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BACKGROUND OF THE INVENTION
1. Field of the Invention
The ability to synthesize oligonucleotide sequences at will and to clone
polynucleotide sequences prepared by synthetic procedures or obtained from
naturally occurring sources has greatly expanded the opportunities for
detecting the presence of specific sequences in an extended
oligonucleotide sequence, e.g., chromosome(s), mixture of sequences,
mRNAs, or the like. Interest in specific sequences may involve the
diagnosis of the presence of pathogens, the determination of the presence
of alleles, the presence of lesions in a host genome, the detection of a
particular mRNA or the monitoring of a modification of a cellular host, to
mention only a few illustrative opportunities. While the use of antibodies
to perform assays diagnostic of the presence of various antigens in
samples has seen an explosive expansion in techniques and protocols since
the advent of radioimmunoassay, there has been until recently no parallel
activity in the area of the DNA probes. Therefore, for the most part,
detection of sequences has involved various hybridization techniques
requiring the binding of a polynucleotide sequence to a support and
employing a radiolabeled probe.
In view of the increasing capability to produce oligonucleotide sequences
in large amounts in an economical way, the attention of investigators will
be directed to providing for simple, accurate and efficient techniques for
detecting specific oligonucleotides sequences. Desirably, these techniques
will be rapid, minimize the opportunity for technician error, be capable
of automation, and allow for simple and accurate methods of detection.
Toward this end, there have already been efforts to provide for means to
label oligonucleotide probes with labels other than radioisotopes and for
improving the accuracy of transfer of DNA sequences to a support from a
gel, as well as improved methods for derivatizing oligonucleotides to
allow for binding to a label. There continues to be a need for providing
new protocols which allow for flexibility in detecting DNA sequences of
interest in a variety of situations where the DNA may come from diverse
sources.
2. Description of the Prior Art
Meinkoth and Wahl, Anal. Biochemistry (1984) 138:267-284, provide an
excellent review of hybridization techniques. Leary, et al., Proc. Natl.
Acad. Sci. USA (1983) 80:4045-4049, describe the use of biotinylated DNA
in conjunction with an avidin-enzyme conjugate for detection of specific
oligonucleotide sequences. Ranki et al., Gene (1983) 21:77-85 describe
what they refer to as a "sandwich" hybridization for detection of
oligonucleotide sequences. Pfeuffer and Helmrich, J. of Biol. Chem. (1975)
250:867-876 describe the coupling of guanosine-5'-0-(3-thiotriphosphate)
to Sepharose 4B. Bauman, et al., J. of Histochem. and Cytochem. (1981)
29:227-237, describe the 3'-labeling of RNA with fluorescers. PCT
Application WO83/02277 describes the addition to DNA fragments of modified
ribonucleotides for labeling and methods for analyzing such DNA fragments.
Renz and Kurz, Nucl. Acids Res. (1984) 12:3435-3444, describe the covalent
linking of enzymes to oligonucleotides. Wallace, DNA Recombinant
Technology (Woo, S., Ed.) CRC Press, Boca Raton, Florida, provides a
general background of the use of probes in diagnosis. Chou and Merigan, N.
Eng. J. of Med. (1983) 308:921-925, describe the use of a radioisotope
labeled probe for the detection of CMV. Inman, Methods in Enzymol. 34B, 24
(1974) 30-59, describes procedures for linking to polyacrylamides, while
Parikh, et al., Methods in Enzymol. 34B, 24 (1974) 77-102, describe
coupling reactions with agarose. Alwine, et al., Proc. Natl. Acad. Sci.
USA (1977 ) 74:5350-5354, describe a method of transferring
oligonucleotides from gels to a solid support for hybridization. Chu, et
al., Nucl. Acids Res. (1983) 11:6513-6529, describe a technique for
derivatizing terminal nucleotides. Ho, et al., Biochemistry (1981)
20:64-67, describe derivatizing terminal nucleotides through phosphate to
form esters. Ashley and MacDonald, Anal. Biochem. (1984) 140:95-103,
report a method for preparing probes from a surface bound template. These
references which describe techniques are incorporated herein by reference
in support of the preparation of labeled oligonucleotides.
SUMMARY OF THE INVENTION
Methods are provided for the detection of specific nucleotide sequences
employing a solid support, at least one label, and hybridization involving
a sample and a labeled probe, where the presence or absence of duplex
formation results in the ability to modify the spatial relationship
between the support and label(s). Exemplary of the technique is to provide
a cleavage site between the label and support through duplexing of a
labeled probe and sample DNA, where the duplex is bound to a support. The
cleavage site may then be cleaved resulting in separation of the support
and the label(s). Detection of the presence or absence of the label may
then proceed in accordance with conventional techniques.
A primary advantage of the invention over the art is that the present
method enables one to distinguish between specific and nonspecific
binding, of the label. That is, in the prior art, label is typically
detected on a solid support, i.e., the sample is affixed to the support
and contacted with a complementary, labeled probe; duplex formation is
then assayed on the support. The problem with this method is that label
can and does bind to the support in the absence of analyte. This direct
binding of the label to the support is referred to herein as "nonspecific"
binding. If any significant amount of nonspecific binding occurs, label
will be detected on the support regardless of the presence of analyte,
giving false positive results.
By contrast, in the present method, label is detected only when the analyte
of interest is present, i.e., only "specific" binding is detected. In a
preferred embodiment, this is done by introducing a cleavage site between
a support and the selected label, through a duplex between the sample and
one or more probes. The cleavage site may be a restriction endonuclease
cleavable site, as described in the parent case hereto, U.S. Application
Ser. No. 06/661,508, or it may be one of a number of types of chemically
cleavable sites, e.g., a disulfide linkage, periodate-cleavable 1,2-diols,
or the like. In an alternative embodiment, specifically bound label is
released by a strand replacement procedure, wherein after binding of the
label to the support through an analyte/ probe complex, a DNA strand is
introduced that is complementary to a segment of the analyte/probe complex
and is selected so as to replace and release the labeled portion thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the difference between specific and nonspecific binding
of a label to a solid support.
FIG. 1a illustrates nonspecific binding in which label is bound directly to
a support, while FIG. 1b illustrates specific binding, i.e., wherein label
is detected only when the analyte of interest is present.
FIGS. 2A through 2D schematically illustrate the preferred method of the
invention, wherein a selectively cleavable site is introduced between a
support and a label through an analyte/probe complex.
More specifically, FIG 2A illustrates the preferred embodiment of the
invention wherein a polynucleotide bound to a support at a first end and
having a detectable label at the opposite end contains a region of at
least four successive nucleotides which homoduplexes with a sequence of
interest, and wherein the sequence of interest includes a restriction
enzyme-cleavable site. FIG. 2B illustrates an alternative embodiment of
the invention in which a two-component reagent is used for the detection
of an oligonucleotide sequence of interest; as illustrated in the figure,
cleavage may take place adjacent the label, approximately midway between
the label and the support, or adjacent the support. FIG. 2C also
illustrates an alternative embodiment of the invention, in which the
analyte is bound to a support and a single-component reagent is used for
detecting an oligonucleotide sequence of interest. FIG. 2D represents
still another embodiment in which, following hybridization, a
support-bound capture probe/label probe/analyte complex is cleaved in its
entirety at a site adjacent the support.
FIG. 3 schematically illustrates an alternative method of the invention,
wherein specifically bound label is released through a strand replacement
technique.
More specifically, FIG. 3A illustrates the complex formed between a capture
probe, a nucleic acid analyte, and a labeling probe. FIG. 3B illustrates a
strand displacement method for releasing label from the complex into
solution.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Detection of specific sequences is achieved using hybridization, whereby
duplexing of the sample DNA and a probe affects the ability to modify the
spatial relationship between a label and a support. In this manner, the
presence or absence of a particular sequence in a sample can be readily
determined by the amount of label which is freed into the medium.
The subject method allows for varying protocols and reagents where the
sample nucleic acid may be bound to a support or free in solution. In a
preferred embodiment, the method involves forming a nucleic acid duplex
where a label is separated from a support by a selectively cleavable bond,
so that the amount of label released under conditions providing selective
cleavage is a measure of the presence and amount of a sequence of interest
in a nucleic acid sample. The selectable cleavage site may be as a result
of formation of a restriction enzyme recognition site through
homoduplexing, or the presence of such selectable cleavage site in the
single-stranded polynucleotide chain may be a result of the prior
introduction of such site into the single-stranded chain.
A reagent will be employed which will include a polynucleotide sequence
having an oligonucleotide sequence of interest that hybridizes to the
nucleic acid analyte. This reagent will sometimes be referred to herein as
a "capture probe", which in the present method, is bound to the selected
solid support. A labeling probe will also be employed, which may or may
not include the sequence of interest.
In the first, preferred embodiment, the subject method involves the forming
of a polynucleotide duplex in a hybridization medium resulting in a label
bound to a support through a selectable cleavage site. Various protocols
may be employed where the sample DNA is bound to a support or dispersed in
a solution.
In order to distinguish the various nucleotide sequences involved, the
following terms will be used:
nucleic acid sample - sample suspected of containing a nucleic acid
sequence having an oligonucleotide sequence of interest;
nucleic acid analyte - DNA or RNA in said nucleic said sample having an
oligonucleotide sequence of interest;
oligonucleotide sequence of interest - a DNA or RNA sequence which may be
all or part of a nucleotide chain, usually at least six bases, more
usually at least about 10 bases, preferably at least about 16 bases, which
may be 5kb or more, usually not more than 0.2kb, which is diagnostic of a
property to be detected, where the property may be a gene or sequence
diagnostic of a hereditary trait, pathogen, etc.;
polynucleotide sequence - DNA or RNA sequences employed as reagents for
detection of the oligonucleotide sequence of interest, which
polynucleotide sequence may be labeled or unlabeled, bound or unbound to a
support, and may or may not include a sequence complementary to the
oligonucleotide sequence of interest. There will be one to two
polynucleotide sequences, which individually or in conjunction with the
nucleic acid analyte will act as a bridge between a label and a support,
with a selectably cleavable site intermediate the label and support; and
selectably cleavable site - a functionality or plurality of functionalities
which can be selectively cleaved and may include restriction sites,
phosphate esters, purines, peptide bonds, etc.
For convenience of description, the preferred embodiment of the subject
invention wherein a selectable cleavage site is created will be divided
into four primary sub-embodiments. In the first of these (see FIG. 2A) the
reagent employed is a single component, which is a polynucleotide joined
proximal to one end to a support and joined proximal to the opposite end
to one or more detectable labels. The polynucleotide will include a region
of at least four successive nucleotides homoduplexing with a sequence of
interest, where such sequence includes a restriction site, which is
intermediate the support and label.
In the second case (See FIG. 2B), the reagent employed will have two
components which will vary with whether the nucleic acid sample is bound
or unbound to a support and the nature of the selectable cleavage site.
Where the nucleic acid sample is bound to the support, the two components
will be (1) a bridging polynucleotide sequence and (2) a polynucleotide
sequence complementary and hybridizing to a portion of the bridging
polynucleotide sequence. Either the bridging or complementary
polynucleotide sequence may be labeled. The presence of the label bound to
the bridging sequence will be limited to when the duplex of the bridging
and analyte polynucleotide sequences define a restriction site as the
selectable cleavage site. Otherwise, only the complementary sequence will
be labeled. Besides having a sequence duplexing with the complementary
sequence, the bridging polynucleotide sequence will have a region
duplexing with the oligonucleotide sequence of interest.
Where the sample nucleic acid is in solution, the two components will be
(1) a first polynucleotide sequence bound to a support, which has a region
complementary to a sequence present in the nucleic acid analyte, which
sequence may or may not define a restriction site and may or may not
define the oligonucleotide sequence of interest; and (2) a labeled second
polynucleotide sequence which as a region complementary to a sequence
present in the nucleic acid analyte, which region is subject to the same
limitations as the region of the first polynucleotide sequence. At least
one of the duplexed regions will define a sequence of interest. In the
absence of one of the regions defining a restriction site or in addition
to the presence of a restriction site, there will be a selectable cleavage
site present with the first or second polynucleotide sequence.
In a third case (see FIG. 2C), the analyte is bound to a support and the
reagent employed is a single component which is a labeled polynucleotide
sequence having a region complementary to the oligonucleotide sequence of
interest which may define a restriction site. The restriction site and/or
a functionality present on the labeled polynucleotide sequence may serve
as a selectable cleavage site.
In a fourth case (see FIG. 2D), a capture probe is provided which is a
polynucleotide chain bound to a solid support via a linkage "Y", and at
its opposing end is complementary to a first sequence present in the
nucleic acid analyte. A labeling probe comprising a labeled second
polynucleotide chain has a region complementary to a second sequence in
the analyte that is distinct from and does not overlap with the first
sequence. The linkage designated "Y" in FIG. 2D represents any
conventional means of binding a probe to a support. The linkage "X" is a
selectable cleavage site, i.e., a chemically cleavable linkage such as a
disulfide bond, periodate-cleavable 1,2-diols, or the like.
The nucleic acid containing sample will be combined with the appropriate
reagent under conditions where duplex formation occurs between
complementary sequences. The mixture is allowed to hybridize under
conditions of predetermined stringency to allow for at least heteroduplex
formation or homoduplex formation over an oligonucleotide sequence of
interest. After a sufficient time for hybridization to occur, the support
may be separate from the supernatant and washed free of at least
substantially all of the non-specifically bound label. The
oligonucleotides bound to the support are then treated with one or more
reagents, which results in cleavage of at least one strand and release of
label bound to support.
Depending upon the presence of a particular sequence in the sample
resulting in duplex formation, release of the label(s) bound to the
support will be observed. Various protocols may be employed, where
normally the supernatant medium may be assayed for the presence of the
label, although in some instances the support may be measured. Protocols
and reagents may be employed, where a physical separation of the support
from the supernatant may or may not be required.
The subject method can be used for the detection of oligonucleotide
sequences, either DNA or RNA, in a wide variety of situations. One
important area of interest is the detection of pathogens, viruses,
bacteria, fungi, protozoa, or the like, which can infect a particular
host. See for example, U.S. Pat. No. 4,358,535. Another area of interest
is the detection of alleles, mutations or lesions present in the genome of
a host, such as involved in amniocentesis, genetic counseling, host
sensitivity or susceptibility determinations, and monitoring of cell
populations. A third area of interest is the determination of the presence
of RNA for such diverse reasons as monitoring transcription, detecting RNA
viruses, differentiating organisms through unexpressed RNA, and the like.
Other areas of interest, which are intended to be illustrative, but not
totally inclusive, include monitoring modified organisms for the presence
of extrachromosomal DNA or integrated DNA, amplifications of DNA
sequences, the maintenance of such sequences.
The physiological samples may be obtained from a wide variety of sources as
is evident from the varied purposes for which the subject method may be
used. Sources may include various physiological fluids, such as excreta,
e.g., stool, sputum, urine, saliva, etc.; plasma, blood, serum, ocular
lens fluids, spinal fluid, lymph, and the like. The sample may be used
without modification or may be modified by expanding the sample, cloning,
or the like, to provide an isolate, so that there is an overall
enhancement of the DNA or RNA and reduction of extraneous RNA or DNA.
Viruses may be plated on a lawn of compatible cells, so as to enhance the
amount of viral DNA; clinical isolates may be obtained by the sample being
streaked or spotted on a nutrient agar medium and individual colonies
assayed; or the entire sample introduced into a liquid broth and the cells
selectively or non-selectively expanded. The particular manner in which
the sample is treated will be dependent upon the nature of the sample, the
nature of the DNA or RNA source, the amount of oligonucleotide sequence of
interest which is anticipated as being present as compared to the total
amount of nucleic acid present, as well as the sensitivity of the protocol
and label being employed.
Either the sample nucleic acid or the reagent polynucleotide may be bound,
either covalently or noncovalently, but in any event non-diffusively, to
the support. (In the case of the embodiment represented by FIG. 2D, the
capture probe alone is bound to the solid support.) Where a sample nucleic
acid is bound to the support, various supports have found particular use
and to the extent, those supports will be preferred. These supports
include nitrocellulose filters, diazotized paper, ecteola paper, or other
support which provides such desired properties as low or no non-specific
binding, retention of the nucleic acid sample, ease of manipulation, and
allowing for various treatments, such as growth or organisms, washing,
heating, transfer, and label detection, as appropriate.
To the extent that a component of the polynucleotide reagent is bound to
the support, the type of support may be greatly varied over the type of
support involved with the sample oligonucleotide. The support may include
particles, paper, plastic sheets, container holder walls, dividers,
millipore filters, etc., where the materials may include organic polymers,
both naturally occurring and synthetic, such as polysaccharides,
polystyrene, polyacrylic acid and derivatives thereof, e.g.,
polyacrylamide, glass, ceramic, metal, carbon, polyvinyl chloride,
protein, and the like. The various materials may be functionalized or
non-functionalized, depending upon whether covalent or non-covalent
bonding is desired.
Where the sample nucleic acid is bound to the support, depending upon the
particular support, heating may be sufficient for satisfactory binding of
the nucleic acid. In other situations, diazo groups may be employed for
linking to the nucleic acid. Where, however, the polynucleotide reagent
component is bound to the support, a wide variety of different techniques
may be employed for ensuring the maintenance of the polynucleotide reagent
bound to the support. For example, supports can be functionalized, to have
active amino groups for binding, resulting from the binding of
alkylamines, hydrazides, or thiosemicarbazides to the support. One can
then add, by means of a terminal transferase, a ribonucleotide to a DNA
polynucleotide reagent. Upon glycol cleavage with an appropriate oxidant,
e.g., periodate, osmium tetroxide plus hydrogen peroxide, lead
tetraacetate, or the like, a dialdehyde is formed, which will then bind to
the amino group on the surface to provide a monosubstituted amino or
disubstituted amino group. Alternatively, one can provide for a maleimide
group which with thiophosphate will form the alkylthioester. Various
techniques described by Parikh, et al., supra and by Inman, supra for
agarose and polyacrylamide may be employed, which techniques may have
application with other materials.
The total number of polynucleotide reagent components on the support
available in the assay medium will vary, for the most part being
determined empirically. Desirably, a relatively high concentration per
unit surface area of polynucleotide to available functional groups on the
support should be employed, so long as the polynucleotide density does not
interfere with hybridization.
The size of the polynucleotide will vary widely, usually being not less
than about 15 bases and may be 50 bases or more, usually not exceeding
about 500 bases, more usually not exceeding 250 bases. There will usually
be a region in the polynucleotide reagent component homologous with a
sequence in the nucleic acid sample, usually the sequence of interest, of
at least six bases, usually at least 12 bases. The region for
hybridization may be 16 bases or more, usually not exceeding about 1 kbp,
where perfect homology is not required, it being sufficient that there be
homology to at least about 50%, more preferably homology to at least 80%.
(By percent homology is intended complementary, ignoring non-complementary
insertions which may loop out, insertions being greater than five bases.)
Particularly, where one is interested in a group of allelic genes, a number
of different strains, or related species, where the messenger RNA or
genomic portion is highly conserved but nevertheless is subject to
polymorphisms, it will frequently be desirable to prepare a probe which
reflects the differences and optimizes the homology for all the sequences
of interest, as against any particular sequence.
The label of the labeled polynucleotide reagent component may be joined to
the polynucleotide sequence through the selectively cleavable site or
through a link which is retained during the assay. A wide variety of
labels may be employed, where the label may provide for a detectable
signal or means for obtaining a detectable signal.
Labels therefore include such diverse substituents as ligands,
radioisotopes, enzymes, fluorescers, chemiluminescers, enzyme suicide
inhibitors, enzyme cofactors, enzyme substrates, or other substituent
which can provide, either directly or indirectly, a detectable signal.
Where ligands are involved, there will normally be employed a receptor
which specifically binds to the ligand, e.g., biotin and avidin,
2,4'-dinitrobenzene and anti(2,4-dinitrobenzene)IgG, etc., where the
receptor will be substituted with appropriate labels, as described above.
In this manner, one can augment the number of labels providing for a
detectable signal per polynucleotide sequence.
For the most part, the labels employed for use in immunoassays can be
employed in the subject assays. These labels are illustrated in U.S. Pat.
Nos. 3,850,752 (enzyme); 3,853,914 (spin label); 4,160,016 (fluorescer);
4,174,384 (fluorescer and quencher); 4,160,645 (catalyst); 4,277,437
(chemiluminescer); 4,318,707 (quenching particle); and 4,318,890 (enzyme
substrate).
Illustrative fluorescent and chemiluminescent labels include fluorescein,
rhodamine, dansyl, umbelliferone, biliproteins, luminol, etc.
Illustrative enzymes of interest include horse radish peroxidase,
glucose-6-phosphate dehydrogenase, acetylcholinesterase,
.beta.-galactosidase, .alpha.-amylase, uricase, malate dehydrogenase, etc.
That is, the enzymes of interest will primarily be hydrolases and
oxidoreductases.
The manner in which the label becomes bound to the polynucleotide sequence
will vary widely, depending upon the nature of the label. As already
indicated, a ribonucleotide may be added to the oligonucleotide sequence,
cleaved, and the resulting dialdehyde conjugated to an amino or hydrazine
group. The permanence of the binding may be further enhanced by employing
reducing conditions, which results in the formation of an alkyl amine.
Alternatively, the label may be substituted with an active halogen, such
as alpha-bromo or -chloroacetyl. This may be linked to a thiophosphate
group or thiopurine to form a thioether. Alternatively, the label may have
maleimide functionality, where a mercapto group present on the
polynucleotide will form a thioether. The terminal phosphate of the
polynucleotide may be activated with carbodiimide, where the resulting
phosphorimidazolide will react with amino groups or alcohols to result in
phosphoramidates or phosphate esters. Polypeptide bonds may be formed to
amino modified purines. Thus, one has a wide latitude in the choice of
label, the manner of linking, and the choice of linking group.
By combining the polynucleotide reagent with the sample, any nucleic acid
analyte present will become bound to the support. The amount of label
released from the support upon cleavage of the selectable cleavage site
will be related to the presence of analyte, where the amount of analyte
may also be determined quantitatively.
The modification of the spatial relationship between the label and the
support can be achieved in a number of ways. As indicated, there can be at
least one recognition site common to the probe and the same
polynucleotide, which can be digested with a restriction enzyme, thus
releasing the probe from the support. A wide variety of restriction
enzymes are available which can detect four base, six base, or eight base
recognition sites, where cleavage can be blunt-ended or staggered, may
occur at the recognition site or distant from the recognition site. In
this manner, the duplex formation of the recognition site(s) provides for
the opportunity to cleave the double strand with release of the label.
The nature of the selective cleavage site may or may not depend upon the
linking group. Where a restriction site is involved, the bonds involved
with the reagent components need only be stable under the assay
conditions. Where a restriction site is not involved, then the site will
involve a bond(s) which allows for separation of the label from the
support.
A phosphodiesterase may be employed where random hydrolysis will separate
the label from the support. The polynucleotide may be tailed with modified
nucleotides which are or may be subsequently labeled.
A wide variety of linking groups can be employed, where the nucleotides may
be modified or unmodified for linkage of the label. WO83/02277 reports the
use of 8-aminoalkyladenosine, where a label can be bound to the amino
group. The DNA polynucleotide reagent may then be tailed with the
ribonucleotides so that a plurality of labels will be present at the
terminus of each labeled polynucleotide. The tailed ribonucleotides may be
selectively cleaved employing an RNase. This will be particularly
advantageous when employing labels which interact to modify the signal.
For example, fluorescers in close proximity tend to self-quench. The
observed fluorescent signal can be greatly enhanced by hydrolyzing the
phosphate bonds, so that the individual fluorescer molecules are randomly
present in the solution. 0f course, fluorescers need not be the only
labels demonstrating this phenomenon, but other of the labels may also
display similar effects. Where enzyme substrates or cofactors are
employed, their presence on a polymer bound to a support will result in
substantial steric interference with enzyme approach. Thus the
depolymerization of the label and release from the support will
substantially enhance the enzyme rate.
Another technique is to add a ribonucleotide to a DNA polynucleotide
reagent and then cleave the ribosyl moiety to produce a dialdehyde. (See,
for example, Lee, et al., Biochemistry (1970) 9:113-118.) The dialdehyde
may be linked to an amino group joined to a label through a selectively
cleavable site. For example, a disulfide link may be present between the
Schiff's base and the label which can be cleaved by reduction, with
Ellman's reagent, or the like, to release the label. Where a restriction
endonuclease will be used to release of the label, then the dialdehyde can
be combined with the amino functionality under reductive amination
conditions. Various amino sources, such as proteins, e.g., enzymes,
phycobiliprotein fluorescers, receptors, such as immunoglobulins or
avidin, or non-proteinaceous labels may be employed.
Another linking method involves activating a terminal phosphate with
carbodiimide to form a phosphorimidazolide. (Chu, et al., Nucleic Acids
Res. (1983) 11:6513-6628.) The phosphorimidazolide may be reacted with
amines to form phosphoramidates. As before, the amino linking group will
include the selectable cleavage site, as appropriate, which could be a
pyrophosphate diester, cleavable by a pyrophosphatase, a short polypeptide
which could be cleaved by a peptidase, a light-sensitive functionality
such as azo, peroxy, or the like.
Another method for attaching the label involves chemical synthesis of
polynucleotides with a modifiable nucleoside derivative such as a cytosine
or uracil containing a 12-atom amine linker arm, followed by initrobenzene
(Ruth, DNA (1984) 3:123).
Ligand substituted nucleotides can be employed where the ligand does not
give a detectable signal directly, but bonds to a receptor to which is
conjugated one or more labels. Illustrative examples include biotinylated
nucleotides which will bind to avidin, haptens which will bind to
immunoglobulins, and various naturally occurring compounds which bind to
proteinaceous receptors, such as sugars with lectins, hormones and growth
factors with cell surface membrane proteins, and the like.
In the embodiment represented by FIG. 2D, the selectable cleavage site may
be introduced in one of two ways.
First, a crosslinking compound may be incorporated into the capture probe 1
itself, i.e., at position "X" as indicated in the figure. Any number of
crosslinking agents may be used for this purpose, the only limitation
being that the cleavage site introduced into the capture probe must be
cleavable with reagents that are compatible with the various probes,
labels, etc., used in the remainder of the method. Examples of suitable
crosslinkers include the following:
N-hydroxy succinimide (NHS), which introduces an amide bond into the probe;
ethylene glycolbis (succinimidylsuccinate) (EGS), which creates a
hydroxylamine-sensitive linkage;
bis[2-succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), which gives a
base-sensitive sulfone linkage; disuccinimidyl tartarate (DST), which
introduces 1,2-diols cleavable by periodate; and
dithiobis(succinimidylpropionate)(DSP), which results in thiol-cleavable
disulfide bonds. The crosslinker is preferably introduced into the capture
probe by (1) preparation of an alkylamine probe as described by Urdea et
al. in Nucleic Acids Research 16 (11):4937-4956 (1988); (2) reaction of
the free amine functionalities on the probe with the selected crosslinking
agent to give probebound crosslinking agent; (3) purification of the
probebound crosslinking agent using chromatographic or other means; and
(4) reaction of the probe-bound crosslinking agent with a solid support
having free reactive moieties, e.g., free amine groups, to provide a
support-bound probe having the desired cleavage site.
The cleavage site may therefore include, for example, the following types
of linkages:
##STR1##
The selectable cleavage site "X" in FIG. 2D may also be introduced by
appropriate modification of the capture probe prior to attachment to the
solid support. This method involves preparation of a polynucleotide having
the structure
##STR2##
where X is or contains the selectable cleavage site as described above. In
a particularly preferred embodiment, the polynucleotide has the structure
##STR3##
This compound may then be attached to a solid support, using conventional
means well known in the art, to give the capture probe illustrated in FIG.
2D. This latter compound is prepared using a reagent derived from tartaric
acid, where the 1,2-diol system is protected as the dibenzoyl compound
during DNA synthesis and which further contains a dimethoxytrityl
(DMT)-protected hydroxyl group and a phosphoramidite-derived hydroxyl
group (wherein "iPr" represents isopropyl):
##STR4##
allowing for incorporation into a DNA fragment using standard
phosphoramidite chemistry protocols. After synthesis and complete
deprotection the DNA/DNA hybrid molecule, as noted above, contains a
1,2-diol, i.e., a linkage that can be cleaved specifically with
NaIO.sub.4. As will be readily appreciated by those skilled in the art,
the DMT protecting group can be replaced with any suitable moiety R.sup.1
that is acid-sensitive and base-stable, e.g., unsubstituted or substituted
aryl or arylkyl groups, where the alkyl is, e.g., phenyl, naphthyl,
furanyl, biphenyl, or the like, and where the substituents are from 0 to
3, usually 0 to 2, and include any non-interfering stable groups, neutral
or polar, electron-donating or withdrawing. Similarly the phosphoramidite
moiety may be replaced with other species R.sup.2 including phosphorus
derivatives (e.g., a phosphotriester, a phosphodiester, a phosphite, an
H-phosphonate, a phosphorothioate, etc.) suitable for polynucleotide
synthesis, or with hydrogen. See, for example, EP Publication No. 0225807
(Urdea et al., "Solution Phase Nucleic Acid Sandwich Assay and
Polynucleotide Probes Useful Therein").
As in the embodiment represented by FIGS. 2A-2C, the embodiment of FIG. 2D
enables detection of specifically bound label in solution (and thus
accurate measurement of analyte 2) while nonspecifically bound label 6
remains bound to the solid suport 5.
In an alternative embodiment of the invention illustrated by FIGS. 3A and
3B, a complex is formed between a capture probe 1 (bound to solid support
5 through linkage Y), the nucleic acid analyte 2, and labeling probe 3, as
in the embodiment of FIG. 2D. The procedure followed to obtain this
hybridization complex is more fully described in EP Publication No.
0225807, cited supra. In order to release the specifically bound label
into solution, a "replacement" polynucleotide strand 4 is introduced,
selected so as to form a more stable hybrid with capture probe 1 than the
analyte forms with the capture probe. Although G/C content is also a
factor, this procedure typically requires that the length "B" of the
replacement strand be somewhat longer than the length "A" of the duplex
formed between the capture probe and the analyte.
A wide variety of supports and techniques for non-diffusive binding of
oligonucleotide chains have been reported in the literature. For a review,
see Meinkoth and Wahl, Anal. Biochem. (1984) 138:267-284. Supports include
nitrocellulose filters, where temperatures of 80.degree. C. for 2 hr
suffices, diazotized papers where bonding occurs without further
activation, ecteola paper, etc. Agarose beads can be activated with
cyanogen bromide for direct reaction with DNA. (Bauman, et al., J.
Histochem. Cytochem. (1981) 29:227-237); or reacted with cyanogen bromide
and a diamine followed by reaction with an .alpha.-haloacetyl, e.g.,
bromoacetyl or with an active carboxylic substituted olefin, e.g., maleic
anhydride, to provide beads capable of reacting with a thiol functionality
present on a polynucleotide chain. For example, DNA can be modified to
form a .alpha.-thiophosphate for coupling. (Pfeuffer and Hilmreich, J.
Biol. Chem. (1975) 250:867-876.) It is also possible to synthesize by
chemical means an oligonucleotide bound to a Teflon support and then fully
deblock the material without removing it (Lohrmann, et al., DNA (1984)
3:122).
In view of the wide diversity of labels and reagents, the common aspects of
the method will be described, followed by a few exemplary protocols.
Common to the procedures will be hybridization. The hybridization can be
performed at varying degrees of stringency, so that greater or lesser
homology is required for duplexing. For the most part, aqueous media will
be employed, which may have a mixture of various other components.
Particularly, organic polar solvents may be employed to enhance
stringency. Illustrative solvents include dimethylformamide,
dimethylacetamide, dimethylsulfoxide, that is, organic solvents which at
the amounts employed, are miscible with water. Stringency can also be
enhanced by increasing salt concentration, so that one obtains an enhanced
ignic strength.... Also, increasing temperature can be used to increase
stringency. In each case, the reverse direction results in reduced
stringency. Other additives may also be used to modify the stringency,
such as detergents.
The period of time for hybridization will vary with the concentration of
the sequence of interest, the stringency, the length of the complementary
sequences, and the like. Usually, hybridization will require at least
about 15 min, and generally not more than about 72 hr, more usually not
more than about 24 hr. Furthermore, one can provide for hybridization at
one stringency and then wash at a higher stringency, so that
heteroduplexes lacking sufficient homology are removed.
The nucleic acid sample will be treated in a variety of ways, where one may
employ the intact genome, mechanically sheared or restriction enzyme
digested fragments of the genome, varying from about 0.5kb to 30kb, or
fragments which have been segregated according to size, for example, by
electrophoresis. In some instances, the sequences of interest will be
cloned sequences, which have been cloned in an appropriate vector, for
example, a single-stranded DNA or RNA virus, e.g., M13.
Included in the assay medium may be other additives including buffers,
detergents, e.g., SDS, Ficoll, polyvinyl pyrrolidone and foreign DNA, to
minimize hon-specific binding. All of these additives find illustration in
the literature, and do not need to be described in detail here.
In accordance with a particular protocol, the sample nucleic acid and
polynucleotide reagent(s) are brought together in the hybridization medium
at the predetermined stringency. After a sufficient time for
hybridization, the support will be washed at least once with a medium of
greater or lesser stringency than the hybridization medium. The support
with the bound polynucleotide and analyte will then be contacted with the
necessary reactants (includes physical treatment, e.g., light) for
cleaving the selectable cleavage site, providing for single- or
double-stranded cleavage. For the most part hydrolase enzymes will be
used, such as restriction endonucleases, phosphodiesterases,
pyrophosphatase, peptidases, esterases, etc., although other reagents,
such as reductants, Ellman,s reagent, or light may find use. After
cleavage, the support and the supernatant may or may not be separated,
depending upon the label and the manner of measurement, and the amount of
label released from the support determined.
To further illustrate the subject invention, a few exemplary protocols will
be described. In the first exemplary protocol, a microtiter plate is
employed, where fluorescent labeled polynucleotides are bound to the
bottom of each well. DNA from a pathogen which has been cloned, is
restricted with one or more restriction enzymes to provide fragments of
from about 0.5-2kb. The fragments are isolated under mild basic conditions
for denaturing and dispersed in the hybridization medium, which is then
added sequentially to the various wells, each of the wells having
different sequences which are specifically homologous with sequences of
different strains of a particular pathogen species.
The wells are maintained at an elevated temperature, e.g., 60.degree. C.,
for sufficient time for hybridization to occur, whereupon the supernatant
is removed and wells are thoroughly washed repeatedly with a buffered
medium of lower stringency than the hybridization medium. Duplex formation
results in a recognition site for a restriction enzyme common to all of
the strains. To each well is then added a restriction enzyme medium for
digestion of double-stranded DNAs which are digested result in the release
of the fluorescent label into the supernatant. The supernatant is
aspirated from each of the wells and irradiated. The amount of
fluorescence is then determined as indicative of the presence of the
sequence of interest. In this manner, one can rapidly screen for which of
the strains is present, by observing the presence of fluorescence in the
liquid phase.
In the second exemplary protocol, one employs a column containing glass
beads to which are bound unlabeled polynucleotide. To the column is then
added the sample nucleic acid containing DNA fragments obtained from
mammalian cells. The fragments range from about 0.5 to 10kb. The sample
DNA is dispersed in an appropriate hybridization medium and added to the
column and retained in the column for sufficient time for hybridization to
occur. After the hybridization of the sample, the hybridization medium is
released from the column and polynucleotide reagent labeled with horse
radish peroxidase (HRP) through a disulfide linkage is added in a second
hybridization medium under more stringent conditions than the first medium
and the second medium released in the column for sufficient time for
hybridization to occur. The labeled polynucleotide has a sequence
complementary to the sequence of interest. The hybridization medium is
evacuated from the column.
The column may then be washed one or more times with a medium of higher
stringency to remove any polynucleotide sequences which have insufficient
homology with the labeled polynucleotide. Ellman's reagent | | |
