dTAG AGG GTT GCT ACT GTA TTA T
N220 5'-dTAG TTC CTG CTA TGT CAC TTC C
N227 5'-dCTG TCT BTAC TTT GAT AAA ACC TC
N233 5'-dTAT TCT TTC CCC TGC ACT GTA
N234 5'-dCTG TAA TAA ACC C~A AAA TTT TGA
A105 5'-dCCC GAG CCG ATG ACT TAC TGG C
A214 5'-dGAT ATC TCA CCC TGG TCG AGG CGG T
A209 5'-dTGT GTG GTG TAG ATG TTC GCG ATT G
N174 5'-dCAG GAG CAG ATG ATA CAG TAT TAG
C103 5'-dAGG CGT TTC CAC ATC TAT ATA GT
C204 5'- dAGT ATC ATC ACC CAC GAT GTG CT
The XYZ pipetter automatically stopped the
rotation of the reaction tubes by tripping a photoswitch
with the sampling tip. Upon cessation of rotation the
paxticles were quickly cleared from solution by the
integral side mounted permanent magnets. The XYZ
pipetter automatically aspirated the buffer from each
reaction tube, washing the tip between each sample, in a
given row. The XYZ pipetter automatically aspirated and
dispensed 200 ~1 wash buffer (4 X SSC, 0.1% SDS) into
each reaction tube, washing the tip between each sample.
The XYZ pipetter then tripped the photoswitch to commence
rotation of the tubes in a given row. The XYZ pipetter
WO91~15768 PCT/US91/02323
~ 2~ 9
proceeded to wash the next row of tubes as described
above until all the samples have been processed. This
process was repeated 2 more times for a total of 3
washes, each at 37~C for 5 minutes.
The above wash process was repeated once more
with the variation that 150 ~l of substrate buffer (30 ~M
4-methylumbelliferyl phosphate (4-MUBP), JBL Scientific,
San Louis Obispo, CA) in 0.l M diethanolamine, (DEA), pH
9.0 (JBL), 5 mM MgCl2,) was dispensed into each reaction
tube rather than wash buffer. Incubation with substrate
proceeded at 37C with rotation for 60 minutes.
Again, the sampling tip pipetter automatically
tripped a photoswitch to stop the rotation of the
reaction tubes in a given row, allowing the particles to
clear from solution. The XYZ pipetter aspirated 40 ~l of
100 mM EDTA from a reagent rack and ll0 ~l of substrate
buffer from the reaction tubes and dispensed the liquid
into a well in a microtiter plate. The XYZ pipetter
performed a tip wash between each sample. The XYZ
pipetter proceeded to quench/transfer the next row of
tubes until all the samples were processed.
At the end of the program, an alarm sounded to
alert the user that the assay was completed. The
microtiter plate was used on a fluorescent plate reader
(Pandex FCA, Baxter Healthcare, Pandex Division,
Mundelein, IL) which automatically scanned each well.
Alternatively, a single well fluorometer can be used
manually. The plate can be read immediately or up to 16
hours after completion. The results are presented in
I Table II.
The results indicate that the minimum
extrapolated detection limit based on a signal to noise
.
WOgl/lS768 PCT/US91/02323
~Q~l9 36 ~
ratio of 2.0 is 4 X 105 copies pMBI107 plasmid target.
The results are linear with respect to target level over
three orders of magnitude. Multiple capture/reporter
probe pairs increase signal without a proportional
increase in noises resulting in better sensitivity
overall.
TABLE II
lo pMBI107 Dilution Series: Multiple Sandwich Pairs
Relative
Fluorescent Signal/
15Copies targetUnits Noise
. ..
4
500,000 10 2.5
5,000,000 66 16.5
2050,000,000 522 130.5
500,000,000 4517 1129.3
EXAMPLE II
Automated Amplification and Detection of Viral DNA
In this example of the invention, it was
demonstrated that it was possible to combine a semi-
automated target amplification assay with the automated
sandwich assay in a HSV model system. It was
demonstrated that open tube amplification could be
performed by the XYZ pipetter without significant
contamination or carryover.
Due to the sensitivity of target amplificaton,
it is extremely susceptible to inter- and intra-assay
contamination. Amplification reactions were set up in a
-, : : .
. . . . , .:
- : : . . -
.. : : ~
- , '~ : ' :;- :'
WO91~15768 PCT/US91~02323
~ 37 2~
clean (i.e. no exposure to amplification products)
biosafety hood in a room separate from the XYZ pipetter.
Clean positive displacement pipettes (PDP) with
disposable pistons and capillaries (Rainin, Woburn, MA),
sterile tissue grade water (Sigma Chemical Co., St.
Louis, M0), sterile light mineral oil (Sigma) and sterile
0.6 ml snap cap tubes (Robbins Scientific, Sunnyvale, CA)
were used to minimize contamination.
Fresh Taq 1 heat stable, recombinant DNA
polymerase (3.1 units AmpliTaq Cetus Corp., Emeryville,
CA) was added to 80 ~1 amplification buffer (62.5 mM KCl,
12.5 mM Tris-HCl, pH 8.3, 1.88 mM MgCl2, 0.13% (w/v)
gelatin, 250 ~M dNTP, 0.31 ~M biotin-A105 and 1.25 ~M
A214). Twenty ~1 water and 20 ~1 template (1 X 104
copies pHSV106 (Bethesda Research Laboratories,
Gaithersburg, MD)) was added to negative and positive
samples, respectively. The final reaction mix contained
50 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgC12, 0.1%
(w~v? gelatin, 200 ~M each dNTP (Pharmacia, Piscataway,
NJ) 7 0.25 ~M biotin-A105 forward primer and l.o ~M A214
reverse primer and 2.5 units AmpliTaq). Samples were
overlaid with 100 ~1 light mineral oil. Amplification
control tubes were capped whereas experimental tubes were
left uncapped and placed on a thermal cycler (Ericomp,
San Diego, CA) which had been modified to fit on the work
table of the XYZ pipetter. An amplification and sandwich
protocol were run concurrently, the sandwich containing a
210 minute timer delay. The amplification protocol
required approximately 200 minutes to complete and
consisted of the following steps: 1 minute denaturation
at 94C; 30 amplification cycles of 1 minute at 55C and
then 1 minute at 94C; 5 minute extension at 72C; 10
minute denaturation at 95 C. The process yielded 1 X 109
fold amplification of target at 100% efficiency.
- . - ~ - . . . . : . . - - :
- . .
WO91/15768 PCT/US91/02323
2~8~9 38 ~
Amplification products were analyzed by agarose
gel electrophoresis, dot blot, Southern blot and
sandwich, the latter as describad in Example I, using as
the detector probe AP-A209. The latter was the only
method that produced quantitative results.
At the end of the amplification program, the
closed tubes were manually uncapped. The XYZ pipetter
automatically inverted the aqueous and oil phases by ! '
dispensing 200 ~l chloroform, equilbrated in lO mM Tris-
Cl, pH 7.5, 1 mM ED~A. The oil/chloroform layer sank to
the bottom and the aqueous layer floated to the top. The
XYZ pipetter then combined 102 ~l hybridization buffer (6
X SSC, pH 8.0, 10% FAM, 0.1% SDS, O.l mg/ml BSA)
l5 containing 3 nM AP-A209 and 20 ~l of amplification
products from the thermal cycler. The automated sandwich
assay proceeded as described in Example I. It is
important that the total amount of biotin (free biotin
primer and biotinylated amplification product) in the -~
sandwich assay does not exceed the binding capacity of
the SA-particles. Twenty ~l of a 0.25 ~M biotin primer
solution is equivalent to 5 pmoles of total biotin which
is 12 times less than the 60 pmoles binding capacity of ;
the particles.
The results indicated that it was pos~ible to
perform target amplification on the worktable of a XYZ
pipetter followed by an automated sandwich assay. The
phase inversion process allowed the sampling tip to
aspirate the sample without contacting the oil layer
which markedly reduced the carryover frequency. The
contamination trequency (cf) was defined as the number of
open negative samples that gave relative fluorescent
units (rfu) greater than twice the rfu from closed
negative samples.
- . - . .- . . . .
- .~ . , - . - . ~ . : ,
- -.: . -
-
WO9~/15768 PCT/US91/02323
~ 3g 2~ 9
C = # o~en neqative rfu > 2 X closed negative rfu
total # open negative
CF = 0/47 (Average results from nine (g)
separate e~periments)
Data from a representative open tube
amplification assay is shown below.
Average Relative Fluorescent
+ or - Units
Tube Status Target _ lactual) -
closed - 20 (22; 18)
- + 22,742 (22,900; 22,S84)
_ 43 (44; 43)
(average closed neg. = 32 rfu)
open - 42 (26; 58)
+ ~3178 (22,610; 23,746) .
_ 50 (56; 44)
t- 229gl (22,952; 23,030) . .
_ 54 (54; 54)
+ 22,694 (22,894; 22,694)
- 36 (32; 40)
cf = # open ne~ative rfu > 64 rfu = 0/4
EXAMPLE III . -
Detection of HIV in Cultured Cells
HIV infected CEM-CM3 cells (ATCC Accession No.
TIB 195) containing approximately 1 to 20 copies of HIV :
virus/cell were harvested and prepared for PCR as :
. - ' : '
.:
. .
.. . . .
~ WO91~15768 PCT/US91/02323
2 ~ 1 9 40 ~
described below. Alternatively, reverse transcriptase
could be used to form cDNA from RNA which could then be
used in PCR.
CEM-CM3 cells were cultured in 75 cc flasks in
RMPI 1640 culture media (M.A. Bioproducts, Walkersville,
MD), 10% fetal calf serum, 1% penicillin-streptomycin, l~
Fungizone (M.A. Bioproducts) and 1% L-glutamine at 37C
in 5% C2- Cells were passed l:6 every 5 to 6 days.
Control cells were passed as follows: 5 mls CEM-CM3:30
mls RMPI media. HIV infected CEM-CM3 cells were passed
as follows: 5 mls HIV infected CEM-CM3 cells: lO mls
CEM-CM3 and 20 mls RMPI media. Uninfected CEM-CM3 cells
were added to infected cells to increase th viral titer.
To maintain a HIV CEM-CM3 continuous cell line without
increasing the viral titer, add 5 mls HIV infected CEM-
CM3 cells to 30 mls RMPI media. Cells were harvested
after 4 to 5 days when titer was maximum. Cells were
pelleted in a clinical centrifuge at lO00 rpm for 15
minutes. The supernatant was discarded and the pellet
was resuspended in 5 mls l X PBS. Cell concent~ation was
quantitated by hemacytometry, usually 2-5 X 106 cells/ml.
Concentration was adjusted to suit.
A dilution series from l X lO1 to l X 105 HIV
infected CEM-CM3 cells were spiked into l X 104 uninfected
CEM-CM3 cells. The cells tVf = 65 ~l) were lysed as
follows: 500 ~l outer membrane lysis buffer (lO mM Tris-
Cl, pH 8.0, 1.5 mM MgCl2, 140 mM NaCl and 0.5~ NP-40) was
added and the mixture was vortexed and centrifuged in a
microfuge. The pellet was resuspended in 50 ~l nuclear
membrane lysis buffer (50 mM KCl, lO mM Tris-Cl, pH 8.3,
2.5 mM MgCl2, 0.1% (w/v) gelatin, 0.05 mg/ml proteinase K,
20 m~ DDT and l.7 ~M SDS) and incubated at 55C for one
hour. The samples were boiled for 20 minutes to
inactivate the proteinase K and centrifuged to collect
- ~ : : - . . .
..
. ~ '- ' .:, ,~ - : ,,
WO91~15768 PCT/US~1/02323
41 2`0~
the condensate. In a laminar flow hood, 25 ~1 of the
sample mix was added to 75 ~1 of amplification buffer (67
mM KCl, 13 mM Tris-Cl, pH 8.3, 2.0 mM MgCl2, 0.13%
gelatin, 267 ~M each dNTP, 0.33 ~M biotin-N174, 1.3 ~M
N224 and 3.3 units freshly added AmpliTaq; Cetus Corp.,
Emmeryville, CA) for a final concentration of 50 mM KCl,
10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl2, 0.1% (w/v) gelatin,
200 ~M each dNTP, 0.25 ~M biotin-N174 forward primer and
1.0 ~M N224 reverse primer and 2.5 units freshly added
heat stable recombinant DNA polymerase (AmpliTaq; Cetus
Corp., Emeryville, CA). The sample tubes were placed on
a thermal cycler and amplified as follows: 1 minute
denaturation at 94OC; 30 amplification cycles of 1 minute
at 55C and then 1 minute at 94C; 5 minute extension at
72C. A 20 ~1 aliquot of the amplified product was
assayed in sandwich using detector probe AP-N227 as
described in Example I and II.
Experimental results indicate that lo ~IV
infected cells could be detected; albeit inconsistently,
by automated sandwich assay after target amplification.
The amount of signal generated is not proportional to the
amount of target present. This may be due to limiting
reaction conditions and/or inhibition by cellular
components during amplification. This latter is
supported by the fact that 1 X 105 infected cells resulted
in complete inhibition. The sandwich assay was not
limiting because there was a 10 fold molar excess of
biotin binding sites on the particles and because
complete turnover of MUBP results in about 70,000 rfu.
WO91/tS768 PCT/US91/02323
2 ~ 8 ~ 42 ~
TABLE III
Relative Average
HIV-CEM per Fluorescent Fluorescent Standard
5 10,000 CEM Units Units Error
(rfu)
1700
28 584 967
24
100 12352
19586 17111 4123
19396
1,000 22060
21592 21825 234
21824
lO,oOo 23470
17494 22041 4028
25160
100,000 8
14 17 11
EXAMPLE IV
Detection of HIV in_Seropositive Blood Sam~les
In this example of the invention, it was
demonstrated that the automated system was able to detect
HIV virus in blood samples from seropositive patients.
Blood was collected from the donor by withdrawal
into a 10.0 ml heparinized or EDTA-treated vacutainer.
The samples were stored at room temperature up to 24
hours after withdrawal.
The blood vacutainer was inverted several times
to mix well. Approximately 7.0 ml whole blood was
removed by volumetric pipette. The blood was transferred
to a cell separation tube (LeukoPrep Cat. No. 2750-2752,
manufactured by Becton-Dickinson, Rutherford, NJ) and
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. :. ',, ~ - :.: :-. -
WO91/15768 PCT/US91/02323
~ 43 2~3~ 9
capped with rubber stopper provided by manufacturer. An
additional 2.0 ml of 1 X PBS (120mM NaCl, 2.7mM KCl, 10mM
phosphate buffered salts) was added to bring the total
volume to 9.0 ml. The LeukoPrep tube was centrifuged at
1600 X g for 20 minutes at room temperature in rotor and
centrifuge designed for containment of infectious
material. After centrifugation, the upper layer of
plasma was removed and discarded. The "buffy coat"
containing mononuclear cells was removed and transferred -
to a 1.5 ml eppendorf tube. From this, 0.5 ml
mononuclear cells was transferred to a 1.5 ml screw-cap
eppendorf tube. The remaining cells were discarded.
Alternate methods of mononuclear cell isolation may be
used, such as Sepracell density gradient method and
Ficoll-Hypague.
One half ml of Lysis Buffer (50mM KCl, 10mM -
Tris-Cl, pH 8.3 (25C), 2.5mM MgCl2, 0.1mg/ml gelatin,
0.45% NP40, 0.45% Tween-20, and 60 ~g/ml proteinase K
added just before use) was added to the nuclear pellets.
The nuclei were resuspended by vortexing and/or
disruption by pipette mixing. Optimal proteinase K -~
activity was promoted by incubation at 55C for one hour.
Proteinase K was inactivated and genomic DNA fully --
denatured by boiling the samples ~or 20 minutes. The
sample was collected by brief centrifugation.
Amplification of target DNA was performed as
follows: Seventy-five microliters of amplification
buffer as described in Example III was added to a 0.6ml
eppendorf tube (Robbins Scientific). Twenty-five
microliters of cell lysate (approximately 105 cells or lug
genomic DNA) was added and pipette mixed. The final
reaction mix contained 10mM Tris-Cl, pH 8.3 (25C), 50mM
KCl, 1.5mM MgCl2, 0.1~ (w/v) gelatin, 200 ~M each dNTP
(Pharmacia), 0.25 ~M ~iotin-N178 forward primer, 1 ~M
~'
:
- . , . . . .. . ~ ~
WO91/15768 PCT/US9~/02323
2~S~ 3 44
N279 reverse primer and 2.5 units AmpliTaq (Cetus). The
sample was overlayed with lOOul oil (Sigma light). The
samples were amplified in an Ericomp Programmable Cyclic
Reactor (San Diego, CA) as described in Example III.
Amplified product for assay in the automated
sandwich hybridization system was prepared as follows:
Twenty-five microliters of HIV amplified product was
added to 25 ~il 10 mM Tris, pH 8.0 in a 2.0 ml microfuge
tube. This dilution provides an analysis of 1/10 of the
PC~ reaction. Sandwich proceded as described in Example
I using AP-N280 as the detector probe. The results are
presented in Table IV.
TABLE IV
Relative
Fluorescent
Patient ID Units
.
106-009 41800
016-167 28422
081-046 24720
106-026 ~5506
neg 1 2986
2970
neg 2 2620
2582
The above data represents a single experiment.
The cutoff for a positive result was made at 2X the
highest negative control for that assay. Even though the
negative controls were unusually high, all 4 results
correlated with serology.
- . . , : , . ,. - ~ - :. . . - - .
.
WO91/15768 PCT/US91/02323
` 45 2 Q~ 0~19
EXAMPLE V
Detectlon of Campylobacter
In this example of the invention, it was
demonstrated that the automated sandwich assay could
detect 5s RNA purified form campylobacter infected stool
samples.
Several campylobacter infected stool samples
were analyzed using conventional dot-blot analysis.
Briefly, 75 ~l denaturation reagent (0.2 N HCl) was added
to the 2 ml eluates from an ion exchang~ column
(Extractor-l0~, Molecular Biosystems, Inc., San Diego,
CA) and was then denatured for 5 minutes at room
temperature. The denatured samples were applied to an
equilibrated centrifuge tube having a transverse membrane
(Gene Screen Plus; DuPont/NEN~ Boston, MA). The device
was spun at 750 X g for 20 minutes. The membrane was
removed and placed in fixing reagent (l M sodium
carbonate) for 30 seconds. It was stirred in distilled
water for 30 seconds and blot dried on Whatman 3 MM
paper. The membrane was transferred to a hybridization ~-
bag and pre-hybridized for 15 minutes at 50C in 2 mls
hybridization buffer (0.75 M sodium citrate, 1%
sarcosine, 0.5% BSAj 0.6% Kathon (Rohm and Haas,
Philadelphia, PA)). The bu~fer was discarded and 2 mls
fresh Hybridization Buffer were added, containing 2.5 nM
AP-Cl03b. Hybridization was allowed to proceed for lS
minutes at sorc. The product was washed for 20 minutes
at 50C in prewarmed working membrane wash buffer l (2 X
SSC, pH 7.0, 0.5% sarcosine, 0.12~ Rathon) and washed l0
minutes at room temperature in working membrane buffer 2
(l X SSC, pH 7.0, 0.5% triton Xl00 0.12% Xathon). The
membrane was placed in a plastic bag and developed
colorimetrically with freshly added (Nitro Blue
Tetrazolium; (NBT; Ameresco, Solon, OH) and 5-Bromo-4-
~ : - : - ':
,,
,
- ~ -, - ' .: ' : '
~ WO91/15768 PCT/US91/02323
2~8~19 46 ~
Chloro-3-indolyl-phosphate, (BCIP; Ameresco) in substrate
buffer (3.7 ~M NBT, 4.6 ~M BCIP, O.l M tris-Cl, pH 7.5,
O.l M NaCl, 0.05 M Mg~l2, 0.02% NaN3) for 4 hours at 37OC.
A weak and a strong positive and a negative control were
identified.
A conventional manual column and a semi-
automated bulk affinity chromatography purification
method were compared by an automated magnetic sandwich
assay. For the manual column format, 200 ~l of sample,
800 ~l diluent reagent (lO~ formalin, O.l M sodium
phosphate, pH 7.0) and 2000 ~l lysis reagent (8 M urea,
0.25% SDS, 0.25~ sarcosine, 0.05 M EDTA) were combined -
and incubated 30 minutes at 50DC. The sample was applied
to a pre-equilibrated extractor column containing l ml of
anion exchange resin. The column was washed with 15 mls
Wash Reagent l (40% ethanol, 0.2 M NaCl, 0.02 M Tris-Cl,
pH 7.5), 5 mls Wash Reagent 2 (0.25 M NaCl, 0.02 M tris-
Cl, pH 7.5, 0.05% NaN3) and eluted in 2 mls Elution
Reagent (0.5 M NaCl, 0.02 M Tris-Cl, pH 7.5, 0.05% NaN3).
The eluent was concentrated to 330 ~l by lyophilization.
The sample was adjusted to the following final
concentrations: 10% formamide, O.l~ SDS, 3.75 nM biotin-
C204b in a final volume of 400 ~l. The sample was
denatured lO minutes at 95 C, chilled in ice water, spun
briefly to collect condensate and placed in the XYZ
pipetter test tube rack.
For the semi-automated bulk format, two hundred
~l of sample, lO0 ~l Diluent Reagent and 600 ~l Lysis
Reagent were combined and incubated 30 minutes at 50C.
A portion of the sample (275 ~l) was applied to 300 ~l of
equilibrated 50% matrix. The sample tubes were placed in
the magnetic rack and agitated lO minutes at 37 C. The
tubes were automatically turned off and the matrix was
allowed to settle ~or 5 minutes. The sampling tip
.
, : .: . ~ ~ : - .: -
: , . .
.- -. . .. ~ ,... . .
: : . ~
WO9l/15768 PCT/US91/02323
47 2~
aspirated 300 ~1 which left some liquid over the matrix
bed. The sampling tip added 300 ~1 Wash Reagent 1, the
tubes were agitated for 5 minutes then turned off, and
the matrix was allowed to settle and 300 ~1 was
aspirated. The procedure was repeated again with Wash
Reagent 1 and repeated twice with Wash Reagent 2. Target
was eluted from matrix with 400 ~1 8.33 X SSC. 330 ~1 of
sample were aspirated. The sample was adjusted to the
following final concentrations: 10% formamide, 0.1~ S~S,
3.75 nM biotin-C204b in a final volume of 400 ~1. The
sample was denatured 10 minutes at 95 D C, chilled in ice
water, spun briefly to collect condensate and placed in
the sample rack.
:
The XYZ pipetter program was loaded and prompted
variables were keyed in by the operator. The sampling
tip automatically combined 100 ~1 hybridization buffer,
containing 15 nM AP-C103b, and 400 ~1 into reaction
tubes. The remainder of the assay proceeds as described
in Example I with the variation that 2 X SSC was
substituted for 1 X SSC in the wash buffer.
Results indicate that a semi-automated sample
preparation and sandwich hybridization method was
feasible using the magnetic rack and a XYZ pipetter. The
bulk chromatography format compares favorably with the
column format. Sample #1270c was more viscous than the
others and this may have contributed to decreased signal
in the bulk format.
- , .: . . . ~ ,. ~ .
. . ~ , ~ . - ~ . - -,
WO91/15768 PCTtUS91/02323
2~ 48 ~
TABLE V
Campylobacter Sandwich-Column vs.
Bulk Sample Purification
Relative Fluorescent
Sample Sample Units (Actual) Bulk/
Type I.D. Column Bulk Column
10 negative 17aO 52(36; 68)35 (34; 36) 67%
weak
positive 1791 176(184; 168)293(252; 334) 167~
15 strong 1270c 5319(5424; 5214~3140(3072; 3208) 59%
positive
EXAMPLE VI
Automated Sample Preparation by
Affinity Capture of HIV Target
In this example of the invention, the sample
preparation and concentration was achieved using the
automated system and an affinity capiure method. The
advantages of the method include target enrichment, which
can obviate the need for amplification, and compatibility
with automation. The example described below detects HIV -
DNA in blood.
Blood samples were collected, transferred to
Leucoprep tubes, and centrifuged as described in Example
IV. The caps were removed and the tubes were placed in a
sample rack on the worXtable of the XYZ pipetter, which
was properly programmed to perform the following steps.
3S The sampling tip transfered 1 ml of mononuclear cells
from the "buffy coat" to a reaction tube in the magnetic
rack. The cells were lysed and the nucleic acid
denatured by adding 1 ml 4.0 M guanidinium thiocyanate,
10 mM EDTA, pH 8.0 and incubated for 10 minutes at 37C
,, - .
- - : - - :
WO91/1S768 PCT/US91/02323
49
with aqitation. Biotin capture probe in hybridization
buffer (15 X SSC, 25% formamide, 0.125% SDS, 0.125%
sarcosin, 0.25 mg/ml BSA and 7.5 nM biotin-N178) is added
~ and the tubes were agitated for 30 minutes at 37~C.
Thirty ~g paramagnetic streptavidin derivatized Fe3O4
particles (Advanced Magnetics) were added and agitated
for 30 minutes at 37OC. Upon cessation of agitation, the
particles containing bound biotin capture probe
hybridized to target nucleic acid were collected to the
sides of the tubes. The supernatant containing
heterologous nucleic acids and cellular debris was
aspirated. The particles were washed twice for 5 minutes
each in 200 ~l of 20 mM Tris, pH 8.3, 250 mM KCl with
agitation. The target nucleic acid was released from the
particles by denaturing the capture probe:target hybrid
with 20 ~1 0.25 M KOH, followed by neutralization with 20
~1 0.25 M HCL, 150 mM Trisl pX 8.3 for 5 minutes each
with agitation. The addition of base and acid was
reversed if RNA was the target of interest. ~he
supernatant (40 ~1) was transferred to a fresh tube for
subsequent use with amplification followed by sandwich
assay, as described in Example IV.
This procedure effectively concentrated the
amount of target approximately 200 fold in an automated
format. The method is compatible with many types of
samples and sample preparation treatments.
Although the invention has been described with
reference to the presently preferred embodiment, it
should be understood that various modifications can be
made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the
following claims.
- . : . ,:, - . . . : . , - :: . : . ,. , : . :- : . . . : . . . -
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