U.S. patent application number 13/269932 was filed with the patent office on 2012-02-02 for particle analysis assay for biomolecular quantification.
Invention is credited to Gang Liu, Tore Straume.
Application Number | 20120028273 13/269932 |
Document ID | / |
Family ID | 35734185 |
Filed Date | 2012-02-02 |
United States Patent
Application |
20120028273 |
Kind Code |
A1 |
Straume; Tore ; et
al. |
February 2, 2012 |
Particle Analysis Assay for Biomolecular Quantification
Abstract
A method is provided for carrying out multi-step separations of
objects bearing at least two binding sites. In the first step, a
first binder/bead composition is bound to objects that bear the
first binding site, and then unbound objects, i.e. objects not
bearing the first binding site, are separated from bound objects.
In the second step, a second binder/bead composition is bound to
the remaining objects that bear the second binding site, and then
the objects that are bound to both beads are removed from those
objects that are bound to only one bead. The beads can differ in
magnetic responsiveness, charge, size, color, and the like, and
these differences can be used to carry out the separation
steps.
Inventors: |
Straume; Tore; (Salt Lake
City, UT) ; Liu; Gang; (Salt Lake City, UT) |
Family ID: |
35734185 |
Appl. No.: |
13/269932 |
Filed: |
October 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11348223 |
Feb 6, 2006 |
8034555 |
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13269932 |
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10089560 |
Apr 1, 2002 |
6994971 |
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PCT/US00/27883 |
Oct 10, 2000 |
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11348223 |
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60158664 |
Oct 8, 1999 |
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Current U.S.
Class: |
435/7.1 ;
436/501 |
Current CPC
Class: |
C12Q 1/6816 20130101;
C12Q 1/6816 20130101; C12Q 2563/143 20130101; C12Q 2545/114
20130101; C12Q 2563/155 20130101; G01N 33/54333 20130101 |
Class at
Publication: |
435/7.1 ;
436/501 |
International
Class: |
G01N 33/82 20060101
G01N033/82; G01N 21/64 20060101 G01N021/64; G01N 33/53 20060101
G01N033/53 |
Claims
1.-20. (canceled)
21. A method of quantifying a desired protein in a sample
comprising the steps of: (a) obtaining a solid support coated with
a first antibody that recognizes the desired protein; (b)
contacting the antibody-coated solid support with the sample to
allow the first antibody to bind to the desired protein; (c)
attaching a second antibody to the desired protein, wherein the
second antibody comprises a modification such that the second
antibody may be attached to a first set of microbeads; (d)
attaching first set of microbeads to the second antibody; (e)
washing to remove microbeads that are not attached to the second
antibody; and (f) counting the microbeads that are attached to the
second antibody to determine the quantity of the desired protein;
and wherein steps (a)-(d) may be performed in any order or
simultaneously.
22. The method of claim 21, wherein the solid support comprises a
second set of microbeads that is distinguishable from the first set
of microbeads by size, charge, color, or attachability to a solid
support.
23. The method of claim 21, wherein the modification comprises a
biotin molecule and the fist set of microbeads comprise
streptavidin.
24. The method of claim 21, further comprising the step of
releasing the microbeads attached to the second antibody by
protease treatment prior to the counting step.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of application Ser. No.
10/089,560, filed Apr. 1, 2002, which was the National Stage of
International Application No. PCT/US00/27883, filed Oct. 10, 2000,
which claims the benefit of U.S. Provisional Application No.
60/158,664, filed Oct. 8, 1999, which applications are incorporated
herein by this reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to methods for isolation,
separation, and detection of selected objects. More particularly,
the invention relates to methods for performing separations of
objects, such as, without limitation, nucleic acids, proteins,
cells, organelles, and the like.
[0003] Separations of biological objects, such as proteins,
chromosomes, nucleic acids, cells, organelles, and the like, and
other types of objects are important in various detection,
isolation, quantification, and diagnostic processes. Specificity
and sensitivity are two important parameters that are generally
desired in these separation schemes.
[0004] Hybridization probes are widely used to detect and/or
quantify the presence of a particular nucleotide sequence in a
mixed sample of nucleotide sequences. Hybridization probes detect
the presence of a particular nucleotide sequence, referred to
herein as a target sequence, through the use of a complementary
nucleotide sequence that selectively hybridizes to the target
nucleotide sequence. For a hybridization probe to hybridize to a
target sequence, the hybridization probe must contain a nucleotide
sequence that is complementary to the target sequence. The
complementary sequence must also be sufficiently long for the probe
to exhibit selectivity for the target sequence over non-target
sequences.
[0005] Hybridization assays can be designed to detect the presence
or absence of a particular nucleotide sequence, for example the
presence of a gene in a DNA sequence. Hybridization assays can also
be designed to detect the movement of a nucleotide sequence
relative to another nucleotide sequence in a sample, for example
the presence of a gene on a chromosome that is known to be normally
located on a different chromosome, e.g., the detection of the abl
gene on chromosome 22 in human leukemia patients (e.g., Tkachuk et
al., 250 Science 559-562 (1990); C-TRAK translocation detection
system commercially available from Oncor, Inc., Gaithersburg, Md.;
U.S. Pat. No. 5,447,841; U.S. Pat. No. 5,731,153; and U.S. Pat. No.
5,783,387).
[0006] As used herein, "nucleotide sequence aberrations" refers to
rearrangements between and within nucleic acids, particularly
chromosomal rearrangements. "Nucleotide sequence aberrations" also
refers to the deletion of a nucleotide sequence, particularly
chromosome deletions. As used herein, the term "nucleic acids"
refers to both DNA and RNA.
[0007] A chromosome translocation is an example of a nucleotide
sequence aberration. A chromosome translocation refers to the
movement of a portion of one chromosome to another chromosome
(inter-chromosome rearrangement), as well as the movement of a
portion of a chromosome to a different location on that chromosome
(intra-chromosome rearrangement). In general, chromosome
translocations are characterized by the presence of a DNA sequence
on a particular chromosome that is known to be native to a
different chromosome or different portion of the same chromosome.
Because chromosome translocations involve the movement of a
nucleotide sequence within a sample, as opposed to the appearance
or disappearance of the nucleotide sequence, it generally is not
possible to detect a chromosome translocation merely by assaying
for the presence or absence of a particular nucleotide
sequence.
[0008] Chromosome translocations are known to increase in frequency
upon exposure to radiation and certain chemicals. Measurement of
the frequency of chromosome translocations after exposure to
radiation or a particular agent is therefore useful for evaluating
the tendency of such agents to cause or increase the frequency of
chromosome translocations. Also, the frequency (translocations per
cell) of chromosome translocations measured in blood lymphocytes
from an individual can be used as a quantitative measure of the
amount of prior exposure to such agents (e.g., T. Straume and J.
Lucas "Validation studies for monitoring of workers using molecular
cytogenetics," Biomarkers in Occupational Health: Progress and
Perspectives (M. L. Mendelsohn, J. P. Peeters, and M. J. Normandt,
Eds.), Joseph Henry Press, Washington D.C., pp. 174-193
(1995)).
[0009] Chromosome translocations are also known to be associated
with specific diseases, including, for example lymphomas and
leukemia, such as Burkitt's lymphoma, chronic myelocytic leukemia,
chronic lymphocytic leukemia, and granulocytic leukemia, as well as
solid tumors such as malignant melanoma, prostate cancer, and
cervical cancer. A method for efficiently detecting a translocation
associated with a disease is needed as a method for diagnosing
disease, follow-up of cancer therapy patients, research, and
population studies.
[0010] Fluorescence in situ hybridization (FISH) using
chromosome-specific composite hybridization probes ("chromosome
painting") was developed as an assay for detecting chromosome
translocations. FISH and selected applications of the FISH method
are described in Pinkel et al., 83 Proc. Nat'l Acad. Sci. USA
2934-2938 (1986); Straume et al., UCRL 93837 (1986); Pinkel et al.,
85 Proc. Nat'l Acad. Sci. USA 9138-9142 (1988); U.S. Pat. No.
5,447,841; Lucas et al., 62 International Journal of Radiation
Biology 53-63 (1992); Straume et al., 62 Health Physics 122-130
(1992); Straume and Lucas, 64 Int. J. Radiat. Biol. 185-187
(1993).
[0011] The fluorescent hybridization probes used in FISH-based
chromosome painting are substantially chromosome-specific, i.e.,
they hybridize primarily to a particular chromosome type. Unique or
substantially unique probes may be used to limit non-specific
hybridization. A discussion of so-called unique, middle repetitive,
and highly repetitive sequences and their implications for
hybridization probes is found in U.S. Pat. No. 5,447,841.
Chromosome translocations are identified in the FISH assay by
visually scanning individual cells for the presence of two
different fluorescent signals on a single chromosome, the two
fluorescent signals originating from two different cocktails of
FISH probes, each probe cocktail having homology to a different
chromosome type.
[0012] Because each FISH probe hybridizes to a specific chromosome
type and not to the chromosome translocation itself, it is not
possible to determine the frequency of chromosome translocations
directly from the fluorescence signal emanating from a FISH probe.
Rather, the frequency of chromosome translocations in a cell sample
must be determined according to FISH assays by visually scanning
individual metaphase cells on slides and identifying whether the
two fluorescent signals appear on the same chromosome. The need to
visually scan such individual cells effectively limits the number
of cells that can be assayed, thereby reducing the sensitivity of
the FISH assay, introducing the possibility of human error, and
greatly increasing cost per analysis.
[0013] Accordingly, a fast, accurate method is needed for
quantifying chromosome translocations and other nucleotide sequence
aberrations. In particular, a method is needed that can isolate and
quantify nucleotide sequence aberrations contained in the nucleic
acid of a sample of cells without the need to analyze each cell
individually.
[0014] U.S. Pat. No. 5,731,153 relates to a two-step separation
procedure that uses two solid supports, each coated with unique
complexing agents that bind to hybridization probes complementary
to different target sequences. This procedure requires detachment
of the target sequence from the first solid support after the first
separation step and reattachment of the target sequence to a second
solid support before the second separation step can be performed.
The requirement for re-attachment of the target sequence is
particularly problematic and would add significantly to the
complexity and cost of commercial separation kits using such
methodology and reduce the precision of the assay because of
variability in the detachment/reattachment step. Further, this
procedure is limited to two types of solid supports, but it would
be useful to have more support options to facilitate multiple
simultaneous analyses. Moreover, the preferable methods for
quantification described in U.S. Pat. No. 5,731,153 require either
very expensive and uncommon equipment (e.g., measure .sup.14C by
accelerator mass spectrometry) or much less quantitative methods
such as the detection of fluorescence labels on reporter nucleic
acid probes. Also, the method in U.S. Pat. No. 5,731,153 is limited
to separation of nucleic acids, whereas it would be advantageous to
separate other types of objects as well.
[0015] Unfortunately, methods available for the quantification of
chromosomal rearrangements are either very costly and inefficient,
e.g., cytogenetic-type analyses (H. J. Evans et al., 35 Chromosoma
310-325 (1971); D. Pinkel et al., 83 Proc. Natl. Acad. Sci. USA
2934-2938 (1986); D. Pinkel et al., 85 Proc. Natl. Acad. Sci. USA
9138-9142 (1988); D. C. Tkachuk et al., 250 Science 559-562
(1990)), or require small sequences such as fusion mRNAs that may
be amplified by PCR and detected (M. H. Delfau et al., 4 Leukemia
1-5 (1990); A. Zippelius & K. Pantel, 906 Annals NY Acad. Sci.
110-123 (2000)). Cytogenetics require a highly trained technician
to visually score metaphase or interphase cells using a microscope
and make judgements about what is observed. PCR is less labor
intensive than cytogenetics but is of limited utility in direct
DNA-based detection of most chromosomal translocations because the
fusion points tend to be variable. D. C. Tkachuk et al., 250
Science 559-562 (1990); E. Solomon et al., 254 Science 1153-1160
(1991). These limitations have essentially restricted PCR to the
detection of fusion mRNAs, which may not always be known, may arise
from ectopic expression, or may be expressed deficiently (A.
Zippelius & K. Pantel, 906 Annals NY Acad. Sci. 110-123
(2000)).
[0016] In view of the foregoing, it will be appreciated that
providing a separation method that does not require reattachment of
the target sequence to a solid support to perform the second step,
does not require PCR, is highly quantitative, can be accomplished
using readily available laboratory equipment, can be used for
multiple simultaneous analyses, and that is applicable to the
isolation and quantization of many different kinds of objects,
including nucleic acids, metaphase chromosomes, proteins, cells,
organelles, and the like, would be a significant advancement in the
art.
[0017] Such methods are disclosed herein.
BRIEF SUMMARY OF THE INVENTION
[0018] The present invention provides methods for separation and
quantization of objects, including nucleic acids, chromosomes,
proteins, organelles, cells, and the like.
[0019] In one preferred embodiment, the present invention relates
to a method for separating nucleotide sequence aberrations from
normal nucleotide sequences and quantification of the frequency of
abnormal sequences. As used herein, "nucleotide sequence
aberration" refers to rearrangements between and within nucleotide
sequences, particularly chromosomes. "Nucleotide sequence
aberration" also refers to the deletion of a nucleotide sequence,
particularly chromosome deletions. As used herein, the term
"nucleic acids" refers to both DNA and RNA of any origin and any
level of organization, e.g., DNA, chromatin, and chromosome.
[0020] A method is provided for separating and quantifying nucleic
acids that include a nucleotide sequence aberration, the nucleotide
sequence aberration being identified by the presence of nucleotide
sequences that include a first, a second, and additional ones if
desired, nucleotide sequence types.
[0021] According to a preferred embodiment of the present
invention, a nucleotide sequence aberration is isolated by
separation of nucleic acids having both a first nucleotide sequence
type (e.g., from a first chromosome) and a second nucleotide
sequence type (e.g., from a second chromosome) from nucleic acid
sequences not having both first and second sequence types. The
presence of the first and the second nucleotide sequence types on
the same nucleic acid indicate the presence of a nucleotide
sequence aberration. Thus, nucleic acids that contain a nucleotide
sequence aberration, characterized by their having nucleic acid
sequences of both a first and a second nucleic acid sequence type,
are selectively isolated. Once isolated, these sequences may be
detected, quantified, and/or characterized.
[0022] In an illustrative embodiment of the invention, a target
nucleic acid is isolated from a mixture of nucleic acids in a
sample by hybridizing two or more hybridization probes to the
mixture of nucleic acids, each probe type being specific for
non-overlapping sequences on the target nucleic acid and containing
complexing agents specific for selected types of solid support
surfaces. Preferred embodiments include two different types of
supports, one for separation (e.g., superparamagnetic microbeads or
the inside surface of microtiter wells) and another for detection
and quantification (e.g., magnetically non-responsive polystyrene
microspheres of selected diameters that can be identified and
counted in a particle size distribution analysis system such as a
Coulter counter). For example, the first complexing agent on the
first hybridization probe is contacted with the second complexing
agent bound to a first bead that is responsive to a magnetic field
(M) either before, during, or after the first and/or second
hybridization probe is hybridized to the sample of nucleic acids.
By contacting the first and second complexing agents, the first
hybridization probe becomes immobilized on the magnetically
responsive bead. This enables the immobilization of any nucleic
acid hybridized to the first hybridization probe, i.e., a nucleic
acid that includes a nucleic acid sequence of the first type. The
magnetically responsive bead enables nucleic acids hybridized to
the first hybridization probe to be separated from nucleic acids
that do not hybridize to the first hybridization probe.
[0023] Similarly, the third complexing agent on the second
hybridization probe is contacted with the fourth complexing agent
bound to a second bead, which is non-responsive to a magnetic field
(NM) but may be of different size than the first bead (and/or
responsive to an electric field), either before, during, or after
the first and/or second hybridization probe is hybridized to the
sample of nucleic acids. By contacting the third and fourth
complexing agents, the second hybridization probe becomes
immobilized on the non-magnetic responsive bead. This enables the
immobilization of any nucleic acid sequence hybridized to the
second hybridization probe, i.e., a nucleic acid sequence that
includes a nucleic acid sequence of the second type. The
magnetically non-responsive bead can then be used as a detectable
marker following magnetic separation for those target nucleic acids
that have both type 1 and type 2 sequences on the same contiguous
molecule. If the magnetically non-responsive bead is responsive to
an electric field (e.g., by coating with carboxylic acid) and
uniquely complexable to a solid support, two additional separation
steps would be possible. For example, Step 1 could be by magnetic
separation, Step 2 by electrophoretic separation, and Step 3 by
complexing the non-magnetic beads to a solid support such as a
glass slide and detection by fluorescence scanning, or to a solid
support such as the inside surface of a well in a 96 well plate.
Also, step 2 separation can be accomplished by filtration if
different size beads are used, or by particle size characterization
if a particle size measurement device is employed (these methods
are taught in Example 2).
[0024] Only nucleic acids containing the first nucleic acid
sequence type, i.e., nucleic acids that hybridize to the first
hybridization probe, will be immobilized onto the magnetically
responsive bead. Of these nucleic acids, only those containing the
second nucleic acid sequence type will hybridize to the second
hybridization probe, which is immobilized onto the magnetically non
responsive bead. Thus, after the first separation step by response
to magnetic force, followed by washing, the remaining target
nucleic acids contain sequences hybridized to the first probe and
sequences hybridized to both the first probe and the second probe.
All target nucleic acid sequences not hybridized to the first
probe, or not hybridized to the same contiguous nucleic acid
molecule as the first probe, are washed out because they are not
immobilized to the magnetic bead.
[0025] The aberrant nucleic acids, which contain sequences
hybridized to the first probe and second probe, can be separated by
exposure to an electric field if the second bead is responsive to
an electric field, or by immobilizing the second bead to a solid
support if the second bead is coated with a member of a third pair
of complexing agents which is capable of specifically complexing
with the complementary member of the third pair of complexing
agents coated on the solid support. The detection and
quantification of nucleic acids containing both type 1 and type 2
sequences, which is directly proportional to the number of nucleic
acid aberrations present in the sample of nucleic acids analyzed,
can be done using a variety of available methods. For example,
various detectable labels can be included on the beads, on the
probes, or on the target nucleic acid, such that they can be
measured by fluorescence, radioactivity, luminescence,
chemiluminescence, electrochemiluminescence, spectrophotometry, and
the like. Colored beads, both fluorescent and non-fluorescent, are
commercially available (e.g., Bangs Labs, Fishers, Ind.) and also
can be used for distinguishing nucleic acid types.
[0026] The method of the present invention increases by orders of
magnitude the speed of detecting nucleotide sequence aberrations,
such as chromosome translocations, over current detection methods,
including FISH assays.
[0027] Since the number of type 1 plus type 2 target sequences
detected in the sample of DNA analyzed would be proportional to the
number of type 1 plus type 2 nucleotide sequences in the cell
extract (e.g., chromosomal DNA from blood lymphocytes), the method
of the present invention can also be used in the early detection
and monitoring of pre-clinical disease progression of malignancies,
such as leukemias that are associated with specific chromosomal
rearrangements, e.g., t(9;22) of human chronic myelogenous
leukemia. According to this embodiment of the method, the first and
second hybridization probes are designed to selectively hybridize
to a first and a second nucleic acid sequence types, the nucleotide
sequence aberration of which is associated with and/or
characteristic of a disease. Only nucleic acids containing both the
first and second nucleotide sequence types, the aberration of which
is associated with and/or characteristic of a disease, will
hybridize to both the first and second hybridization probes. As a
result, after the first separation by magnetic force followed by
washing, the separation of the beads complexed to the second
hybridization probe, either by electric force, immobilization on a
solid support, bead filtration, or particle size analysis, may be
used to diagnose a disease associated with the particular nucleic
acid aberration being detected. Examples of diseases that may be
detected include (but are not limited to) cancers such as leukemia,
lymphoma, melanoma, prostate, and cervical cancer.
[0028] It is within the scope of the present invention that
probe-bead attachments and hybridizations of probes to target
nucleotide sequences can be performed in any order, as well as
simultaneously.
[0029] In another preferred embodiment of the invention, the
present method can be used for separating and rapidly quantifying
objects such as proteins, cells, organelles, and the like. Instead
of using hybridization probes for binding to the target object,
antibodies or other binding molecules, such as lectins, are used.
It is merely required that there be at least two binding sites on
the target for which there is a corresponding number of binding
molecules. For example, a protein having two epitopes can be
separated from other proteins provided that an antibody for binding
each epitope is available for carrying out the separation. Example
4 teaches the separation of cells using the methods of the present
invention.
[0030] In another preferred embodiment of the invention, beads of
different sizes can be used for carrying out separation steps. For
example, if a small magnetically non-responsive bead is coupled to
an antibody that recognizes one epitope and a larger magnetically
responsive bead is coupled to an antibody that recognizes another
epitope, then nucleic acids, proteins, cells, organelles, and the
like that bear both epitopes can be separated from other objects
that lack both epitopes. A description of this embodiment of the
invention is provided in Example 2 for nucleic acids, Example 4 for
cells, and Example 7 for proteins.
[0031] It will be recognized by those skilled in the art that at
least the following differences among beads can be used for
carrying out multi-step separations: the degree of magnetic
responsiveness, the degree of charge responsiveness, selected bead
size differences, selected bead color differences, and selected
complexing agents on beads and supports.
[0032] The present invention also relates to a kit for separating
and quantifying nucleic acid aberrations and diagnosing disease
according to the methods of the present invention. In general, the
kits of the present invention include beads with complexing agents
and hybridization probes (e.g., magnetically responsive beads
coated with type 1 probes and magnetically non-responsive beads
coated with type 2 probes). The kits may also include vials with
reagents, suitable solid supports, instructions for using the kit,
and a calibration curve (or suitable internal control) relating the
measured quantity to the frequency of nucleic acid sequence
aberrations in the target sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIGS. 1A, 1B, 2A, and 2B show exemplary methods for
separating and isolating a chromosome translocation. FIG. 1 shows
the isolation of DNA from a sample of cells, hybridization of the
DNA to first and second hybridization probes, attaching the
hybridized DNA to a first bead that is magnetically responsive and
specifically complexes with the first hybridization probe and a
second bead that is magnetically non-responsive and specifically
complexes with the second hybridization probe, and a first
separation step accomplished by subjecting the mixture of
bead-attached hybridized DNA to a magnetic force followed by
washing. At this stage, the beads can could be detached from the
target DNA (e.g., by DNase treatment) and directly analyzed as
described in Example 6. FIG. 1B depicts a similar procedure in
which the probes are attached to a non-magnetic solid support such
as the inside surface of a well of a microtiter plate, permitting
step 1 separation followed by bead counting or particle size
distribution analysis. FIG. 2A depicts the second separation step
of DNA that contains both type 1 and type 2 sequences from a sample
of cells, wherein the second bead is magnetically non-responsive
but is electrically responsive, after the first step separation,
the DNA containing both type 1 and type 2 sequences is then
separated by application of an electric force. FIG. 2B shows the
second separation step of DNA that contains both type 1 and type 2
sequences from a sample of cells, wherein the second bead is
capable of specifically complexing with the second hybridization
probe and a solid support, after the first separation step, the DNA
containing both type 1 and type 2 sequences is separated by
complexing the second bead to a solid support, followed by
washing.
[0034] FIGS. 3A-B illustrate a pUC 19 plasmid and a 18.2 kb DNA
insert used here as a model system to demonstrate the feasibility
of the separation method presented in this invention.
[0035] FIG. 4 shows the sequences of the terminal ends (SEQ ID NO:1
and SEQ ID NO:2) of the DNA insert seen in FIG. 3B and the 50mer
probes (SEQ ID NOS:3-10) selected to be complementary for type 1
(one of the ends) and type 2 DNA (the other end).
[0036] FIG. 5 shows Dynal and Bangs beads observed using a light
microscope at 1000.times. magnification. In this case, beads were
taken from the stock solutions, mixed, placed on a glass microscope
slide, and viewed under oil immersion.
[0037] FIG. 6 shows a microscope image of beads deposited on a
glass slide after the bead solution (Dynal+Bangs) had been
subjected to magnetic separation, but without hybridization to the
18.2 kb DNA. Note that only Dynal beads are seen (the few small
dots are dust on lens as they can also be seen on the other
images).
[0038] FIG. 7 shows a microscope image following hybridization of
beads to the 18.2 kb target DNA and magnetic separation. In this
case, 1 .mu.l of Solution B was deposited on a slide and HA on the
Bangs beads complexed with the anti-HA on the surface of the slide.
After washing, only the Bangs beads attached to the slide. The
Dynal beads, which were connected to the Bangs beads because they
were both hybridized to the same DNA molecule, are also present on
the slide. The presence of both Dynal and Bangs beads on this slide
demonstrates that both were hybridized to the target DNA and that
the separation procedure was successful.
[0039] FIG. 8 illustrates the particle size distribution obtained
for the two bead types following magnetic separation of the 18.2 kb
target DNA sequence. In contrast with the results in Table 1, in
this case, the larger non-magnetic beads were selected to place the
non-magnetic bead peak in a region of lower background counts. The
materials and methods used to obtain these results were the same as
those used for Table 1 with the following differences: FIG. 8 used
4.4 .mu.m diameter magnetically non-responsive polystyrene beads
coated with streptavidin (Bangs Labs, Fisher, Ind.) whereas Table 1
used 0.94 .mu.m diameter magnetically non-responsive polystyrene
beads coated with streptavidin (Bangs Labs); FIG. 8 used 5 .mu.g of
18.2 kb target DNA whereas Table 1 used 20 .mu.g of 18.2 kb target
DNA; partial magnetic separation was done for FIG. 8 following
DNase treatment to reduce (but not fully eliminate) the number of
superparamagnetic beads to provide a lower background level while
at the same time provide a 2.8 .mu.m peak for
comparison/illustration purposes; and for FIG. 8 the final bead
concentration was diluted two-fold just before generating the bead
size distributions using the Coulter Multisizer II. It is clear
that separation and quantification of these bead types can be
accomplished using our methods and commercially available particle
analyzers.
[0040] FIG. 9 illustrates Type 1 and Type 2 DNA and the beads with
ECL labels and probes for detection of the non-magnetic beads by
electrochemiluminescence (ECL). Note that the hybridization could
occur in any order, prior to, during, or after complexing the
probes with the beads. If attachment of probes to the beads is
performed first in separate solutions, then both bead types can use
the same complexing agents (e.g., avidin-biotin). However, if the
probes are hybridized first (which is the preferred method) then
the complexing agents binding the beads to the respective probes
would be unique and specific for each bead-probe complex. In this
case, we could use streptavidin coated magnetic beads that would
complex with biotinylated probes and antidigoxigenin coated
non-magnetic beads that would complex with digoxigenin labeled
probes. As depicted, a magnet may be used to hold the complex in
place during measurement.
[0041] FIG. 10 shows the separation of erythrocytes containing the
glycophorin MN surface proteins from all other erythrocytes.
[0042] FIG. 11 illustrates a method for evaluating for the
presence, absence, or amplification of nucleic acid sequences in a
sample of nucleic acid. The examples of complexing agents are as
follows: 1=biotin; 2=digoxigenin; 3=estradiol; 4=fluoresceine;
5=anti-digoxigenin; 6=anti-estradiol; 7=anti-fluoresceine; and
8=avidin.
[0043] FIGS. 12A-D illustrate particle size spectra obtained using
the particle counting assay to detect bcr/abl fusions in genomic
DNA isolated from human CML cells. FIGS. 12A-C are the spectra of
microparticles observed in 500 .mu.l samples with 165 ng, 16.5 ng,
and 0 ng of genomic K-562 DNA, respectively. FIG. 12D is the result
obtained with 16.5 ng genomic K-562 DNA when the probes were not
present during hybridization.
[0044] FIG. 13 illustrates a method to simultaneously separate and
quantify selected target molecules (e.g., proteins) using
antibodies and beads of different diameters. Magnetic (M) beads and
non-magnetic (N) beads are complexed with selected antibodies that
permit their unique attachment to different antigenic sites on the
same target molecule. Note that the N-beads will remain after
magnetic separation only if both antigenic sites are present on the
same contiguous molecule.
[0045] FIG. 14 shows the particle size distribution obtained using
the described method to separate ferritin.
[0046] FIG. 15 shows the results obtained by repeating the
separation in FIG. 14, but without ferritin in the target
solution.
DETAILED DESCRIPTION OF THE INVENTION
[0047] Before the present compositions and methods for separation
and quantization of aberrant nucleic acid sequences, other
molecules, cells, and the like are disclosed and described, it is
to be understood that this invention is not limited to the
particular configurations, process steps, and materials disclosed
herein as such configurations, process steps, and materials may
vary somewhat. It is also to be understood that the terminology
employed herein is used for the purpose of describing particular
embodiments only and is not intended to be limiting since the scope
of the present invention will be limited only by the appended
claims and equivalents thereof.
[0048] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
[0049] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out herein.
[0050] In one illustrative embodiment, the present invention
relates to a multi-step method for detecting and separating nucleic
acid sequence aberrations. As used herein, the term "nucleotide
sequence aberration" refers to rearrangements between and within
nucleotide sequences, particularly chromosomes. Nucleotide sequence
aberration also refers to the deletion of a nucleic acid sequence,
particularly chromosome deletions. As used herein, the term
"nucleic acids" refers to DNA and RNA of any origin, and any level
of organization, e.g., DNA molecule, chromatin, chromosome.
[0051] According to the method of the present invention, a
nucleotide sequence aberration is detected by isolating and
quantifying nucleotide sequences having both a first nucleotide
sequence type (e.g., from a first chromosome) and a second
nucleotide sequence type (e.g., from a second chromosome). The
presence of the first and the second nucleotide sequence types on
the same nucleic acid indicating the presence of a nucleotide
sequence aberration.
[0052] The method is referred to as a multi-step method because it
involves at least two sequential separation steps. According to the
method of the present invention, as illustrated in FIG. 1, the
nucleic acid sample comprises chromosomal DNA isolated from a
sample of cells. Chromosomal DNA may be isolated by any of the
variety of methods known in the art. For example, the DNA may be
isolated by the method taught in U.S. Pat. No. 5,447,841 and
references therein; in Vooijs, et al., Am. J. Hum. Genet.
52:586-597 (1993); or by using the GIBCO BRL TRIzol.TM. Reagent
(Life Technologies, Gaithersburg, Md.), or by using a QIAprep kit
(Qiagen, Inc., Valencia, Calif.).
[0053] Chromosomal DNA may be analyzed as whole chromosomes,
chromosome fragments, chromatin fragments, or chromosomal DNA
fragments, all of which are hereinafter referred to as chromosomal
DNA. When analyzing chromosomal DNA for the presence of nucleotide
sequence aberrations, the chromosomal DNA may be organized as an
extended double strand, as extended nucleosomes, as chromatin
fiber, as folded fiber, and as interphase, prophase, or metaphase
DNA. Sandberg, "The chromosomes in human cancer and leukemia",
Elsevier; New York (1980), pp. 69-73.
[0054] The preferred chromosome organization for assaying
chromosomal DNA for the presence of a nucleotide sequence
aberration depends on the number of bases separating the first and
second nucleotide sequence types being recognized by the first and
second hybridization probes used to identify the aberration. The
preferred size of beads is a function of the size of the piece of
target DNA or RNA to be evaluated. For example, target pieces of
DNA can range from less than a micrometer to several millimeters in
length depending on the level of organization used and the degree
to which the chromosomes are fractionated. For example, accurate
quantification of the frequency of t(9;22) fusions in chronic
myelogenous leukemia (CML) patients would require target DNA pieces
on the order of a few hundred kilobases (less than 1 mm) if the DNA
molecules are fully extended and only a few micrometers if the
chromosomes are in the interphase level of organization. (Note that
the position of the fusion point in CML patients can vary by about
225 kb.)
[0055] Although it is within the scope of the present invention
that probe-bead attachments and hybridizations of probes to target
nucleotide sequences can be performed in any order, as well as
simultaneously, as illustrated in FIG. 1, it is a preferred
embodiment of this invention that the following order be used to
reduce the number of complexing agents required (e.g., see Example
1). Two different probe-bead complexes are prepared in separate
solutions. Solution I would contain the first hybridization probe
with a member of a pair of first complexing agents capable of
attaching to a complementary member of the first pair of complexing
agents. Also contained in Solution I would be magnetically
responsive beads coated with the complementary member of the pair
of first complexing agents. The beads and hybridization probes
would be combined in Solution I such that complexing of the pair of
first complexing agents takes place. Subsequent washing would
remove all probes and reagents not complexed with the beads.
Solution II would contain the second hybridization probe with a
member of a second pair of complexing agents capable of attaching
to a complementary member of the second pair of complexing agents.
Also contained in Solution II would be magnetically non-responsive
beads coated with the complementary member of the second pair of
complexing agents. The beads and hybridization probes would be
combined in Solution II so as to permit complexing of the second
pair of complexing agents. Subsequent washing would remove all
probes and reagents not complexed with the beads. The magnetically
non-responsive beads in Solution II may also be coated with a
member of a third pair of complexing agent to facilitate a
particular Step 2 separation embodiment, if desired (see
below).
[0056] The two types of probe-bead complexes are then combined in
one solution and contacted with a sample of target nucleic acid
sequences under conditions favorable for hybridization. The first
hybridization probe includes a nucleotide sequence probe that is at
least partially complementary to a first target nucleotide sequence
type. The second hybridization probe includes a nucleotide sequence
that is at least partially complementary to a second nucleotide
sequence type and that selectively hybridizes to the second
nucleotide sequence type over the first nucleotide sequence
type.
[0057] In the case of detecting and separating chromosomal
translocations, the first hybridization probe is preferably a
chromosome-specific probe such that it selectivity hybridizes to a
particular chromosome type. In the case of detecting
inter-chromosomal rearrangements, "chromosome type" refers to
individual chromosomes. In the case of detecting intrachromosomal
rearrangements, "chromosome type" refers to different portions of
an individual chromosome since intra-chromosomal rearrangements
involve the movement of a sequence to a different portion of the
same chromosome.
[0058] Any hybridization probe that preferentially hybridizes to a
particular nucleotide sequence may be used as the first
hybridization probe and is intended to fall within the scope of the
present invention. In the case of detecting chromosome
translocations, a large number of both chromosome-specific painting
probes and unique sequence probes are available commercially (e.g.,
Oncor, Inc., Gaithersburg, Md.; or Vysis, Inc. (www.vysis.com)). In
addition, methods are broadly available for anyone skilled in the
art to prepare nucleic acid probes that are complementary to any
known sequence. For example, thousands of human genes have now been
mapped to specific regions of chromosomes and sequenced. These
sequences are now available on the Internet and can be used to make
biotinylated type 1 and type 2 probes for use in the present
invention to quantify DNA rearrangements (see Example 1). Exemplary
methods for preparing DNA probes for chromosome translocation
detection are given in U.S. Pat. No. 5,447,841, which is hereby
incorporated herein by reference. A preferred modification to the
method of U.S. Pat. No. 5,447,841 is the use of biotin TEG
phosphoramidite to attach individual biotin molecules to the 5' end
of the probes (see Example 1).
[0059] Because the first hybridization probe is selective for a
first nucleotide sequence type as opposed to the nucleotide
sequence aberration itself, the first hybridization probe
hybridizes to all nucleotide sequences containing the first
nucleotide sequence type. For example, with regard to detecting a
chromosome translocation, the first hybridization probe may be a
chromosome-specific probe. Thus, the first hybridization probe does
not by itself detect the nucleotide sequence aberration. Rather,
the method of the present invention relies on the second
hybridization probe to identify those nucleotide sequences isolated
by the first hybridization probe that also have a nucleotide
sequence of a second type. By contrast, in most prior art
hybridization assays using two hybridization probes, the first
hybridization probe selectively isolates the nucleic acid being
detected while the second hybridization probe serves to enable
detection of the nucleotide sequence isolated by the first
hybridization probe.
[0060] The first hybridization probe also includes a complexing
agent that is configured for binding to a complementary complexing
agent for forming a first pair of complexing agents. The
complementary complexing agent is attached to a first bead, which
is responsive to magnetic force, thereby enabling the
immobilization of the first hybridization probe on the magnetically
responsive bead. The second hybridization probe includes another
complexing agent that is configured for binding to another
complexing agent for forming a second pair of complexing agents.
The complementary complexing agent of the second pair of complexing
agents is attached to a second bead, which is non-responsive to
magnetic force, but is either electrically responsive, is coated
with a complexing agent that is configured for binding to still
another complementary complexing agent for forming a third pair of
complexing agents, and/or is of different size than the first bead.
The complementary complexing agent of the third pair of complexing
agents is attached to a solid support, thereby enabling the
immobilization of the second hybridization probe on the solid
support.
[0061] The first, second, and third pairs of complexing agents may
be any pair of complexing agents that form a strong binding pair.
Since elevated temperatures may be required for hybridization, the
binding pair should preferably be stable at temperatures at least
up to about 40.degree. C.
[0062] Examples of suitable binding pairs of complexing agents
include biotin-avidin, and antibody-antigen pairs, such as
hemagglutinin and anti-hemagglutinin, and digoxigenin and
anti-digoxigenin. Avidin-biotin and analogues and derivatives
thereof are particularly preferred as binding pairs due to their
enhanced thermal stability.
[0063] Magnetically responsive and magnetically non-responsive
beads suitable for the present invention are commercially available
(Dynal AS, Oslo, Norway; Bangs Labs, Fishers, Ind.). Preferably the
first bead is magnetically responsive and the second bead is
non-responsive to magnetic force, but is either responsive to
electric force, coated with an additional complexing agent (e.g., a
peptide for step 2 antibody separation), or smaller in size than
the first bead, thus facilitating step 2 separation by filtration
or by particle size distribution analysis (see Example 2).
Preferably, both bead types are coated with avidin as a complexing
agent for attachment to uni-labeled biotinylated probes (i.e., only
one biotin per probe). The magnetically non-responsive beads may
also be coated with, e.g., carboxylic acid, which provides a
negative surface charge and which would make them responsive to
electric force.
[0064] Any solid support to which a complexing agent may be
attached may be used in the present invention. Examples of suitable
solid support materials include, but are not limited to, silicates
such as glass, plastics, polyethylene, cellulose and
nitrocellulose, polymethacrylate, latex, rubber, fluorocarbon
resins such as TEFLON, metals, nylon, polystyrene, and the
like.
[0065] The solid support material may be used in a wide variety of
shapes including, but not limited to microscope slides,
microspheres, and microtiter wells. Examples are provided herein of
attachments to glass microscope slides and quantification by
fluorescence scanning or microscopy (Example 1), and also of the
use of different bead sizes, filtering, and quantification by bead
counting or size characterization (Example 2).
[0066] Preferably, avidin or an avidin derivative is used in both
the first and second pairs of complexing agents. Magnetically
responsive and magnetically non-responsive microbeads labeled with
streptavidin may be obtained from Dynal AS, Oslo, Norway, or Bangs
Labs, Fishers, Ind. (see Example 1).
[0067] The first and second hybridization probes may be immobilized
to the first bead or second bead either before, during, or after
the first and second hybridization probes are hybridized to the
sample of target nucleic acids. The first and second hybridization
probes are preferably attached to the beads in separate solutions
before the probes are hybridized to the sample of nucleic acids.
This permits the use of biotin-avidin complexing agents for both
bead types. Note, in this case, only one biotin is attached to each
probe such that after binding to the beads and washing there is no
unbound biotin on the probes to permit cross reaction between the
beads (see Example 1).
[0068] Once target nucleic acids have been hybridized to the first
and second hybridization probes immobilized on the first and second
beads, the nucleic acids that hybridized with the first
hybridization probe may be isolated by subjecting the hybridization
mixture to a magnetic force followed by washing. Any non-hybridized
nucleic acids and nucleic acids not containing type 1 sequence are
washed out because they are not complexed with the magnetically
responsive bead.
[0069] The second hybridization probe includes a nucleotide
sequence that does not hybridize to nucleic acids of the same type
as the first hybridization probe. Any nucleotide sequence that does
not hybridize to the first target nucleotide sequence type may be
used in the second hybridization probe and is intended to fall
within the scope of the present invention. Both hybridization
probes may include analytically detectable markers that could be
used to quantify the frequency of the nucleotide sequence
aberration being detected. Furthermore, both types of beads can be
tagged with detectable markers using methods available to someone
with ordinary skill in the art. Also, beads are now available
commercially in a wide selection of colors, e.g., from Bangs Labs,
Fishers, N.
[0070] Optionally, the second hybridization probe may hybridize to
more than one chromosome type other than the chromosome type to
which the first hybridization probe hybridizes, e.g., a
heterogeneous mixture of unique sequences selected from many
non-type 1 chromosomes (see U.S. Pat. No. 5,447,841). In this
embodiment the second hybridization probe would permit the
detection of a larger fraction of the nucleotide sequence
aberrations involving the chromosome identified by the first
hybridization probe.
[0071] Where possible, the hybridization probe preferably includes
a nucleotide sequence that is uniquely specific to the nucleotide
sequence aberration being detected. The use of uniquely specific
hybridization sequences is preferred since it minimizes the
occurrence of background noise due to nonspecific hybridization. It
is also possible to use a composite second hybridization probe that
includes a series of sequences that are all either unique or
chromosome specific for the aberration being detected. Such a
composite "cocktail" of probes, if each with detectable markers,
would enhance the signal to be detected and thus potentially
decrease the limit of detection for the assay.
[0072] After the first step of magnetic force separation, the
remaining target nucleic acids contain type 1 sequence complexed
with the magnetic responsive beads, type 1+non complexed with
magnetic responsive beads, or type 1+type 2 sequences if a
rearrangement (fusion) between type 1 and type 2 nucleic acids has
occurred (see FIG. 1). FIGS. 2A and 2B illustrate two embodiments
of the second separation step of the present invention. According
to FIG. 2A, the second bead used is non-responsive to magnetic
force, but responsive to electric force. After hybridization and
the first step of magnetic force separation described above, the
nucleic acids that contain both type 1 and type 2 sequences can be
separated by application of an electric force, because only nucleic
acids containing both type 1 and type 2 can be complexed with the
second bead, which is electrically responsive. It should be noted
that since the nucleic acids containing both type 1 and type 2
sequences are responsive to both magnetic and electric forces, they
can be purified through additional cycles of application of
magnetic and electric forces.
[0073] According to FIG. 2B, the second bead used is non-responsive
to magnetic force, but is coated with another complexing agent in
addition to complexing agent from the second pair of complexing
agents. This additional complexing agent is configured for forming
a specific complex with a complementary complexing agent that is
coated on a solid support. The combination of these two complexing
agents forms a third pair of complexing agents. After hybridization
and the first step of magnetic force separation described above,
the nucleic acids containing both type 1 and type 2 sequences can
be separated by immobilization on the solid support, because only
nucleic acids containing both type 1 and type 2 can be complexed
with the solid support by forming a complex between the third pair
of complexing agents. After washing to remove unattached bead-DNA
complexes, the immobilized nucleic acids containing type 1 and type
2 sequences may be detected by a variety of methods known in the
art including, but not limited to, automated fluorescence scanning
(e.g., tagged beads in a miniarray format), microscopy, etc. (see
Example 1).
[0074] According to the method of the present invention, the first
separation step enables the separation of nucleotide sequences of a
first nucleotide sequence type. Since the second hybridization
probe is designed so that it does not hybridize to nucleotide
sequences of the first nucleic acid type, the second hybridization
probe does not bind to nucleic acids immobilized by the first
hybridization probe that do not contain a nucleotide sequence
aberration. As a result, the number of target nucleic acid
fragments (or metaphase chromosomes) that contain both type 1 and
type 2 sequences isolated after the second step is proportional to
the number of nucleotide sequence aberrations in the sample of
nucleic acids being analyzed.
[0075] Because the magnetic separation step resulted in the
elimination of all nucleic acids that did not contain at least some
type 1 DNA, the result of the second separation step would be to
end up with only nucleic acids that contain both type 1 and type 2
DNA. The second step could also be accomplished using other
separation detection methods, such as bead size characterization,
or filtration and bead counting, taught in Example 2. Also, if the
second step was electrophoresis separation, a third step could be
added for separation and quantification such as depositing the
electrophoretically-separated bead complexes onto a miniarray on a
glass slide and detection of fluorescence labels (e.g., Cy-3) on
the beads by automated fluorescence scanning. This is a sensitive
and efficient method that, theoretically, can detect as little as
one bead per array spot.
[0076] Another format claimed in the present invention which
facilitates automated processing and detection of nucleic acid
aberrations is 96 well (or other format) plates.
[0077] If non-unique "painting probes" are used, nonspecific
binding by the non-unique hybridization probes to the nucleic acid
sample may be minimized through the use of non-specific sequence
blocking techniques such as those disclosed by U.S. Pat. No.
5,447,841, and Pinkel et al., 85 Proc. Natl. Acad. Sci. USA
9138-9142 (1988), which are hereby incorporated herein by
reference.
[0078] The first and second hybridization probes may include RNA or
DNA sequences such that the complementary nucleotide sequences
formed between the hybridization probes and the target sequence may
be two DNA sequences or an RNA and a DNA sequence.
[0079] The detection and quantification of isolated sequences
containing type 1+2 sequences can be done using a variety of
available methods, such as total DNA measured by spectrophotometry,
various labels on the beads or probes such that they can be
measured by chromatography, fluorescence, isotopes, and the like.
Any analytically detectable label that can be attached to or
incorporated into a hybridization probe or bead may be used in the
present invention. An analytically detectable label refers to any
molecule, moiety, or atom that can be analytically detected and
quantified. Methods for detecting analytically detectable labels
include, but are not limited to, radioactivity, fluorescence,
absorbance, mass spectroscopy, EPR, NMR, XRF, luminescence, and
phosphorescence. For example, any radiolabel that provides an
adequate signal and a sufficient half-life may be used as a
detectable label.
[0080] Fluorescent molecules, such as fluorescein and its
derivatives, rhodamine and its derivatives, cyanide and its
derivatives, dansyl, umbelliferone and acridimium, and
chemiluminescent molecules such as luciferin and
2,3-dihydrophthalazinediones may also be used as detectable labels.
As discussed herein, the nucleotide sequences used in hybridization
probes may themselves function as detectable labels where the bases
forming the nucleotide sequence are quantified using techniques
known in the art.
[0081] Also, beads can be detected by directly counting as in
Example 2 using different bead sizes, or by fluorescence intensity
emitted from minispots, on a glass slide detected by automated
fluorescence scanning. A large number of fluorescent tags and
methods for attaching to nucleic acid probes and beads are now
available commercially.
[0082] A nucleotide sequence aberration frequency (e.g., number of
aberrant sequences per total number of cells from which the DNA
sample was obtained) may be determined based on the signal
generated from the detectable marker using a calibration curve. The
calibration curve may be formed by analyzing a sample of cells
having a known nucleotide sequence aberration frequency. For
example, the FISH method for detecting chromosome translocations
may be used to determine the nucleotide sequence aberration
frequency rate of a sample of cells. Then, by serially diluting the
sample of cells and assaying the cells according to the method of
the present invention, a calibration curve may be generated.
Alternative methods for generating a calibration curve are within
the level of skill in the art and may be used in conjunction with
the method of the present invention.
[0083] Interchromosomal rearrangements typically are of two types:
translocations and dicentrics. Translocation are rearrangements
that result in two derivative chromosomes that have one centromere
each, whereas dicentrics are rearrangements that result in one
derivative chromosome with two centromeres and another with no
centromeres. When quantifying the frequency of interchromosomal
rearrangements, it is often useful to know whether they are
translocations or dicentrics. Translocations persist for a
lifetime, while dicentrics diminish with time. Dicentric
chromosomes may be identified according to the method of the
present invention by using first and second hybridization probes
that each hybridize to the centromere of a different chromosome.
DNA probes specific to the centromeres for almost all human
chromosomes are now commercially available for (e.g., Ventana
Medical Systems, Inc., Tucson, Ariz.; Oncor, Inc., Gaithersburg,
Md.).
[0084] The present invention also relates to a kit for separating
and quantifying nucleic acid aberrations and diagnosing diseases
according to the methods of the present invention. In general, the
kits of the present invention include a first hybridization probe
and a second hybridization probe as described herein. The kits may
also include a complexing agent bound to a magnetic responsive
bead, one or more complexing agents bound to a bead that is
magnetically nonresponsive. The kit may also be designed for a
96-well plate format, either with or without magnetic beads, as
described herein as well as instructions for using the kit. The
kits may also include beads of different sizes, colors, and
detectable markers.
[0085] All publications, patents, patent applications, and
commercial materials cited herein are hereby incorporated by
reference.
EXAMPLES
[0086] The following examples are given to illustrate various
embodiments which have been made within the scope of the present
invention. It is to be understood that the following examples are
neither comprehensive nor exhaustive of the many types of
embodiments which can be prepared in accordance with the present
invention.
Example 1
[0087] This is an example of a method that can be used to separate
and quantify DNA with two unique and non-overlapping sequences. In
this example, the sequences (identified here as Type 1 and Type 2
DNA) span 0.8 kb each and are about 17 kb apart on a contiguous
18.2 kb double stranded DNA molecule. Importantly, these sequences
are too far apart for detection by PCR, which is limited to less
than about 10 kb, and the 18.2 kb extended DNA molecule is too
small for detection by FISH which is generally limited to the
detection of condensed DNA such as that in metaphase chromosomes.
Hence, the separation and detection demonstrated in the present
example could not have been accomplished using available methods.
It is also important to note that the DNA molecule selected for
this example could just as well have been the result of a
rearrangement between Type 1 and Type 2 DNAs, where, for example,
Type 1 DNA is from one chromosome and Type 2 DNA is from another
chromosome. Hence, the method of the present example can be used to
separate and quantify any rearrangement for which complementary
Type 1 and Type 2 DNA probes are available or obtainable. A large
number of probes are now available for sequences that flank DNA
fusion points associated with cancer-related chromosomal
rearrangements, such as the t(9;22) observed in human myelogenous
leukemia and described in Tkachuk et al., 250 Science 559-562
(1990), and U.S. Pat. No. 5,487,970.
[0088] It is also possible to use the method of the present example
to separate and quantify random rearrangements that may be induced
by clastogenic agents such as radiation and certain chemicals. In
this case, the object would be to sample as large a fraction of the
genome as possible by selecting the largest chromosomes as
hybridization targets. For example, if Type 1 DNA probes were
selected to be unique to chromosome 1 (e.g., a sequence, or
cocktail of sequences, complementary to one or more genes located
on chromosome 1, or a human chromosome 1 centromeric probe
available from Oncor, Inc., Gaithersburg, Md.), and Type 2 DNA
probes were selected to be unique to chromosomes other than
chromosome 1 (e.g., a cocktail of composite probes complementary to
gene sequences on chromosomes 2 through 4), any chromosome 1 with
Type 2 DNA probes attached would be the result of a translocation
between chromosome 1 and chromosomes 2, 3, and/or 4. The present
method would quickly separate and quantify such events. A large
number of chromosome-mapped gene sequences are now available from
human genome projects. In fact, a chromosome-specific physical map
now exists for over 30,000 human genes (Deloukas et al., Science
282, 744-746; 1998) and a large number of these genes have
sequences published on the Internet (e.g., www.ncbi.nlm.nih.gov).
Importantly, anyone skilled in the art can use available methods
(described below) and these published sequences to synthesize
biotinylated probes that are complementary to the available gene
sequences and thus make biotinylated probes (or cocktails of
composite biotinylated probes) that are unique to any human
chromosome, even unique to many regions of human chromosomes. The
probes could also be painting probes (e.g., commercially available
from Vysis, Inc., or Ventana Medical Systems, Inc. (Tucson, Ariz.).
For painting probes, non-specific hybridization can be reduced
using available non-specific hybridization blocking methods (Pinkel
et al., Proc. Natl. Acad. Sci. USA 9138-9142, 1988; U.S. Pat. No.
5,447,841). By using chromosome-specific (unique) probes (e.g.,
complementary to gene coding sequences) for hybridization to Type 1
DNA, the first separation step would be assured to be very clean.
For the special case of separation and quantification of dicentric
chromosomes (i.e., interchromosomal rearrangements resulting in a
chromosome with two centromeres), centromeric probes can be used
for both Type 1 and Type 2 DNA. These probes are available
commercially for essentially all human chromosomes (e.g., Oncor,
Inc).
[0089] Target DNA
[0090] The DNA used as hybridization target for this example of the
present method is an 18.2 kb insert in a pUC19 plasmid (FIGS.
3A-B). The double-stranded DNA insert was positioned at the
plasmid's SalI site so that it could be removed from the plasmid by
digestion with SalI restriction enzyme.
[0091] The DNA insert was amplified by growing the plasmid in E.
coli bacteria as follows. First, 4.2 .mu.g of plasmid DNA was
diluted in 1000 .mu.l distilled deionized water. One .mu.l of this
solution was then used to electroporate the DNA into E. coli using
a Bio-Rad electroporation apparatus (Bio-Rad, Inc.). The E. coli
were immediately collected and placed in an incubator at 37 C for
30 min, followed by plating on agar medium (Luria-Bertani with
ampicillin), and incubating at 37 C overnight. The next day,
individual colonies were collected and grown overnight in 5 mL of
liquid medium (Luria-Bertani with ampicillin).
[0092] The plasmid DNAs were purified using a QIAprep-spin plasmid
kit (#27104) according to manufacturer's protocol in QIAprep
Miniprep Handbook, April 1998, pp. 18-19 (Qiagen, Inc., 28159
Stanford Ave., Valencia, Calif. 91355).
[0093] The purified plasmid DNAs were then digested with SalI to
permit extraction of the 18.2 kb target DNA insert. The Sal I
restriction enzyme was obtained from Sigma at a stock concentration
of 10,000 units/mL. To the plasmid solution (30 .mu.g/132 .mu.l TE)
was added 15 .mu.l SalI stock solution, 30 .mu.l Sal I buffer from
Sigma, and 135 .mu.l distilled deionized water. After mixing, the
reaction mixture was incubated at 37 C for 1 hour, then maintained
at 4.degree. C. overnight. Before use, solutions were changed to
fresh aliquots of B&W buffer (10 mM Tris-HCl, pH 7, 1 mM EDTA,
2 M NaCl).
[0094] Then, the 18.2 kb target DNAs were extracted using
phenol/chloroform/isoamyl alcohol (25:24:1; v:v:v) as described in
"Molecular Cloning: A Laboratory Manual", Second Edition, Sambrook
et al., Eds. (1989) and washed with ether. DNAs were precipitated
with 100% ethanol after adding 3 M NaOAc to final concentration 0.3
M. The precipitated solutions were then stored at 0 C for 2 hours,
centrifuged, and the supernates removed. Residues were washed with
70% ethanol and dried at room temperature in air.
[0095] The extracted 18.2 kb DNAs were verified by agarose gel
electrophoresis using standard methods.
[0096] DNA Probes
[0097] For the present example, the DNA sequences (50mers) used as
hybridization probes were synthesized using an Applied Biosystems
Model 3948 synthesizer (Perkin-Elmer, Applied Biosystems Division,
Foster City, Calif.) and standard methods known to those skilled in
the art of DNA oligonucleotide synthesis to be uniquely homologous
with the terminal ends of the 18.2 kb insert. First, about 1 kb of
DNA was sequenced at each terminal end of the 18.2 kb insert. Then,
four 50 bp complementary sequences, each with uniquely different
sequences, were synthesized with homology to one end of the DNA
insert (Type 1 Probes) and another four 50 bp complementary
sequences synthesized with unique homology to the other end of the
DNA insert (Type 2 Probes).
[0098] The probes were then biotinylated by covalently attaching
biotin TEG phosphoramidite (Cat. #10-1955-02, Glen Research,
Sterling, Va.) to the 5' end of the DNA probes via a 15 atomic bond
spacer arm. The result is that no probe has more than one biotin
attached to it, and hence there is no unbound biotin on the probe
after complexing with avidin on the solid support and washing. This
is useful in the present invention because all probe types (as well
as any other attachments to the beads such as probes for detection
and peptides for other unique complexing agents) can use biotin as
complexing agents to attach them to the avidin-coated beads as long
as the attachments to the beads of different probe types are
performed in separate solutions. Using a spacer arm greater than 10
to 12 atomic bonds eliminates potential binding problems between
the biotinylated probes and the beads, and also facilitates
subsequent hybridization of the probe to the target DNA (A. J.
Ninfa and D. P. Ballou, "Fundamental laboratory approaches for
biochemistry and biotechnology", pp. 102-103, 1998, Fitzgerald
Science Press, Inc., Bethesda, Md.).
[0099] The sequences of the terminal ends of the 18.2 kb DNA and
the unique 50mer probes are shown in FIG. 4. Sequencing of the ends
of the 18.2 kb DNA was performed using ABI Prism BigDye Terminators
and cycle sequencing with Taq FS DNA Polymerase. DNA sequences were
collected and analyzed on an ABI Prism 377 automated DNA sequencer
(Perkin-Elmer, Applied Biosystems Division, Foster City,
Calif.).
[0100] Beads
[0101] Two types of beads were used in this example,
magnetically-responsive beads purchased from Dynal AS, Oslo,
Norway, and magnetically non-responsive beads purchased from Bangs
Labs, Fishers, Ind.
[0102] The magnetically responsive beads were "Dynabeads
Streptavidin" (Product #112.05), which are 2.8 .mu.m in diameter
and coated with streptavidin. The magnetically non-responsive beads
were from Bangs Labs (Catalog #CP0 IN), which are 0.94 .mu.m in
diameter and also coated with streptavidin. The two bead types are
seen in FIG. 5 (1000.times. magnification, light microscope).
[0103] The two bead sizes selected provided a simple and very
useful means to verify results as the technology was being
developed. That is, hybridization and separation efficiencies are
rapidly determined by observing bead types at various stages using
a light microscope.
[0104] Attach Probes to Beads
[0105] The biotinylated probes were then attached to the Dynal
avidinylated beads as follows. First, 0.2 mL TE buffer solution (10
mM Tris-HCl, pH 7.5, 1 mM EDTA) containing 2.8 .mu.g Probe 1, 2.8
.mu.g Probe 2, 2.8 .mu.g Probe 3, 2.8 .mu.g Probe 4, and 1.3 .mu.g
of a biotinylated T7 22mer (Cat. #300322, Stratagene.com) was
prepared. Probes 1 through 4 were the 50mers that were synthesized
to be complementary to the DNA on one end of the 18.2 kb target DNA
(Type 1 DNA). The T7 22mer is a unique probe used here for
detection purposes only, i.e., it provides an option for subsequent
hybridizations with a fluorescent marker to detect the presence of
the Dynal beads.
[0106] Next, the Dynal beads (2 mg/0.2 mL) were washed once with 1
mL of B&W buffer solution and resuspend in 0.2 mL B&W
buffer. For the magnetically-responsive Dynal beads, washing was
accomplished using the Magnetic Particle Concentrator (MPC) (Dynal
AS).
[0107] Finally, the solution of Probes 1 through 4 and T7 was mixed
with the solution of Dynal beads. This mixture was then gently
shaken for 1 hour at room temperature. The beads were then washed
four times with B&W buffer, 0.4 mL each. Next, the probe-coated
Dynal beads were resuspended in 0.4 mL B&W and stored at 4
C.
[0108] The biotinylated probes were attached to the Bangs
avidinylated beads as follows. Similar to the preparation of the
Dynal beads described above, 0.2 mL TE buffer solution was prepared
containing 2.8 .mu.g Probe I, 2.8 .mu.g Probe II, 2.8 .mu.g Probe
III, 2.8 .mu.g Probe IV, and 12 .mu.g of a biotinylated
hemagglutinin (HA) peptide. Probes I through IV were the 50mers
that were synthesized to be complementary to the DNA on the other
end of the 18.2 kb target DNA (Type 2 DNA). The HA peptide was
synthesized using an Advanced ChemTech Model 348 peptide
synthesizer (Advanced ChemTech, Inc., www.peptide.com) and was
biotinylated by attaching a single biotin molecule to the terminal
amino-end of a 6 carbon spacer molecule that was then attached via
its carboxyl end to the terminal amino end of the HA peptide. The
biotinylated HA peptide was then attached to the Bangs beads to be
used subsequently as a complexing agent for stage 2 separation
involving anti-HA antibody.
[0109] Next, the Bangs beads (2 mg/0.2 mL) were washed once with 1
mL of B&W buffer followed by one wash with 1 mL of TE. The
beads were resuspended in 0.2 mL B&W solution. For the Bangs
beads (which were non-magnetic), washing was accomplished using
centrifugation.
[0110] Finally, the solutions of biotinylated probes I through IV
and biotinylated HA peptide were mixed with the solution of Bangs
beads. This mixture was gently shaken for 1 hour at room
temperature, and then the reaction solution was removed by
centrifugation. The beads were then washed once with 1:1 (v/v)
B&W buffer and TE, 0.4 mL each. The probe-coated Bangs beads
were then resuspended in 0.4 mL B&W and stored at 4 C.
[0111] Hybridization and Magnetic Separation (Usually Stage 1
separation)
[0112] The 18.2 kb target DNA was hybridized to probes on beads.
Selected amounts (typically .mu.g quantities) of the 18.2 kb DNA
were dissolved in 300 .mu.l 70% formamide denaturing solution (2.10
.mu.l formamide, 30 .mu.l 20.times.SSC, 60 .mu.l distilled
deionized water), then heated to 70 C for 5 min. The hot solution
was immediately added to the cooled solution containing 10 .mu.l
Dynal beads (2 mg/0.2 mL TE), 10 .mu.l Bangs beads (2 mg/0.2 mL
TE), 75 .mu.l 20.times.SSC, and 16.6 .mu.l distilled deionized
water. After mixing, 4.2 .mu.l 10% SDS and 4.2 .mu.l salmon sperm
DNA were added. Hybridization was carried out in an incubator for
15 hours at 40 C with constant rotation of about 1 rpm. After
cooling to room temperature, MPC was used to remove hybridization
solution and unhybridized Bangs beads. The remaining beads were
then washed once with 600 .mu.l 1.times.SSC, 0.2% SDS, for 6 min at
room temperature. The MPC washing step was then repeated. The beads
were next washed with 600 .mu.l solution of 0.1.times.SSC, 0.2%
SDS, for 10 min at room temperature. The MPC step was repeated, and
then the beads were washed three times with distilled deionized
water. The beads were next resuspended in 500 .mu.l PBS. A 50 .mu.l
aliquot was taken as solution B (1.34.times.10.sup.4 Dynal
beads/L), from which 5 .mu.l was mixed with 45 .mu.l PBS to become
solution C (1.34.times.10.sup.3 Dynal beads/L). The remaining 450
.mu.l solution was removed by MPC and 45 .mu.l PBS was added to
become solution A (1.34.times.10.sup.5 Dynal beads/1 L). Note: PBS
buffer is 0.14 .mu.g NaH.sub.2PO.sub.4, 0.79 .mu.g
Na.sub.2HPO.sub.4, 8.1 .mu.g NaCl in total 1000 mL distilled
deionized water.
[0113] Filtration Separation
[0114] If the magnetically responsive beads in solutions A, B, and
C above were selected to be larger than the magnetically
non-responsive beads, then Stage 2 separation could be accomplished
by cutting the DNA connecting the two bead types (i.e., the 18.2 kb
DNA molecule hybridized to both bead types can be digested by DNase
or released from the magnetically-responsive bead via a cleavable
linker) and filter the solution through a filter selected to permit
only the smaller non-magnetic beads to pass through. The number of
small beads could then be quantified using available methods such
as a Coulter counter. Also, if a cleavable linker is used to
release the DNA molecule from the magnetic bead then the DNA would
be pulled along with the small non-magnetic bead through the filter
and would thus permit recovery of the target DNA. Importantly, the
number of small beads recovered after filtration should be
proportional to the number of target DNA molecules (i.e., Type
1+Type 2 DNA) in the hybridization solution. An example of the
filtration method is described in Example 2 below.
[0115] Antibody-Peptide Separation
[0116] Attach antibody to glass slides. Streptavidin coated
microscope slides obtained from Cell Associates, Inc.
(www.cel-1.com) were washed 3 times with B&W buffer solution,
then immersed in 20 mL solution containing 100 .mu.g anti-HA-biotin
(i.e., 10 mL TE, 9.5 mL Tris, 0.5 mL of 200 .mu.g
anti-HA-biotin/mL). The slides were shaken for 30 min at room
temperature, then allowed stand at room temperature for 18 hours.
Next, the slides were washed five times (2 min each) with PBS.
After air drying at room temperature, the slides were stored at 4
C. The anti-HA-biotin was purchased from Boehringer Mannheim
Corporation (Indianapolis, Ind.). Then, 1 .mu.l each of solutions
A, B, and C (from Stage 1 separation described above) was placed on
the anti-HA coated slide at room temperature. After about 30 min,
but before the 1 .mu.l spots were dry, the slide was transferred
into a slide box with a small amount of water in the bottom of the
box (the water should not come in direct contact with the slide).
The slides were incubated at 4 C overnight, then washed twice with
PBS and once with distilled deionized water. The slides were then
dried at room temperature.
[0117] Detection and Quantification
[0118] Light microscopy. The beads deposited on the slides were
then viewed using a Nikon microscope at 1000.times. total
magnification (100.times. objective and 10.times. eyepiece). FIG. 6
shows a microscope image of a bead solution B (described above)
deposited on a slide after probe attachment and magnetic
separation, but without hybridization to the 18.2 kb target DNA.
Note that no Bangs beads are observed. This demonstrated that Bangs
beads are washed away during the magnetic separation step if not
connected to the Dynal beads via hybridization to the 18.2 kb DNA
molecule.
[0119] In contrast, FIG. 7 shows a microscope image of bead
solution B which was hybridized to the 18.2 kb DNA. In this case,
the bead-probe complexes were hybridized to the 18.2 kb DNA
followed by magnetic separation as described above under
"Hybridization and Magnetic Separation." Note that many of the
smaller Bangs beads were present in FIG. 7 and were pulled along
during the magnetic separation step by their attachment via
hybridization to the same DNA molecule that the Dynal beads were
hybridized.
[0120] Fluorescence scanning. In addition to light microscopy
analysis, beads deposited on glass slides have also been analyzed
using a fluorescence scanner. Fluorescence intensities were
measured of 0.1 .mu.l spots of beads deposited in an array format
on a glass slide. The measurements were made using a Molecular
Dynamics fluorescence scanner (Avalanche model). Each spot contains
an average of about 33 Dynal beads and was about 1 mm in diameter.
After depositing on the slide, the beads were hybridized with a
Cy-3 labeled 22mer complementary to the T7 22mer that was
previously attached to the Dynal beads. The complementary 22mer was
synthesized using standard methods and a Cy-3 molecule was attached
to the 5' end of the 22mers using Cy3-CE-Phosphoramidite (Glen
Research, Sterling, Va.). Based on the present results, the fact
that each streptavidin-coated Dynal bead can attach about 500,000
oligonucleotides (Dynal AS, Oslo, Norway), and the detection limit
of commercially available fluorescence scanners which are on the
order of 1 to 10 Cy-3 per m.sup.2 (Bowtell, 21 Supplement, Nature
Genetics page 31, 1999), the method described herein should be able
to detect as little as a single bead deposited on a glass slide
using Cy-3 labeling and a commercially-available fluorescence
scanner. Similar detection limits would be expected for other
common fluorescence labels. In practice, each bead type could be
tagged in advance and thus identified at any stage of separation
using a fluorescence scanner.
[0121] Particle counting. As described below in Example 2, if beads
of different sizes are used with the present technology, each size
coated with a unique probe, then the different sized beads can be
selected by filtration and counted using a particle counter (e.g.,
from Coulter). This is a very quick, accurate, and low-cost method
to quantify the number of beads which, after hybridization and
magnetic separation, would be proportional to the number of target
DNA molecules (or other target objects) in the analysis solution
(see Example 2 for details).
[0122] It is also possible (as described in Example 2) to eliminate
the filtration step by using a particle counter that measures both
number of particles and their sizes (e.g., the Multisizer II by
Coulter). In this case, distributions of particle sizes would be
obtained and the number of beads of any selected size can be
quantified by integrating the distributions (e.g., see Examples 6
and 7).
[0123] Electrophoresis Separation
[0124] It is also possible to perform separation using
electrophoresis of the magnetically non-responsive beads, if such
beads are selected to be electrically charged (e.g., this would
permit the use of electrophoresis for Stage 2 separation and
antibody-peptide separation for Stage 3). An experiment was
performed in which two bead types were used,
magnetically-responsive beads coated with amino groups (positive
surface charge) and magnetically non-responsive beads coated with
carboxylic acid (negative surface charge). The beads were from
Bangs Labs (#MC05N, magnetic; #DC04, non-magnetic). The beads were
selected to have two different colors, the magnetic beads were
brown and the non-magnetic beads were green. Both bead types were
about 1 .mu.m diameter.
[0125] A 2 mm hole was drilled through a 10 mL plastic pipette (the
hole was drilled at the 5 mL mark through one side only) and the
pipette submerged in a standard TAE electrolyte buffer (pH 8.3) of
a standard electrophoresis apparatus. The two types of beads were
then mixed 1:1 in TAE buffer (pH 8.3) and 1 mL injected through the
hole in the center of the pipette. The electrophoresis was carried
out at 5 Volts per cm and a photograph taken at the start of the
electrophoresis and again at +20 min. The results clearly showed
that the green and brown beads were mixed at T=0 but were separated
by about 1.5 cm at T=20 min (i.e., the negatively charged
non-magnetic beads moved about 0.75 mm per min). This demonstrated
the feasibility of using electrophoresis to separate non-magnetic
beads from magnetic beads.
[0126] Based on these electrophoresis results, the magnetic
separation results provided in this example, and the
electrophoretic mobility measurements of similar beads made by
Ottewill et al., Kolloid Zh., 218, 34 (1967), it is apparent that a
magnetic bead with essentially neutral surface charge should be
pulled along with the electrophoretically-responsive non-magnetic
beads if the beads were connected to the same DNA molecule in a
buffered solution. Magnetically-responsive beads with various
surface charges (including essentially neutral surface charge) are
commercially available (e.g., Bangs Labs, Fishers, Ind.).
Example 2
[0127] As discussed above, Stage 2 separation and quantification
can also be accomplished by particle counting and. size
distribution analysis.
[0128] Filtration. For example, if Stage 1 separation is by
magnetic force and Stage 2 is by filtration of beads, then the
magnetically responsive beads would simply be selected to be larger
than the non-magnetic beads. This would permit rapid separation of
magnetically-responsive beads from non-magnetic beads. Because the
only magnetically non-responsive beads remaining after Stage 1
separation are those complexed with magnetically-responsive beads
via hybridization to the same contiguous nucleic acid molecule, the
number of non-magnetic beads after Stage 1 would be proportional to
the number of target nucleic acid molecules in the hybridization
mix.
[0129] To facilitate separation of the beads by filtration, Stage I
could be followed by detaching DNA from beads (e.g., via DNase
treatment or cleavable linker) and filtering the beads through a
filter that only permits the smaller non-magnetic beads to pass
through. The smaller beads could then be counted using available
particle counting technologies (e.g., Coulter counter) or
quantified using available technologies such as fluorescence, flow
cytometry, spectrophotometry, etc. Commercially available beads
have a large number of colors and fluorescence wavelengths (e.g.,
Bangs Labs.).
[0130] FIGS. 5, 6, and 7, and Table I provide results that
demonstrate the successful separation and quantification of target
DNA molecules using a combination of magnetically-responsive and
magnetically non-responsive beads, each type differing in size.
FIG. 5 is a photomicrograph of the two bead types used in this
example. The magnetically-responsive beads are 2.8 .mu.m diameter
and the non-magnetic beads are 0.94 .mu.m diameter (beads are
described in Example 1). FIG. 6 shows that all of the magnetically
non-responsive beads are eliminated (washed away) during magnetic
separation if hybridization of the DNA molecule is not performed to
both the magnetic and the non-magnetic beads. In contrast, if
hybridization is performed then some of the small beads are pulled
along with the large beads during magnetic separation, as seen on
the glass slide in FIG. 7.
[0131] If instead of depositing on a glass slide after magnetic
separation (as was done in FIG. 7), the beads are separated from
each other by detaching the hybridized DNA by, e.g., DNase
treatment or a detachable linker between the magnetic bead and the
hybridized DNA, and pass the beads through a 2 .mu.m filter, the
2.8 .mu.m magnetic beads would be too large to pass through the
filter resulting in a filtered solution of only the non-magnetic
beads (see data in Table I). These are the magnetically
non-responsive beads that were previously hybridized to the same
contiguous piece of DNA as the magnetic beads and thus were pulled
along during magnetic separation. Note that after magnetic and
filtration separation the number of these small beads recovered
would be proportional to the number of Type 1+Type 2 DNA in the
hybridization mix (this would be true for the present example as
well as for target DNAs obtained from a human blood sample). The
methods used to obtain the results in FIGS. 5, 6, and 7, are
identical to those described in Example 1. The methods used to
obtain the data in Table I are also identical to the methods in
Example 1 for DNA target, DNA probes, beads, attachment of probes
to beads, hybridization, and magnetic separation.
[0132] In Example 2, i.e., where Stage 2 separation is by
filtration or size distribution analysis, the magnetic separation
step in Example 1 is followed by detaching the DNA from the beads
using DNase treatment (DNase I from Gibco BRL, Frederick, Md.)
resulting in the beads becoming free in solution. One .mu.l of the
DNase I stock solution was diluted in 405 .mu.l glycerol and 405
.mu.l NTB buffer. The NTB buffer comprises 0.5 M Tris-HCl pH 7.5,
0.1 M MgSO.sub.4, 1 mM dithiothreitol, and 500 g/mL bovine serum
albumin (Fraction V, Sigma). Then, 22 .mu.l of the diluted DNase
solution was added to 44 .mu.l of the bead solution to be DNA
digested (i.e., the beads were in 44 .mu.l 3.times.NTB). The
solution of beads was then passed through a filter that permits
only the small beads to pass through, in this example, a filter
with 2 .mu.m diameter circular pores (polycarbonate membrane filter
available commercially from Millipore, Fisher Scientific Catalog
#MP 013 00). Associated syringe filter holders are also
commercially available to perform the filter operation (used for
the present example, Millipore Stainless Steel 13 Filter Holder,
Fisher Scientific Catalog Number XX-30 012 00).
[0133] The number of beads in the filtered solution can then be
quantified using available methods, e.g., a Coulter counter,
microscopy, fluorescence scanning, flow cytometry,
spectrophotometry, etc. A preferred method of bead quantification
is by counting in a Coulter counter. At this stage, only the small
beads that were hybridized to a Type 1+Type 2 DNA are present in
the solution, hence the number of these beads are proportional to
the number of Type 1+Type 2 DNA sequences in the initial sample
(e.g., a blood sample). The proportionality constant can be
obtained from laboratory measurements using standard mixes of
target DNA and bead-probe complexes. That is, just as routinely
done for most kinds of measurement technologies, a standard curve
would be used to convert the measured number of beads to the number
of target DNAs in the original sample.
[0134] Table I presents results obtained from a filtration
experiment. In this case, three different solutions were filtered
using the 2.0 .mu.m Millipore filter system described above: (1)
saline only without beads, (2) a solution containing both Dynal and
Bangs beads which has been subjected to the complete hybridization
conditions and reagents except that the 18.2 kb DNA was not
included in the hybridization mix, and (3) a solution containing
both Dynal and Bangs beads and hybridized with 20 .mu.g of the 18.2
kb target DNA. The results demonstrate that without hybridization
to the 18.2 kb DNA target molecule, the Bangs beads are not pulled
along with the Dynal beads during magnetic separation. That is,
without hybridization there is no significant difference between
the bead counts and the saline background. This is due to the
removal of all Bangs beads during the magnetic separation step and
subsequent removal of all Dynal beads during the filtration
step.
TABLE-US-00001 TABLE I Coulter Counter Results for Three Types of
Filtered Solutions Mean Counts/100 L SD Filtered Saline 145 109
Filtered beads*, w/o 159 20 hybrid Filtered beads**, with 2910 300
hybrid Table I shows particle counter results for three types of
filtered solutions, filtered using 2.0 .mu.m Millipore filters
(Fisher Scientific Cat. No. TTTP 013 00), wherein a Coulter counter
Model Zf was used with a 30 .mu.m aperture tube to measure the
indicated solutions. The means and SDs are based on multiple
measurements.
[0135] In contrast, when hybridization is done then the Bangs beads
are detected after filtration. This is due to the fact that both
Dynal and Bangs beads are hybridized to the same 18.2 kb DNA
molecule. Hence, the Bangs beads are pulled along by the Dynal
beads during the magnetic separation step, released from the Dynal
beads by DNase treatment, and finally separated from the Dynal
beads by filtration through a 2.0 m filter that only permits the
Bangs beads to pass through. The results for the conditions of the
present experiment show a count signal that is 20 times above
background, providing a very accurate measurement.
[0136] The data in Table I suggest a detection limit for this
method of about 5 ng target DNA or about 1000 cell equivalents of
DNA. This is highly competitive with available detection methods
(Duggan et al., 21 Nature Genetics (Supplement) 10-14 (1999)).
[0137] More generally, this approach can be used to detect
rearrangements in any nucleic acid molecule larger than about 10 bp
for which suitable hybridization probes are available or
obtainable, including DNA at any level of organization from single
stranded to metaphase chromosomes. For example, DNA probes for abl
and bcr (which are available) could be used together with the
technology described here to rapidly separate and quantify marker
chromosomes for human chronic myelogenous leukemia, i.e., so-called
Philadelphia chromosomes involving a very specific translocation
between chromosomes 9 and 22. The abl and bcr genes flank the
fusion point on chromosome 22 (Tkachuk et al., 250 Science 559-562
(1990)). To quantify these types of rearranged chromosomes using
the present invention, a uni-biotinylated probe (only one biotin
per probe) homologous to bcr is complexed to the avidin-coated
Dynal paramagnetic beads and a uni-biotinylated probe homologous to
abl to the avidin-coated Bangs non-magnetic beads and then perform
the separation and quantification procedures as described
above.
[0138] It is also an embodiment of the present invention to employ
methods for the specific detachment of the DNA from the
magnetically-responsive bead prior to filtration. If the detachment
site was between the DNA and the bead then the complete DNA
molecule would remain attached to the small non-magnetic bead and
would therefore pass through the filter and be isolated together
with the small beads. This would permit further
evaluations/diagnostics of the isolated target DNA. Site-specific
detachment can be accomplished using available cleavable
linkers.
[0139] Both specific and random rearrangements can be quantified
using this approach. The 9;22 translocation described above is an
example of a specific rearrangement. An example of the
quantification of random rearrangements is if we selected a probe
with unique homology to chromosome 1 (i.e., Probe 1) and a cocktail
of composite probes with homology to other chromosome(s) but not to
chromosome 1 (i.e., Probe 2). The more sequences of
non-chromosome-1 targets covered by Probe 2, the more
translocations would be detectable. Probe 1 would then be complexed
with the 2.8 .mu.m paramagnetic beads and Probe 2 would be
complexed with the 0.94 .mu.m non-magnetic beads. If, in this
example, Probe 2 hybridizes to chromosome 1 then interchromosomal
rearrangement has in fact occurred. These translocated chromosomes
can then be separated and quantified using the method described
above. It is expected that standard calibration curves would be
obtained for each particular kind of detection kit, i.e., one for
each specific rearrangement and one for a particular class of
random rearrangements.
[0140] Size distribution analysis. If, after magnetic separation
and DNA detachment (described above), the resultant solution of
beads is analyzed using a particle sizer/counter (e.g., a
Multisizer II by Coulter), the two bead types can be separated by
their size distributions and the number of beads in each
distribution quantified. This is illustrated in FIG. 8. It is seen
that the distributions for the 4.4 .mu.m beads and the 2.8 .mu.m
beads are well separated and that the number of beads in each
distribution can be quantified by integration of the peaks. In
contrast to Table I, the distributions in FIG. 8 show the results
of a DNA separation experiment in which the magnetically
non-responsive beads were larger than the superparamagnetic beads.
In this case, the larger non-magnetic beads were selected to place
the peak in a region of lower background counts. The materials and
methods used to obtain the results in FIG. 8 were the same as those
used for Table 1 with the following differences: FIG. 8 used 4.4
.mu.m diameter magnetically non-responsive polystyrene beads coated
with streptavidin (Bangs Labs, Fisher, Ind.) whereas Table 1 used
0.94 .mu.m diameter magnetically non-responsive polystyrene beads
coated with streptavidin (Bangs Labs); FIG. 8 used 5 .mu.g of 18.2
kb target DNA whereas Table I used 20 .mu.g of 18.2 kb target DNA;
partial magnetic separation was done for FIG. 8 following DNase
treatment to reduce (but not fully eliminate) the number of
superparamagnetic beads to provide a lower background level while
at the same time provide a 2.8 .mu.m peak for
comparison/illustration purposes; and for FIG. 8 the final bead
concentration was diluted two-fold just before generating the bead
size distributions using the Coulter Multisizer H. Note that when
accounting for the differences in the amount of target DNA
hybridized and final two-fold dilution, the results of Table 1 and
FIG. 8 agree very well, i.e., 2910.+-.300 non-magnetic beads were
recovered in the experiment reported in Table I, while 322
non-magnetic beads were recovered in the experiment shown in FIG.
8. If the two experiments are normalized by the target DNA (5 .mu.g
v. 20 .mu.g) hybridized and the two-fold dilution, the result in
FIG. 8 of 322 non-magnetic beads would become
322.times.4.times.2=2576 non-magnetic beads, not significantly
different from 2910 in Table I.
[0141] It should also be noted that the intention of the present
invention is not to be limited by two sizes of beads, but rather
that many beads sizes may be used in combination in a more complex
and multiplexed system of separation and detection.
Example 3
[0142] This is an example of a method that can be used to separate
and quantify nucleic acids (or chromosomes) with two unique and
non-overlapping sequences, both on a contiguous nucleic acid
molecule or chromosome. In this example, the two different
sequences are identified as Type 1 and Type 2 nucleic acid
sequences.
[0143] The method of the present disclosure includes, but is not
limited to, the use of two kinds of microbeads. One kind of
microbead is responsive to a magnetic field (e.g., Dynabeads
Streptavidin, Product #112.05 from Dynal A/S, Oslo, Norway) and
could be coated with biotinylated nucleic acid probes complementary
to Type 1 nucleic acid sequences by attaching to the avidin on the
surface of the magnetically-responsive beads. The other kind of
microbead is not responsive to a magnetic field (e.g.,
streptavidin-coated polystyrene beads from Bangs Labs, Fishers,
Ind.) and could be coated with biotinylated nucleic acid probes
complementary to Type 2 nucleic acid sequences by attaching to the
avidin on the surface of the magnetically non-responsive beads. The
attachment of the two probe types to their respective kinds of
beads would be performed in separate solutions. The magnetically
non-responsive beads would also be coated with an
electrochemiluminescence (ECL) marker, e.g., ruthenium (II)
tris-bipyridine NHS ester. See Blackburn, et al. Clin. Chem. 37(9),
1534-1539 (1991) for a description of detection using ECL
labels.
[0144] In the present invention, the ECL marker would be attached
to the magnetically non-responsive beads by, e.g., biotinylating
the marker and attaching it to the beads either separately or in
competition with the biotinylated probes. An example of DNAs and
coated beads is illustrated in FIG. 9.
[0145] After coating the respective kinds of beads and washing away
unbound reagents, the coated beads are mixed together in a
hybridization solution containing the target nucleic acid (e.g.,
isolated DNA or metaphase chromosomes). Using hybridization
reagents and conditions available in the art (e.g., see Example 1
for isolated DNA; the Example in U.S. Pat. No. 5,731,153 for
metaphase chromosomes; and the hybridization methods in U.S. Pat.
No. 5,447,841 for "painting" probes) the result is the
hybridization of the probes on the microbeads to their respective
homologous sequences on the target nucleic acid, which produces the
hybridized complexes shown in FIG. 9.
[0146] The next step is magnetic separation. This is accomplished
by first gently shaking the vial to obtain a homogeneous suspension
of beads and then placing the vial in the Dynal magnet stand (Dynal
A/S, Oslo, Norway) for 2 minutes to allow beads to migrate to the
side of the tube. This is followed by removing the supernatant by
aspiration with a pipette while the tube remains in the magnetic
stand. The tube is then removed from the magnetic stand and fresh
buffer is added. This separation step can be repeated and results
in the removal of all DNA and beads that are not connected to
magnetically-responsive beads via a contiguous target molecule,
i.e., only the kinds of complexes shown in FIG. 9 should remain in
the sample tube after magnetic separation.
[0147] Importantly, following magnetic separation, the only complex
with the ECL label is in fact the one containing both Type 1 and
Type 2 DNA. This could, for example, be a fusion between two
different chromosomes such as those resulting from interchromosomal
translocations, or it could be any two uniquely different sequences
on a contiguous target nucleic acid molecule.
[0148] The next step is detection by ECL. This would be
accomplished by adding vast molar excess of tripropylamine (TPA) to
the buffer containing the magnetically separated bead complexes and
then placing aliquots of the solution onto an electrode surface. A
low voltage is applied to the electrode which triggers a cyclical
oxidation and reduction reaction of the ruthenium metal ion which
generates the emission of 620 nm photons. Methods and devices that
can be used to detect and quantify the ECL label on the
non-magnetic beads are described in Blackburn, et al. Clin. Chem.
37(9), 1534-1539 (1991), and also in www.igen.com (i.e., the home
page of IGEN International, Inc., which provides a commercially
available instrument to detect ECL signals). Importantly, by
keeping our magnetic-non magnetic bead complexes intact following
magnetic separation (i.e., no DNase treatment) will permit direct
measurement using IGEN's automatic sample processing system which
employs a magnet to immobilize each sample during ECL measurement.
The magnetic bead in our complex will serve as the immobilizing
particle while the attached non-magnetic bead will contain the ECL
label and hence produce the ECL signal.
[0149] Detection is illustrated in FIG. 9, which includes the Type
1+Type 2 DNA bead complex deposited on the electrode surface. A
photon is emitted and detected by a photomultiplier tube (PMT) or
other suitable detection system. Such detection systems are now
available commercially (e.g., IGEN International, Inc.). For a
given number of ECL labels per bead (which can be selected) the
number of photons is proportional to the number of non-magnetic
beads, which for a given probe/target nucleic acid protocol, is
proportional to the number of contiguous Type 1+Type 2 target
sequences in the sample.
[0150] More generally, this approach can be used to detect
rearrangements in any nucleic acid molecule of sufficient size to
hybridize one or more microbeads to each type of nucleic acid, and
for which suitable hybridization probes are available or
obtainable, including DNA at any level of organization from single
stranded to metaphase chromosomes. Presently, commercially
available microbeads range in diameter from about 50 nm to several
mm. Fifty nm is equivalent to the length of about 50 bp of the DNA
molecule and would probably be near the minimum distance required
to hybridize one bead. Of course, the maximum distance will simply
depend on the size of the target sequence. Alternatively, one could
hybridize the magnetically-responsive microbead to the Type 1
nucleic acid and hybridize a nucleic acid probe with the ECL label
attached directly to the probe without a non-magnetic bead to the
Type 2 nucleic acid target. This should permit unique hybridization
to target nucleic acid sequences larger than about 10 bp.
[0151] Both specific and random rearrangements can be quantified
using this approach. For example, DNA probes for abl and bcr (which
are available) could be used together with the technology described
here to rapidly separate and quantify marker chromosomes for human
chronic myelogenous leukemia, i.e., so-called Philadelphia
chromosomes involving a very specific translocation between
chromosomes 9 and 22. The abl and bcr genes flank the fusion point
on the Philadelphia chromosome. To quantify these types of
rearranged chromosomes using the present invention, we would attach
the probe homologous to bcr to the superparamagnetic beads and the
probe homologous to abl and the ECL label to the non-magnetic beads
and then perform the separation and quantification procedures as
described above.
[0152] An example of the quantification of random rearrangements is
if we selected a probe with unique homology to chromosome #1 (i.e.,
Probe 1) and one or more probes with homology to any other
chromosome but not to chromosome #1 (i.e., Probe 2). Probe 1 would
then be complexed with the paramagnetic beads and Probe 2 (which
could be a composite of several probes, including painting probes)
would be complexed with the non-magnetic beads containing the ECL
label. If both Probe 1 and Probe 2 are hybridized to the same
chromosome then interchromosomal rearrangement has in fact
occurred. These translocated chromosomes can then be separated and
quantified using the method described above. Standard calibration
curves would be obtained for each particular kind of detection kit,
e.g., one for each specific kind of rearrangement and one for a
particular class of random rearrangements. For example, ECL
intensity vs. rearrangements per cell, could be measured in
standard samples of known rearrangement frequencies using
standardized kit formats with demonstrated reproducibility.
Example 4
[0153] Avidin-coated magnetically responsive microbeads are
complexed with biotinylated antibodies specific for the M allelic
GPA protein (FIG. 10). Magnetically non-responsive microbeads are
complexed with biotinylated antibodies specific for the N allelic
GPA protein. The two types of bead-antibody complexes are then
incubated with blood erythrocytes (the attachment of beads and
complexing agents can be done in any order as long as they are
unique). The resulting products are of three types: (1)
erythrocytes bearing the M protein complexed to beads coupled to
the anti-M antibody; (2) erythrocytes bearing the N protein
complexed to bead coupled to the anti-N antibody; and (3)
erythrocytes bearing both the M and N proteins complexed to both
the beads coupled to the anti-M antibody and the beads coupled to
the anti-N antibody.
[0154] Magnetic separation results in washing away all erythrocytes
without magnetic bead attachments. Next, the complexing agents are
simply digested with protease and the beads analyzed using the
particle size distribution using the Multisizer II (Coulter, Inc.).
Using different size particles for M and N results in a readily
quantifiable number of non-magnetic particles which would be
proportional to the number of MN erythrocytes. Information on GPA
mutations and the methods to complex antibodies with the M and N
allelic proteins on the surface of human red blood cells is
available in Langlois, et al., 236 Science 445-448 (1987), and
references therein, herein incorporated by reference. The methods
to biotinylate the antibodies and attach them to the avidin coated
beads are available to those skilled in the art.
[0155] The beads may be freed from the cells by digesting the
peptide complexing agents with proteinase to remove the beads from
the cell membrane. Then, one may obtain a bead size distribution
using the Multisizer II and determine the number of large
non-magnetic beads by peak integration. The number of N beads would
be proportional to the number of MN cells.
Example 5
[0156] Here we describe a new technology that provides a method for
efficiently evaluating for the presence, absence, or amplification
of nucleic acid sequences in a sample of nucleic acid. An example
of the method is illustrated in FIG. 11.
[0157] In this method, the target nucleic acid can be DNA or RNA,
single stranded or double stranded, fully purified, or as chromatin
or chromosomes. Preferably, the nucleic acid would be extracted as
purified nucleic acid (e.g., from cells) and evaluated as genomic
or fractionated to segments of sizes, suitable for the particular
evaluation being performed (e.g. by restriction enzyme digestion).
The minimum length of the target nucleic acid is on the order of 10
bp (it has to be sufficiently long for near unique hybridization).
There is no maximum limit for the length of the target nucleic
acid, i.e., it could be the whole genome.
[0158] Materials:
[0159] (A) Probes. Nucleic acid probes are made that are
complementary to specific regions of the target nucleic acid to be
evaluated. The individual probes can be of various lengths
(typically 50 bp to 1000 bp) and each probe with two complexing
agents, typically incorporated into the nucleic acid probe by
available nick translation methods.
[0160] (B) Antibodies. Antibodies are biotinylated and complexed
with streptavidin-coated microspheres. The biotinylation includes a
spacer between the antibody and the biotin molecule to limit
interference with antibody-antigen binding.
[0161] (C) Microspheres. Microspheres (typically, 1 to 20 .mu.m
diameter polystyrene beads) are coated with streptavidin. Each bead
size would be coated with only one kind of complexing agent.
[0162] Methods:
[0163] (A) Extract nucleic acids (e.g., from cells) and process as
desired using available methods.
[0164] (B) Insert-peptide-dUTP into probes by available nick
translation methods.
[0165] (C) Hybridize the nucleic acid probes to the target nucleic
acid in solution and purify to remove unhybridized probes.
[0166] (D) In separate solutions, attach the biotinylated
antibodies to their respective beads (one kind of antibody for each
bead size).
[0167] (E) Simultaneously, react the coated beads with the target
DNA-probe complexes to attach the beads to their respective
complexing agents on the probes.
[0168] (F) Place the solution into a vial coated with a unique
complexing agent that complexes with the probes hybridized to the
target DNA. After attachment to the vial surface, wash several
times to remove unattached beads.
[0169] (G) Separate the beads from the nucleic acid by protease
treatment and quantify size spectrum using a Multisizer II
(Coulter, Inc.)
[0170] (H) The number of beads of a particular size would be
proportional to the number of target sequences in the nucleic acid
sample. Hence, e.g., gene amplification would result in more beads
of a particular size than expected from a control sample.
Similarly, Downs Syndrome (an extra chromosome 21) would result in
50% more beads attaching to probes complementary to chromosome 21.
In contrast, a deletion of a particular target sequence would
result in fewer beads than expected. Using many different bead
sizes would permit the evaluation of many target nucleic acid
sequences simultaneously by using calibration curves, one for each
target sequence, to translate the number of beads in each peak into
the concentration of the corresponding target sequence in the
sample.
Example 6
[0171] In this example, we describe results using our particle
analysis assay to rapidly quantify bcr/abl chromosomal fusions in
genomic DNA extracted from a human chronic myelogenous leukemia
cell-line. It is demonstrated that our assay makes possible very
rapid and low cost quantification of rearrangements in genomic DNA
without the need for cell culturing, microscope scoring, or
sequence amplification. The assay requires only seconds to "score"
the number of chromosomal translocations typically taking weeks or
even months of very costly technician time by available cytogenetic
methods. The principle of the assay is to use the diameter of
microparticles as the detectable marker in a sandwich-type assay
and the number of such particles as the quantitative measurement.
The position of particle attachment to the target DNA can be
selected by the sequences of the hybridization probes and the
unique complexing agents on the particles. The "scoring" of these
detectable markers is accomplished by automated size-distribution
analysis that requires only 10 to 15 seconds per sample regardless
of how many cell-equivalents are being evaluated.
[0172] The particle analysis assay was used to detect Ph.sup.1
chromosome translocations in isolated genomic DNA from a human CML
cell line known to be Ph.sup.1 positive, K-5628, and from a control
cell line (H-1395) known to be Ph.sup.1 negative (American Type
Culture Collection, ATCC Cell-Line Number CRL-5868,
http://www.atcc.org; M. R. Speicher et al., 80 Laboratory
Investigation 1031-1041 (2000)). Commercially-available painting
probes for the bcr/abl fusion region were used to capture the
target sequences on the solid support surface (in this case,
magnetic beads) and to attach the 5.7 .mu.m diameter non-magnetic
particles used here for quantification. The probe hybridization
cocktail contained digoxigenin-labeled bcr probe and biotinylated
abl probe. Following simultaneous hybridization of the probes to
the genomic target DNA, the beads were added to the solution
resulting in the anti-digoxigenin-coated magnetic beads complexing
with the bcr probes and the streptavidin-coated non-magnetic beads
complexing with the abl probes. This was followed by magnetic
separation to remove unattached non-magnetic beads, DNase treatment
to cut the fusion DNA connecting the magnetic and non-magnetic
beads, another magnetic separation step to remove the magnetic
beads, and then the acquisition of a particle-size spectrum and
counting the number of 5.7 .mu.m particles. The magnetic beads were
removed, in this case, because their size distribution was very
broad and would have interfered with the size distribution of the
non-magnetic particles used here for detection. In practice,
removal of the magnetic beads would not be required because their
size distribution would be selected to prevent interference with
the size distribution of the non-magnetic particles. For the
present evaluation, these particular magnetic beads were used
simply because they were available commercially with
anti-digoxigenin coating.
[0173] Particle-size spectra obtained using this method are
illustrated in FIG. 12A-D. The number of particles counted is
presented as a function of particle diameter. The total number of
particles of a particular size (in this case, the observed peak) is
obtained automatically by integration using a computer interface
with the particle counter. It is observed in FIG. 12 that the level
of background noise outside the peak is relatively low resulting in
a high signal-to-noise ratio. It is also observed that the size of
the peak decreases with decreasing target DNA (i.e., decreasing
number of bcr/abl fusions) present in the sample solution. The
result in FIG. 12A was obtained using 165 ng genomic DNA from the
human CML cell line. A single large peak is observed with a mean
diameter of 5.7 .mu.m. This peak is composed of the non-magnetic
microparticles recovered after hybridization and magnetic
separation. In FIG. 12B, a smaller peak is observed at 5.7 .mu.m
particle diameter. This peak includes the non-magnetic
microparticles recovered using 16.5 ng target DNA. In FIG. 12C, the
hybridization solution contained only probes and beads, but no
genomic DNA from the CML cells. In this case, only a very small
peak is observed at 5.7 .mu.m. Although small, the fact that a
detectable peak exists at all in this size interval indicates that
some of the microparticles must have become attached to the
magnetic beads during the hybridization procedure even though
target DNA was not present. In order to determine how the particles
may have become attached, we performed an experiment where all
conditions were identical to those in FIG. 12B except that we did
not include probes. These results are seen in FIG. 12D and show
that the peak in FIG. 12B disappeared when the probes were not
included. Based on these results, it is clear that the
non-unique-sequence "painting" probes used here for abl and bcr
exhibit some non-specific inter-probe hybridization, which resulted
in some, albeit small, cross-attachment of magnetic and
non-magnetic beads. Importantly, it is expected that this can be
eliminated or at least substantially reduced by using
unique-sequence probes. The small "background" seen in FIG. 12D
from .about.0.2 .mu.m to about .about.8 .mu.m is the residual from
the broad size distribution of the magnetic beads used for these
experiments. As mentioned above, the magnetic beads would not be
present in the non-magnetic particle interval if they were selected
to have a non-overlapping size distribution.
[0174] The numerical results are presented in Table II. These
values are the integrals of the peaks. The genomic K-562 DNA per
500 .mu.l sample analyzed were 0, 0.165, 1.65, 16.5, and 165 ng.
Given that the K-562 cells are essentially triploid8, the number of
cell-equivalents per sample ranged from 0 to 18,000. Also, based on
the frequency of Ph.sup.1 chromosomes in this cell line8, we
estimate that the bcr/abl fusions would range from 0 to 2700 per
sample. It is observed that there is a clear relationship between
the number of beads counted per sample and the number of expected
bcr/abl fusions in the sample. It appears from these initial
results that on the order of 1 microparticle is counted per bcr/abl
fusion in the sample.
TABLE-US-00002 TABLE II Quantification of Ph.sup.1 fusions in human
genomic DNA. Genomic DNA bcr/abl probe Expected number Number of
beads Counting time per sample # cell per sample of Ph.sup.1 in
counted per per sample (ng) equivalent (.mu.l) sample.sup.a
sample.sup.b (sec) K-562 cells.sup.c 165 18,000 0.5 2700 1430 .+-.
38 12.8 16.5 1800 0.5 270 779 .+-. 28 12.7 1.65 180 0.5 27 151 .+-.
12 12.8 0.165 18 0.5 2.7 138 .+-. 12 12.7 0.sup.d 0 0.5 0 108 .+-.
10 19.1 16.5.sup.e 1800 0 270 49 .+-. 7 12.6 H-1395 cells.sup.c
16.5 2700 0.5 0 58 .+-. 8 12.8 0.sup.d 0 0.5 0 53 .+-. 7 12.9
Separated magnetic beads only.sup.f 17 .+-. 4 12.6 Saline
only.sup.g 0 12.8 .sup.aBased on 15% Ph.sup.1 chromosome frequency
in the K-562 cell culture used here.sup.8. .sup.bMean .+-. 1 s.d.
based on counting statistics only. .sup.cCells are described under
experimental protocol. .sup.dRepeated the procedure as above, but
without genomic DNA. .sup.eRepeated the procedure as above with
16.5 ng genomic DNA, but without probe. .sup.fMagnetic beads were
placed in a saline solution without DNA, probes, or non-magnetic
beads. The magnetic beads were then removed by magnetic separation
and the residual number of beads in the relevant interval counted.
The initial number of magnetic beads were the same as in all of the
above measurements. .sup.gPure saline solution was counted, without
any beads or other reagents present.
[0175] The results in Table II also include several controls. When
K-562 DNA was not present in the sample, then 108 particles were
counted in the 5.0-6.8 .mu.m diameter interval. As indicated above,
we believe that these beads resulted from non-specific
hybridization between the commercial painting probes used here as
well as from residual magnetic beads that were not fully removed by
the second magnetic separation step. This conclusion is supported
by the 49 beads observed when no probes were present in the K-562
experiment suggesting that the probe-probe non-specific
hybridization contributed about half of the 108 beads observed. We
have also observed that the number of residual magnetic beads
remaining after magnetic removal can vary somewhat between
experiments and can account for a substantial fraction of the 49
beads observed without probes. Seventeen beads were observed in the
5.0-6.8 .mu.m interval following the separation of magnetic beads
alone, i.e., without the presence of probes, DNA, or non-magnetic
beads. These were residual magnetic beads that remained after the
magnetic bead removal step. Taken together, it appears that the
background level for the PCA is on the order of 100 particles when
using commercially available painting probes and the magnetic beads
employed here. Given that the background of the counting instrument
itself (using pure saline) is essentially zero in the relevant size
interval, it is expected that substantial reductions in background
are possible by using unique-sequence probes instead of the
"painting" probes employed in the present experiments and by using
magnetic beads (or other solid support) that will not interfere
with the size distribution of the non-magnetic particles.
[0176] Also presented in Table II are results for a human cell line
(H-1395) which does not have Ph.sup.1 chromosomes. Genomic DNA from
these cells was used to measure the non-specific hybridization
between the bcr/abl probes used here and human genomic DNA. In this
case, we repeated the 16.5 ng DNA measurement by adding H-1395
genomic DNA instead of K-562 genomic DNA in the sample. We also
included a simultaneous control, i.e., no genomic DNA. The results
are clear. Only 58 beads were counted when we used H-1395 DNA
compared with 779 beads counted when we used K-562 DNA. This
demonstrates that non-specific hybridization is relatively low and
contributes less than 8% of the beads counted with 16.5 ng genomic
DNA. Given that 53 beads were counted with probe alone, most of
this 8% was actually from inter-probe hybridization. This is
consistent with the 3:1 probe-to-genomic DNA in the sample.
[0177] The counting times per sample are also listed in Table II.
These counting times should be compared with the efforts required
for microscopic analysis of cytogenetic preparations. The sample
measured here of 165 ng genomic DNA is equivalent to about 18,000
cells. Using FISH cytogenetics, a technician typically scoring
about 200 cells per day would require about 4 months to score
18,000 cells. In contrast, our bead counting method completed the
"scoring" in only 12.8 seconds. Clearly, this dramatic advance in
the quantification speed of chromosomal rearrangements opens new
possibilities in medicine, health, and research hitherto not
possible due to the inefficiencies and inadequacies of available
assays.
[0178] Finally, the PCA approach has many other potential
applications as well. For example, multiple nucleic acid
rearrangements could be quantified in the same sample
simultaneously by selecting beads of various diameters, each
diameter representing a particular type of rearrangement. This
would result in several peaks in FIG. 13A, each peak with a
different mean particle diameter. Also, random inter-chromosomal
rearrangements, such as may be caused by certain environmental
agents, could be rapidly quantified by using a cocktail of
whole-chromosome painting probes, each probe complementary to a
different chromosome. In this case, the largest chromosomes would
preferably be used to maximize the fraction of the genome
evaluated. Because of it's intrinsic quantitative nature, the PCA
method should also be a powerful tool for the detection of more
subtle alterations such as deletions, gene amplifications, and a
variety of other genetic abnormalities. We have also shown that the
PCA method can be used to quantify specific proteins. For protein
detection, the complexing agents are antibodies instead of nucleic
acid probes and the microparticles are used as the detectable
marker instead of, for example, a radioisotope in the case of
radio-immunoassays (RIAs). It is instructive to note that automated
particle counting detects 100% of the detectable marker in the
sample in about 13 seconds while scintillation counting of a
radiolabel such as .sup.125I would require 40 days (the
radiological half life of .sup.125I) to detect at most 50% of the
marker. Furthermore, as with DNA rearrangements, the PCA approach
can be used to quantify multiple proteins simultaneously in a
single sample by generating a spectrum of different size particles,
each size unique for a particular protein.
[0179] Experimental Protocol
[0180] Target DNA. Genomic DNA from two different human cell lines
were used in these experiments. Purified genomic DNA from the K-562
human chronic myelogenous leukemia cell-line was purchased from the
American Type Culture Collection (ATCC; Rockville, Md.). These
cells have been determined to contain an average of about 69
chromosomes per cell and have a Ph1 chromosome frequency of=15%
(Culture B in ref. 8). Purified genomic DNA from the H-1395 human
lung adenocarcinoma cell-line was also purchased from ATCC. This is
a near-normal cell-line without any apparent Ph1 chromosomes
(American Type Culture Collection, ATCC Cell-Line Number CRL-5868,
http://www.atcc.org; M. R. Speicher et al., 80 Laboratory
Investigation 1031-1041 (2000)).
[0181] Probes. The m-bcr/abl (minor breakpoint, Catalog Number
P5120) translocation DNA probe was obtained from Ventana Medical
Systems, Inc. (Tucson, Ariz.). The probe is a mixture of
digoxigenin-labeled DNA probes specific for the minor breakpoint
region of the bcr locus on chromosome 22, and biotin-labeled DNA
probes specific for the abl locus on chromosome 9. The probes
specific for the m-bcr gene are proximal to the translocation
breakpoint on chromosome 22 and the probes specific for the abl
gene are distal to the translocation breakpoint on chromosome 9.
The DNA probe solution is premixed with blocking DNA in 50%
formamide and 2.times.SSC.
[0182] Microparticles. The streptavidin-coated non-magnetic
particles (5.7 .mu.m diameter, s.d. .+-.0.3 .mu.m) were purchased
from Bangs Laboratories, Inc. (Fishers, Ind.). These were
monodisperse polystyrene microspheres suspended in a stock solution
of 9.4.times.107 beads per mL. The anti-digoxigenin-coated magnetic
particles (about 1 .mu.m mean diameter, but with a broad
distribution of sizes) were purchased from Roche Molecular
Biochemicals (Indianapolis, Ind.). These were superparamagnetic
polystyrene particles with no residual magnetism of the particles
after removal of the magnet. The magnetic particles were suspended
in a stock solution of 1.5.times.1010 particles per mL.
[0183] Hybridization. A 10 .mu.l solution consisting of 3.3-.mu.g
genomic DNA in Tris buffer was added to 40 .mu.l of denature
solution with final concentration of 70% (vol/vol) formamide and
2.times.SSC. This solution was heated to 70.degree. C. in a water
bath for 5 min and then transferred to a 30 .mu.l hybridization
solution containing 10 .mu.l probe. The final hybridization
solution contained 50% (vol/vol) formamide, 2.times.SSC, 1%
(vol/vol) salmon sperm DNA (Sigma, St. Louis, Mo.), and 1%
(vol/vol) SDS (10% Sodium dodecyl sulfate solution, Sigma, St.
Louis, Mo.). The mixture was then incubated with gentle agitation
overnight at 40.degree. C. in a Lab-Line Environ-Shaker.
[0184] Particle attachment to probes. After hybridization, 200
.mu.l TE buffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA) and 230 .mu.l
B&W buffer (2 M NaCl; 10 mM Tris-HCl, pH 7.5; 1 mM EDTA) were
added to the sample and then 25 .mu.l of magnetic particle solution
and 75 .mu.l non-magnetic particle solution were added. The
magnetic particles were pre-washed with B&W twice and the
non-magnetic particles with B&W/TE (1:1 vol/vol) twice. The
mixture was rotated gently at room temperature for 1 h.
[0185] Magnetic separation. The magnetic particles, and those
non-magnetic particles cross-linked with magnetic particles via
hybridization to the same contiguous target DNA sequence, were
collected with a magnetic particle concentrator (MPC, Dynal, Inc,
Lake Success, N.Y.) and the supernatant was removed. The particles
collected were washed once with 1.times.SSC, 0.2% SDS solution and
once with 0.1.times.SSC, 0.2% SDS solution (10 min each), followed
by washing with B&W three times and TE once, each with 200
.mu.l.
[0186] Particle counting. The magnetically separated particles were
digested using DNase I (Gibco BRL, Grand Island, N.Y.) according to
the manufacturer's protocol to detach all particles that may be
cross-linked by hybridization. The magnetic beads were collected by
MPC and the supernatant transferred to a counting cuvette (Beckman
Coulter, Inc, Miami, Fla.) containing Isoton II diluent (Beckman
Coulter, Inc, Miami, Fla.). Final volume was 10 mL. The particles
remaining in the solution were counted using a Coulter Multisizer
II (Beckman Coulter, Inc, Miami, Fla.). The measurement volume was
500 .mu.l.
Example 7
[0187] This new technology can be used to quantify many different
kinds of target molecules simultaneously and can be used to detect
any molecule for which there exists two non-cross reacting
complexing agents. For example, proteins of importance in health
and nutrition such as ferritin, transferrin, transferrin receptor,
folic acid, vitamin-B12, vitamin-A retinol binding protein,
insulin, cortisol, estradiol, FSH, LH, progesterone, T3, T4, and
TSH have two commercially available antibodies that bind to
different sites on the same molecule and would therefore be
detectable individually or simultaneously using the present method.
Antibodies have been developed for a large number of other
molecules as well which can also be quantified using the present
method. In addition, modern antibody production can now be used to
develop low-cost antibodies against almost any desired target
molecule.
[0188] The protein separation and quantification technology is
illustrated in FIG. 13. For the detection of proteins, one antibody
type (preferably a monoclonal antibody) would be attached to a
solid support, e.g., to the surface of a super-paramagnetic
microbead (M), and another antibody type (usually a polyclonal
antibody attaching to several sites on the same protein) attached
to the magnetically non-responsive microbeads (N). In this example,
the antibodies are coated directly onto the surface of carboxylic
acid coated beads. Other attachment methods are also possible such
as biotin/avidin, digoxygenin/anti-digoxygenin, and other
antibody/antigen complexing reactions.
[0189] We have successfully performed experiments using our
Particle Analysis Assay to detect protein ferritin. We performed
our initial tests using this protein because it is very important
in the evaluation of iron status in humans. Our results of
separation and particle size distribution analysis are illustrated
in FIG. 14. Two peaks are seen, one with a mean particle diameter
of 2.8 .mu.m (the super-paramagnetic beads) and another with a mean
particle diameter of 4.45 .mu.m (the magnetically non-responsive
beads). It is clear that these two peaks are easily resolved and
that a much larger number of peaks could also be resolved. The
total number of beads of each kind can be obtained from the
integral of each peak. In this case, there were a total of 808
non-magnetic beads observed in a 0.5 mL aliquot of the 10 mL final
sample solution. This translates into a total of
808.times.20=16,160 non-magnetic beads recovered following the
magnetic separation step. Based on a total of 0.27 .mu.g ferritin
in the initial test solution, we calculate a proportionality
constant of 3.75.times.10.sup.17 mole of ferritin per non-magnetic
bead recovered. The minimum detection limit of this method is only
a few beads. For identical protocol conditions, this calibration
constant could be multiplied by the number of non-magnetic beads
recovered from an unknown sample of ferritin to obtain the ferritin
concentration in the unknown sample from the number of non-magnetic
beads counted in the unknown sample.
[0190] Experiments were also performed to measure the number of
counts in the relevant size windows when ferritin was not present
in the solution. FIG. 15 shows that when ferritin is not included
the peak for non-magnetic beads is not present, demonstrating that
there is no detectable non-specific cross-reactions between
beads.
[0191] Based on these results, and the fact that we have not yet
optimize and fine tuned the method, we estimate the detection limit
for this method to be in the amole range, considerably lower than
available radio-immunoassays and ELISAs, which are typically in the
pmole to fmole range.
[0192] Experimental Protocol
[0193] Coat beads with ferritin-specific antibodies.
Magnetically-responsive beads (Dynabeads M-270) with carboxylic
acid surface coating (Catalog #A143.05) were purchased from Dynal
A/S, Oslo, Norway. These were 2.8 .mu.m mean diameter microspheres
in 2.times.10.sup.9 beads per mL stock solution. Suspend First, the
Dynabeads M-270 were fully suspended in the stock solution by
pipetting and vortexing for 1 min. Immediately pipette 100 .mu.l
into a 1.5 mL Eppendorf tube. Place the tube in a Dynal magnet
stand (MPC) for 4 min and remove the supernatant. Resuspend beads
in 100 .mu.l of 0.01 M NaOH. Mix well for 5 min and repeat once.
Wash beads twice with 100 .mu.l of 0.1 M MES
(2-[N-morpholino]ethane sulfonic acid) buffer pH 5.0 and once 100
.mu.l of cold Milli-Q water. Add 200 .mu.l of 0.005 M CMC
(N-cyclohexyl-N-(2-morpholinoethyl)carbodimde
methyl-p-toluensulfonate) in cool Mili-Q water to beads and vortex
to mix properly. Incubate for 10 min at 4.degree. C. with slow tilt
rotation. Remove supernatant using magnet stand. Add 120 .mu.l of
0.005 M CMC (Catalog #C1011, Sigma) and 80 .mu.l of 0.3 M MES
(Catalog #M2933, Sigma). Vortex and incubate as above for 30 min.
Wash beads twice with cold 200 .mu.l of 0.1 M MES as quickly as
possible. Resuspend Dynabeads M-270 in 150 .mu.l of 10 mM MES
containing 60 .mu.g of monoclonal anti-ferritin (Catalog #M94157,
Fitzgerald Industries International, Inc, Concord, Mass.). Vortex
to ensure good mixing of protein and beads. Incubate for 20 min at
4.degree. C. with slow tilt rotation. Add BSA (Sigma) to final
concentration 0.1% and incubate as above for 4 h. Wash with 120
.mu.l of PBS containing 0.1% BSA and 0.1% Tween 20 (Sigma) four
times. Resuspend beads in 200 .mu.l of PBS with 0.1% BSA, 0.1%
Tween 20, and 0.02% NaN.sub.3 and store at 4.degree. C.
[0194] Non-magnetically-responsive beads (carboxylic acid coated
polystyrene beads, Catalog #PC05N, Lot #1193) were purchased from
Bangs Labs, Fishers, Ind. These were 4.45 .mu.m mean diameter
microspheres in 1.988.times.10.sup.9 beads per mL stock solution.
The protocol for coating the Bangs beads with antibody was the same
as described above for the Dynabeads M-270 with the exceptions that
the Bangs beads were coated with polyclonal anti-ferritin (Catalog
#70-XG50, Fitzgerald Industries International, Inc, Concord, Mass.)
and employed centrifugation for the washing steps instead of the
Dynal magnet stand.
[0195] Magnetic separation. Mix well 2 million (2 .mu.l) monoclonal
antibody coated Dynal beads with 0.27 .mu.g ferritin (Catalog
#30-AF10, Fitzgerald Industries International, Inc, Concord, Mass.)
in TBST (10 mM Tris, 50 mM NaCl, and 0.1% Tween 20, pH 7.5) buffer
(total 150 .mu.l). Rotate gently at room temperature (r. t.) for 1
h. Wash with 150 .mu.l of TBST three times using Dynal magnet stand
(4 min each). Mix with 2 million (2 .mu.l) polyclonal coated Bangs
beads in 150 .mu.l of TBST. Again, rotate gently at r. t. for 1 h.
Wash with B&W 4 times and TE once using Dynal magnet stand (4
min each). Digest with 1 .mu.l Proteinase K (from 23 mg/mL stock
solution, Catalog #P2308, Sigma) in 150 .mu.l of 0.01 M Tris and
0.005 M EDTA at 40.degree. C. for 4 hours. Place tube into Dynal
magnet stand (2 min) and transfer supernatant into Beckman-Coulter
counting cuvette (Catalog #8320592). Wash the tube 3 times with
saline (Isoton II, Beckman-Coulter, Inc.) and then dilute with
Isoton II diluent (Beckman Coulter, Inc.) to 10 mL. The same
procedure was followed for the control experiment except that
ferritin was not added to the solution.
[0196] Particle size analysis and bead counting. Obtain particle
size distribution for the beads that remain in the solution using
the Coulter Multisizer II (Beckman-Coulter, Inc) It is seen in FIG.
14 that the number of non-magnetic particles is readily obtained
and the concentration of the target protein (ferritin in this
example) can be quickly determined from the number of non-magnetic
beads using standard calibration data. For example, the
non-magnetic beads counted in FIG. 14 result in
3.75.times.10.sup.-17 mole of ferritin per non-magnetic bead
recovered. For identical protocol conditions, this calibration
constant could be multiplied by the number of non-magnetic beads
recovered from an unknown sample of ferritin to obtain the ferritin
concentration in the unknown sample from the number of non-magnetic
beads counted in the unknown sample.
[0197] Multiple Molecular Detection and Quantification Using the
Method of the Present Invention
[0198] Target Molecules (antigens): Ferritin, transferrin receptor,
TSH, retinol binding protein, and folic acid. In human bood
serum.
[0199] Microbeads: Beads of different sizes and with various
chemical and physical functional surfaces are available
commercially (e.g. Dynal AS, and Bangs Labs, Inc.). Beads of
different diameters can be selected to serve as unique identifiers
for each of the five target molecules. The various chemical and
physical functional surfaces provide the opportunity for selecting
and optimizing bead-antibody immobilization strategies. A preferred
approach is to use streptavidin-coated beads as the basic substrate
upon which the selected biotinylated antibodies can be
attached.
[0200] The magnetically-responsive (M) beads can be Dynabeads
M-280, 2.8 .mu.m mean diameter. These beads are commercially
available with streptavidin surface coating from Dynal AS, Oslo,
Norway.
[0201] The non-magnetic (N) beads can be selected to be five
different diameters, one diameter for each of the five target
molecules. For this example, bead diameters in the 4 to 20 .mu.m
range, (a much broader range of sizes can be used) would be
selected to eliminate significant overlap of the peaks.
Streptavidin coated beads of these sizes (and almost any other
diameter if special order) are available commercially from Bangs
Labs, Fishers, Ind. The main selection criterion for bead size is
to make sure that we can adequately resolve the peaks in the
measured bead size distribution. We have performed several
experiments with various size beads from Bangs Labs that provide
assurance that the bead sizes selected here should be
resolvable.
[0202] Antibodies: The antibodies used in this example would be
obtained from commercial vendors. Monoclonal and polyclonal
antibodies for ferritin, transferrin receptor are available from
Fitzgerald Industries International, Inc, Concord, Mass. A
monoclonal antibody for retinol binding protein is available from
Fitzgerald. A polyclonal antibody for retinol binding protein is
available from US Biological, Swampscott, Mass. Both monoclonal and
polyclonal antibodies for TSH are available from Fitzgerald.
Monoclonal and polyclonal antibodies for folic acid are also
available from Fitzgerald.
[0203] Biotinylation of Antibodies: A biotin molecule would be
conjugated with each antibody using a "BiotinTag Micro
Biotinylation Kit" (B-TAG) from Sigma chemical company. It should
be noted that this procedure also adds a 12 atom spacer between the
biotin molecule and the antibody to facilitate protein binding.
Briefly, the biotinylated antibodies would be synthesized by adding
10 .mu.L (5 .mu.g/.mu.L) of
Biotinamidocaproate-N-hydroxysulfosuccinimide ester (BAC-SulfoNHS)
in 0.1 M sodium phosphate buffer, pH 7.2 to 0.1 mL of antibody (10
mg/mL) in same sodium phosphate buffer. Then, reacted for 2 hours
with gentle shake at 4.degree. C. The biotinylated antibody will
then be purified by applying the biotinylation reaction mixture to
a pretreated Micro-spin column G-50 packaged with Sephadex G-50.
Spin the column for 2 min at 700.times.g. The purified sample is
collected at the bottom of the support tube. Place the column into
another tube and add 0.2 mL PBS (0.01 M, pH 7.4). Spin the column
for 1 min at 700.times.g and repeat. Pool the fractions containing
the purified antibody which is now ready to use.
[0204] Immobilizing Antibodies on Beads: To immobilize the
biotin-labeled antibodies on the streptavidin coated beads, the
beads are first washed with PBS twice and suspended in the same
buffer (0.5 mg beads per mL PBS buffer). To this solution, we will
add 5 .mu.g biotinylated antibody and then react at room
temperature (.about.22.degree. C.) for 30 min with gentle mixing.
The beads will then be washed three times with PBS and resuspended
in the same buffer for use.
[0205] NOTE: The preceding bead and antibody preparations can all
be done in advance, and for commercial applications, would be part
of a kit. The separation and detection steps listed below can be
simplified and some steps (e.g., incubation) shortened
substantially.
[0206] Magnetic Separation: React the antibody-coated magnetic
beads with the target antigen (for this example, with human serum).
This is accomplished as follows: To antigen-containing matrix (0.5
mL), add 0.25 mg/0.5 mL (PBS buffer supplemented with 0.2% BSA)
magnetic beads, and incubate for 1 hour at room temperature.
Collect the beads with a magnetic particle concentrator (MPC) and
wash three times with PBS. Resuspend the beads with 0.5 mL of the
same buffer. To this solution, add non-magnetic beads coated with
the second antibody (0.25 mg/0.5 mL, PBS buffer supplemented with
0.2% BSA) and incubate for 1 hour at room temperature. Collect the
magnetic beads with MPC and wash 5 times with buffer. In this
procedure, non-magnetic beads are removed by the magnetic washes
except those that crosslink with the magnetic beads through
antibody-antigen-antibody coupling. The beads are then suspended in
0.5 mL PBS. Note that separation could also be done using a solid
support surface other than magnetic beads, e.g., the inside of a
microtiter well, as taught in the body of the patent.
[0207] As an example, the separation from human serum of ferritin,
transferrin receptor, TSH, retinol binding protein, and folic acid,
is accomplished using the magnetic separation procedure described
above. In this case, 100 .mu.L serum is diluted (1/10) with
phosphate buffer saline total 1 mL (pH 7.2 containing 0.5 mL/L
Tween 20). To this solution are added Dynal beads, each with only
one of the five kinds of monoclonal antibodies used in this project
(0.25 mg beads/25 .mu.l PBS). The mixture is incubated for 1 hour
at room temperature with gentle rotation. Collect the beads with a
magnetic particle concentrator (MPC) and wash three times with PBS.
The beads are resuspended with 1 mL of the same buffer supplemented
with 0.1% BSA. To this solution are added non-magnetic beads coated
with a polyclonal antibody, in this case a unique bead size for
each kind of antibody (0.3 mg beads/0.3 mL), and incubated for 1
hour at room temperature. Collect the complexes with MPC and wash
with buffer five times. In this procedure, non-magnetic beads are
removed by multi-wash except those that crosslink with the magnetic
beads through antibody-antigen-antibody coupling. The beads are
then suspended in 0.5 mL PBS. The proteins on beads are then cut by
use of proteinase K (15 .mu.L from 23 mg/mL stock solution), then
diluted with Isoton II diluent (Beckman Coulter Counter) to 10 mL
and analyzed using the Coulter Multisizer II. Initially, we will
perform the magnetic separation and non-magnetic bead attachment in
two separate steps. This approach will remove essentially all of
the non-target proteins from the solution and hence reduce any
possible non-specific reactions with the non-magnetic beads used
here as our detectable marker.
[0208] Simultaneous Quantification of Target Molecules: Determine
the target protein concentration by counting the number of
non-magnetic beads after magnetic separation. The quantification of
target protein concentration will be accomplished by digesting the
proteins on the beads using proteinase K (2.5 .mu.L from 23 mg/mL
stock solution), allow to react for 30 min, and then dilute with
Isoton II diluent (Beckman Coulter Counter) to 10 mL and size/count
using the Coulter Multisizer II. Generation of a bead size
distribution requires only 10 to 15 seconds.
[0209] The invention may be embodied in other specific forms
without departing from its essential characteristics. The described
embodiments are to be considered in all respects only as
illustrative and not restrictive. The scope of the invention is,
therefore, indicated by the appended claims rather than by the
foregoing description. All changes that come within the meaning and
range of equivalency of the claims are to be embraced within their
scope.
Sequence CWU 1
1
101880DNAArtificial SequenceSynthetic Oligonucleotide 1tngttctcca
gcttgcatgc ctgcaggtcg acgcccctag atctgtctcc taaaatggct 60ccccagacac
agcacagtgt tcctggggtc gttcaggacg gaaggcagcg gcgccccccc
120ccaatctttg catgtcttgg gatgcaaaac aatttcccca ccttctctct
gctcacccca 180ccgaccgtcg cccctaaagt gaagtctgct ggctgccgaa
aagggaaatg gaaaggagga 240accattcaag ttcaacgaca tggcgacggc
agctccggcg ggagccgcgc tttggcaggg 300gagggtgcgc catctgcagc
agcgcgctag cacatagggg aaggggcgat gggcccccct 360ccacgcctta
gcgtgcaact cgcccccata ttctccccac agcattcatc cttgacccaa
420cccgctttgc tctttagccc cagctctctg ctttggtcat caccccgaaa
acctatgaaa 480atccagagcc cctgcacccg cgcgttccgc tagagaacct
accgtgaaga cccgagcgtt 540gtgtccttgt ccttgcttat tcgatcctac
ttgaaacact ggcagcactc acggccttcg 600gggctcggcc agcagcttcc
gagaacgata gctttcttgc gcagcgcgta gacgcgatgc 660ggtaattttg
agccacccaa gataagacac taacttgacc ttaactttgt cagggcgccc
720ctggtatctg gagaacgtga acagacactt gtctggcagc ttctcgtaaa
aactgactgg 780ggaagggatt ctgagtcatt tcatttatta ccccttacaa
gttttgcaag aaaagcnttt 840tcttccttgn ccaaacttta attattttat
tgctcntttt 8802795DNAArtificial SequenceSynthetic oligonucleotide
2ttnnnttntt cgnctcggta cccggggatc ctctagagtc gacgcggccg cggaattaac
60cctcactaaa gggaacgaat tcggatctac cttctgaaga ccagagaacc cctggggaat
120tgccccgccc ctttaaggaa acctcctaca cagagagctt tggtaattgt
tcatggttta 180tacttatctc caataatgga tgtcatgggg ggttgaaagt
tttgcataac cggttttttt 240tttcttcatg ttacctgtct tatttaaagg
caggcctacc tcaaaaacat tacaccagtg 300gaggagagag agagagagag
agagagagag agagagagag agagttacat ttgttgaaaa 360aatagtcatt
tcatatcctt tccagaaagg agaggatgaa attagaaatg gacccagttt
420tcagtttctg atatcttcaa agtaccatca ccaagaacaa gaacactcag
acaaaaatct 480aacccaaacc ccatgccttc aaagggcatc ttccacctat
gcgaagggca tgccaaattt 540ttaagattgg gagtgaggtg acatacagga
aaaaatttct ctgtattacc caaaaagaaa 600gttttgctgg caagaatgat
gtaaacaaag caagggcatt ttcttttcct ccttttcttt 660ttctccttcc
ttcctttctt ccttccttcc ttccttcctt ccttcctttc ttacttcttt
720ctttctttct ttcttctttc tttctttctc ctggggnggg ggtagactgc
caaactaagt 780atttgtttct tgtaa 795350DNAArtificial
SequenceSynthetic oligonucleotide 3tgccttccgt cctgaacgac cccaggaaca
ctgtgctgtg tctggggagc 50450DNAArtificial SequenceSynthetic
oligonucleotide 4cggcagccag cagacttcac tttaggcgcg acggtcggtg
gggtgagcag 50550DNAArtificial SequenceSynthetic oligonucleotide
5gctgcagatg gcgcaccctc ccctgccaaa gcgcggctcc cgccggagct
50650DNAArtificial SequenceSynthetic oligonucleotide 6gggttgggtc
aaggatgaat gctgtggcga gaatatgggg gcgagttgca 50750DNAArtificial
SequenceSynthetic oligonucleotide 7cgaattcgga tctaccttct gaagaccaga
gaacccctgg ggaattgccc 50850DNAArtificial SequenceSynthetic
oligonucleotide 8catggtttat acttatctcc aataatggat gtcatggggg
gttgaaagtt 50950DNAArtificial SequenceSynthetic oligonucleotide
9agtcatttca tatcctttcc agaaaggaga ggatgaaatt agaaatggac
501050DNAArtificial SequenceSynthetic oligonucleotide 10tcagacaaaa
atctaaccca aaccccatgc cttcaaaggg catcttccac 50
* * * * *
References