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Description  |
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TECHNICAL FIELD OF INVENTION
The present invention relates generally to the detection of genetic
material by polynucleotide probes. More specifically, it relates to a
method for quantifiably detecting a targeted polynucleotide sequence in a
sample of biological and/or nonbiological material employing a probe
capable of generating a soluble signal. The method and products disclosed
herein in accordance with the invention are expected to be adaptable for
use in many laboratory, industrial, and medical applications wherein
quantifiable and efficient detection of genetic material is desired.
BACKGROUND OF THE INVENTION
In the description, the following terms are employed:
Analyte--A substance or substances, either alone or in admixtures, whose
presence is to be detected and, if desired, quantitated. The analyte may
be a DNA or RNA molecule of small or high molecular weight, a molecular
complex including those molecules, or a biological system containing
nucleic acids, such as a virus, a cell, or group of cells. Among the
common analytes are nucleic acids (DNA and RNA) or segments thereof,
oligonucleotides, either single- or double-stranded, viruses, bacteria,
cells in culture, and the like. Bacteria, either whole or fragments
thereof, including both gram positive and gram negative bacteria, fungi,
algae, and other microorganisms are also analytes, as well as animal
(e.g., mammalian) and plant cells and tissues.
Probe--A labelled polynucleotide or oligonucleotide sequence which is
complementary to a polynucleotide or oligonucleotide sequence of a
particular analyte and which hybridizes to said analyte sequence.
Label--That moiety attached to a polynucleotide or oligonucleotide sequence
which comprises a signalling moiety capable of generating a signal for
detection of the hybridized probe and analyte. The label may consist only
of a signalling moiety, e.g., an enzyme attached directly to the sequence.
Alternatively, the label may be a combination of a covalently attached
bridging moiety and signalling moiety or a combination of a non-covalently
bound bridging moiety and signalling moiety which gives rise to a signal
which is detectable, and in some cases quantifiable.
Bridging Moiety--That portion of a label which on covalent attachment or
non-covalent binding to a polynucleotide or oligonucleotide sequence acts
as a link or a bridge between that sequence and a signalling moiety.
Signalling Moiety--That portion of a label which on covalent attachment or
non-covalent binding to a polynucleotide or oligonucleotide sequence or to
a bridging moiety attached or bound to that sequence provides a signal for
detection of the label.
Signal--That characteristic of a label or signalling moiety that permits it
to be detected from sequences that do not carry the label or signalling
moiety.
The analysis and detection of minute quantities of substances in biological
and non-biological samples has become a routine practice in clinical,
diagnostic and analytical laboratories. These detection techniques can be
divided into two major classes: (1) those based on ligand-receptor
interactions (e.g., immunoassay-based techniques), and (2) those based on
nucleic acid hybridization (polynucleotide sequence-based techniques).
Immunoassay-based techniques are characterized by a sequence of steps
comprising the noncovalent binding of an antibody and antigen
complementary to it. See, for example, T. Chard, An Introduction To
Radioimmunoassay And Related Techniques (1978).
Polynucleotide sequence-based detection techniques are characterized by a
sequence of steps comprising the non-covalent binding of a labelled
polynucleotide sequence or probe to a complementary sequence of the
analyte under hybridization conditions in accordance with the Watson-Crick
base pairing of adenine (A) and thymine (T), and guanine (G) and cytosine
(C), and the detection of that hybridization. [M. Grunstein and D. S.
Hogness, "Colony Hybridization: A Method For The Isolation Of Cloned DNAs
That Contain A Specific Gene", Proc. Natl. Acad. Sci. U.S.A., 72, pp.
3961-65 (1975)]. Such polynucleotide detection techniques can involve a
fixed analyte [see, e.g., U.S. Pat. No. 4,358,535 to Falkow et al], or can
involve detection of an analyte in solution [see U.K. patent application
No. 2,019,408 A].
The primary recognition event of polynucleotide sequence-based detection
techniques is the non-covalent binding of a probe to a complementary
sequence of an analyte, brought about by a precise molecular alignment and
interaction of complementary nucleotides of the probe and analyte. This
binding event is energetically favored by the release of non-covalent
bonding free energy, e.g., hydrogen bonding, stacking free energy and the
like.
In addition to the primary recognition event, it is also necessary to
detect when binding takes place between the labelled polynucleotide
sequence and the complementary sequence of the analyte. This detection is
effected through a signalling step or event. A signalling step or event
allows detection in some quantitative or qualitative manner, e.g., a human
or instrument detection system, of the occurrence of the primary
recognition event.
The primary recognition event and the signalling event of polynucleotide
sequence based detection techniques may be coupled either directly or
indirectly, proportionately or inversely proportionately. Thus, in such
systems as nucleic acid hybridizations with sufficient quantities of
radiolabeled probes, the amount of radio-activity is usually directly
proportional to the amount of analyte present. Inversely proportional
techniques include, for example, competitive immuno-assays, wherein the
amount of detected signal decreases with the greater amount of analyte
that is present in the sample.
Amplification techniques are also employed for enhancing detection wherein
the signalling event is related to the primary recognition event in a
ratio greater than 1:1. For example, the signalling component of the assay
may be present in a ratio of 10:1 to each recognition component, thereby
providing a 10-fold increase in sensitivity.
A wide variety of signalling events may be employed to detect the
occurrence of the primary recognition event. The signalling event chosen
depends on the particular signal that characterizes the label or
signalling moiety of the polynucleotide sequence employed in the primary
recognition event. Although the label may only consist of a signalling
moiety, which may be detectable, it is more usual for the label to
comprise a combination of a bridging moiety covalently or non-covalently
bound to the polynucleotide sequence and a signalling moiety that is
itself detectable or that becomes detectable after further modification.
The combination of bridging moiety and signalling moiety, described above,
may be constructed before attachment or binding to the sequence, or it may
be sequentially attached or bound to the sequence. For example, the
bridging moiety may be first bound or attached to the sequence and then
the signalling moiety combined with that bridging moiety. In addition,
several bridging moieties and/or signalling moieties may be employed
together in any one combination of bridging moiety and signalling moiety.
Covalent attachment of a signalling moiety or bridging moiety/signalling
moiety combination to a sequence is exemplified by the chemical
modification of the sequence with labels comprising radioactive moieties,
fluorescent moieties or other moieties that themselves provide signals to
available detection means or the chemical modification of the sequence
with at least one combination of bridging moiety and signalling moiety to
provide that signal.
Non-covalent binding of a signalling moiety or bridging moiety/signalling
moiety to a sequence involve the non-covalent binding to the sequence of a
signalling moiety that itself can be detected by appropriate means, i.e.,
or enzyme, or the non-covalent binding to the sequence of a bridging
moiety/signalling moiety to provide a signal that may be detected by one
of those means. For example, the label of the polynucleotide sequence may
be a bridging moiety non-covalently bound to an antibody, a fluorescent
moiety or another moiety which is detectable by appropriate means.
Alternatively, the bridging moiety could be a lectin, to which is bound
another moiety that is detectable by appropriate means.
There are a wide variety of signalling moieties and bridging moieties that
may be employed in labels for covalent attachment or non-covalent binding
to polynucleotide sequences useful as probes in analyte detection systems.
They include both a wide variety of radioactive and non-radioactive
signalling moieties and a wide variety of non-radioactive bridging
moieties. All that is required is that the signalling moiety provide a
signal that may be detected by appropriate means and that the bridging
moiety, if any, be characterized by the ability to attach covalently or to
bind non-covalently to the sequence and also the ability to combine with a
signalling moiety.
Radioactive signalling moieties and combinations of various bridging
moieties and radioactive signalling moieties are characterized by one or
more radioisotopes such as .sup.32 P, .sup.131 I, .sup.14 C, .sup.3 H,
.sup.60 Co, .sup.59 Ni, .sup.63 Ni and the like. Preferably, the isotope
employed emits .beta. or .UPSILON. radiation and has a long half life.
Detection of the radioactive signal is then, most usually, accomplished by
means of a radioactivity detector, such as exposure to a film.
The disadvantages of employing a radioactive signalling moiety on a probe
for use in the identification of analytes are well known to those skilled
in the art and include the precautions and hazards involved in handling
radioactive material, the short life span of such material and the
correlatively large expenses involved in use of radioactive materials.
Non-radioactive signalling moieties and combinations of bridging moieties
and non-radioactive signalling moieties are being increasingly used both
in research and clinical settings. Because these signalling and bridging
moieties do not involve radioactivity, the techniques and labelled probes
using them are safer, cleaner, generally more stable when stored, and
consequently cheaper to use. Detection sensitivities of the
non-radioactive signalling moieties also are as high or higher than
radio-labelling techniques.
Among the presently preferred non-radioactive signalling moieties or
combinations of bridging/signalling moieties useful as non-radioactive
labels are those based on the biotin/avidin binding system. [P. R. Langer
et al., "Enzymatic Synthesis Of Biotin-Labeled Polynucleotides: Novel
Nucleic Acid Affinity Probes", Proc. Natl. Acad. Sci. U.S.A., 78, pp.
6633-37 (1981); J. Stavrianopoulos et al., "Glycosylated DNA Probes For
Hybridization/Dection Of Homologous Sequences", presented at the Third
Annual Congress For Recombinant DNA Research (1983); R. H. Singer and D.
C. Ward, "Actin Gene Expression Visualized In Chicken Muscle Tissue
Culture By Using In Situ Hybridization With A Biotinated Nucleotide
Analog", Proc. Natl. Acad. Sci. U.S.A., 79, pp. 7331-35 (1982)]. For a
review of non-radioactive signalling and bridging/signalling systems, both
biotin/avidin and otherwise, see D. C. Ward et al., "Modified Nucleotides
And Methods Of Preparing And Using Same", European Patent application No.
63879.
Generally, the signalling moieties employed in both radioactive and
non-radioactive detection techniques involve the use of complex methods
for determining the signalling event, and/or supply only an unquantitable
positive or negative response. For example, radioactive isotopes must be
read by a radioactivity counter; while signalling moieties forming
insoluble "signals", i.e., precipitates, certain fluorescers, and the like
[see, e.g., David et al., U.S. Pat. No. 4,376,100] only provide detection
not quantitation of the analyte present in the tested sample.
One step toward facilitating rapid and efficient quantitation as well as
detection of the hybridization event was the work of Heller et al. in
European Patent Application Nos. 70685 and 70687 which describe the use of
a signalling moiety which produces a soluble signal for measurable
detection by a spectrophotometer. These European patent applications
disclose the use of two different probes complementary to different
portions of a gene sequence, with each probe being labelled at the end
which will abut the other probe upon hybridization. The first probe is
labelled with a chemiluminescent complex that emits lights of a specific
wavelength. The second probe is labelled with a molecule that emits light
of a different wavelength measurable by spectrophotometry when excited by
the proximity of the first signalling moiety. However, this technique is
performed in solution and can generate false positive results in the
absence of the analyte if the two probes happen to approach too closely in
solution and react with each other.
Similarly, U.K. Patent Application Ser. No. 2,019,408A, published Oct. 31,
1979, discloses a method for detecting nucleic acid sequences in solution
by employing an enzyme-labelled RNA or DNA probe which, upon contact with
a chromogen substrate, provides an optically readable signal. The analytes
may be separated from contaminants prior to hybridization with the probe,
or, alternatively, the hybrid probe-analyte may be removed from solution
by conventional means, i.e., centrifugation, molecular weight exclusion,
and the like. Like Heller's technique, this method is performed in
solution.
There remains therefore a need in the art for a reliable, simple and
quantifiable technique for the detection of analytes of interest in
biological and non-biological samples.
SUMMARY OF THE INVENTION
The invention provides a solution for the disadvantages of presently
available methods of detecting analytes by a novel combination of
hybridization and immunological techniques. In the present invention,
chemically labelled polynucleotide or oligonucleotide probes are employed
to detect analytes by having the capacity to generate a reliable, easily
quantifiable soluble signal.
Analytes to be detected by the detection processes of this invention may be
present in any biological or non-biological sample, such as clinical
samples, for example, blood urine, feces, saliva, pus, semen, serum, other
tissue samples, fermentation broths, culture media, and the like. If
necessary, the analyte may be pre-extracted or purified by known methods
to concentrate its nucleic acids. Such nucleic acid concentration
procedures include, for example, phenol extraction, treatment with
chloroform-isoamyl alcohol or chloroform-octanol, column chromatography
(e.g., Sephadex, hydroxyl apatite), and CsCl equilibrium centrifugation.
The analyte, separated from contaminating materials, if present, is
according to the present invention, fixed in hybridizable form to a solid
support.
Analytes in a biological sample are preferably denatured into
single-stranded form, and then directly fixed to a suitable solid support.
Alternatively, the analyte may be directly fixed to the support in
double-stranded form, and then denatured. The present invention also
encompasses indirect fixation of the analyte, such as in situ techniques
where the cell is fixed to the support and sandwich hybridization
techniques where the analyte is hybridized to a polynucleotide sequence
that is fixed to the solid support. It is preferred that the solid support
to which the analyte is fixed be non-porous and transparent, such as
glass, or alternatively, plastic, polystyrene, polyethylene, dextran,
polypropylene and the like. Conventional porous materials, e.g.,
nitrocellulose filters, although less desirable for practice of the method
of the present invention, may also be employed as a support.
It is also highly desirable that the analyte be easily fixed to the solid
support. The capability to easily fix the analyte to a transparent
substrate would permit rapid testing of numerous samples by the detection
techniques described herein.
Chemically-labeled probes are then brought into contact with the fixed
single-stranded analytes under hybridizing conditions. The probe is
characterized by having covalently attached to it a chemical label which
consists of a signalling moiety capable of generating a soluble signal.
Desirably, the polynucleotide or oligonucleotide probe provides sufficient
number of nucleotides in its sequence, e.g., at least about 25, to allow
stable hybridization with the complementary nucleotides of the analyte.
The hybridization of the probe to the single-stranded analyte with the
resulting formation of a double-stranded or duplex hybrid is then
detectable by means of the signalling moiety of the chemical label which
is attached to the probe portion of the resulting hybrid. Generation of
the soluble signal provides simple and rapid visual detection of the
presence of the analyte and also provides a quantifiable report of the
relative amount of analyte present, as measured by a spectrophotometer or
the like.
The method of the present invention involving the colorimetric or
photometric determination of the hybridized probes employs as the
signalling moiety reagents which are capable of generating a soluble
signal, e.g., a color change in a substrate in solution. Preferable
components of the signalling moiety include enzymes, chelating agents and
co-enzymes, which are able to generate colored or fluorescent soluble
signals. Specifically, certain chromogens upon contact with certain
enzymes are utilizable in the method of the present invention. The
following Table I lists exemplary components for the signalling moiety of
the present invention. Each chromogen listed is reactive with the
corresponding enzyme to produce a soluble signal which reports the
presence of the chemically-labeled probe analyte hybrid. The superscript
notation (*) indicates that the chromogen fluoresces, rather than produces
a color change.
TABLE I
______________________________________
ENZYME CHROMOGEN
______________________________________
alkaline phosphatase
*4-Methylumbelliferyl
or phosphate
acid phosphatase *bis (4-Methylumbelli-
feryl phosphate
3-0-methylfluorescein.
*Flavone-3-diphosphate
triammonium salt
p-nitrophenyl phosphate
2Na.
peroxidase *Tyramine hydro-
chloride
*3-(p-hydroxyphenyl)
Propionic acid
*p-Hydroxyphenethyl
alcohol
2,2'-Azino-Di-3-
Ethylbenzthiazoline
sulfonic acid
(ABTS)
ortho-phenylenedia-
mine 2HCl
0-dianisidine
*5-aminosalicylic acid
p-cresol
3,3'-dimethyloxy-
benzidine
3-methyl-2-benzo-
thiazoline hydra-
zone
tetramethyl benzidine
.beta.-D-galactosidase
0-nitrophenyl .beta.-D-
galactopyranoside
4-methylumbelliferyl-
.beta.-D-galactoside
glucose-oxidase ABTS
______________________________________
As another aspect of the present invention, the signalling moiety may be
attached to the probe through the formation of a bridging entity or
complex. Likely candidates for such a bridging entity would include a
biotin-avidin bridge, a biotin-streptavidin bridge, or a sugar-lectin
bridge.
Once the fixed probe-analyte hybrid is formed, the method may further
involve washing to separate any non-hybridized probes from the area of the
support. The signalling moiety may also be attached to the probe through
the bridging moiety after the washing step to preserve the materials
employed. Thereafter, another washing step may be employed to separate
free signalling moieties from those attached to the probe through the
bridging moiety.
Broadly, the invention provides hybridization techniques which provide the
same benefits as enzyme linked immunosorbent assay techniques, i.e., the
qualitative and quantitative determination of hybrid formation through a
soluble signal. Various techniques, depending upon the chemical label and
signalling moiety of the probe, may be employed to detect the formation of
the probe-analyte hybrid. It is preferred, however, to employ
spectrophotometric techniques and/or colorimetric techniques for the
determination of the hybrid. These techniques permit not only a prompt
visual manifestation of the soluble signal generated by the signalling
moiety on the double-stranded hybrid, but also permit the quantitative
determination thereof, i.e., by the enzymatic generation of a soluble
signal that can be quantitatively measured.
Yet another aspect of the method of the present invention involves
generating the soluble signal from the probe-analyte hybrid in a device
capable of transmitting light therethrough for the detection of the signal
by spectrophotometric techniques. Examples of devices useful in the
spectrophotometric analysis of the signal include conventional apparatus
employed in diagnostic laboratories, i.e., plastic or glass wells, tubes,
cuvettes or arrangements of wells, tubes or cuvettes. It may also be
desirable for both the solid support to which the analyte is fixed and the
device to be composed of the same material, or for the device to function
as the support in addition to facilitating spectrophotometric detection.
A further aspect of the present invention provides products useful in the
disclosed method for detection of a polynucleotide sequence. Among these
products is a device containing a portion for retaining a fluid. Such
portion contains an immobilized polynucleotide sequence hybridized to a
polynucleotide or oligonucleotide probe. The probe, as described above,
has covalently attached thereto a chemical label including a signalling
moiety capable of generating a soluble signal. Also part of the device is
a soluble signal, preferably a colored or fluorescent product, generatable
by means of the signalling moiety. The portion of the device for
containing the fluid is desirably a well, a tube, or a cuvette. A related
product of the invention is an apparatus comprising a plurality of such
devices for containing a fluid, in which at least one such device contains
the above-described immobilized polynucleotide sequence, polynucleotide or
oligonucleotide probe, signalling moiety, and soluble signal. Additionally
the present invention provides for the novel product of a non-porous solid
support to which a polynucleotide is directly fixed in hybridizable form.
Such a fixed sequence may be hybridized to another polynucleotide sequence
having covalently attached thereto a chemical label including a signalling
moiety capable of generating a soluble signal. As indicated above, the
support is preferably transparent or translucent. Such products could be
advantageously employed in diagnostic kits and the like.
Other aspects and advantages of the present invention will be readily
apparent upon consideration of the following detailed description of the
preferred embodiments thereof.
DETAILED DESCRIPTION
The following examples are illustrative of preferred embodiments of the
method of the present invention. Specifically referred to therein are
methods for fixing the analyte to a non-porous solid support, as well as
illustrations of the use of soluble signals in polynucleotide probes as
discussed above.
EXAMPLE 1
For purposes of the present invention, an analyte is immobilized on a solid
support, preferably a non-porous translucent or transparent support. To
effect easy fixing of a denatured single-stranded DNA sequence to a glass
support, an exemplary "fixing" procedure may involve pretreating the glass
by heating or boiling for a sufficient period of time in the presence of
dilute aqueous nitric acid. Approximately forty-five minutes in 5% dilute
acid should be adequate to leach boron residues from a borosilicate glass
surface. The treated glass is then washed or rinsed, preferably with
distilled water, and dried at a temperature of about 115.degree. C., for
about 24 hours. A 10 percent solution of gamma-aminopropyltriethoxysilane,
which may be prepared by dissolving the above-identified silane in
distilled water followed by addition of 6N hydrochloric acid to a pH of
about 3.45, will then be applied to the glass surface. The glass surface
is then incubated in contact with the above-identified silane solution for
about 2-3 hours at a temperature of about 45.degree. C. The glass surface
is then washed with an equal volume of water and dried overnight at a
temperature of about 100.degree. C. The resulting treated glass surface
will now have available alkylamine thereon suitable for immobilizing or
fixing any negatively charged polyelectrolytes applied thereto. [See
Weetal, H. H. and Filbert, A. M., "Porous Glass for Affinity
Chromatography Applications", Methods in Enzymology, Vol. XXXIV, Affinity
Techniques Enzyme Purification: Part B. pp. 59-72, W. B. Jakoby and M.
Wilchek, eds.]
Such treated glass could then be employed in the method of the invention.
For example, glass plates provided with an array of depressions or wells
would have samples of the various denatured analytes deposited therein,
the single-stranded analytes being fixed to the surfaces of the wells.
Thereupon, polynucleotide probes provided with a chemical label may be
deposited in each of the wells for hybridization to any complementary
single-stranded analyte therein. After washing to remove any
non-hybridized probe, the presence of any hybrid probe-analyte is then
detectable. One detection technique as described herein involves the
addition of an enzyme-linked antibody or other suitable bridging entity of
the label for attachment to the probe. Subsequently a suitable substrate
is added to elicit the soluble signal, e.g., a color change or chemical
reaction, which is then measured colorimetrically or photometrically.
EXAMPLE 2
A glass surface treated as described in Example 1 can be employed in the
method of the present invention, wherein glucosylated DNA is employed as
the labelled probe, and the signalling moiety comprises the combination of
acid phosphatase and its substrate paranitrophenylphosphate.
In this procedure, glucosylated bacteriophage T.sub.4 DNA, isolated from E.
coli CR63 cultures infected with phage T.sub.4 AM82 [44.sup.- 62.sup.- ]
and purified to be free of chromosomal DNA, or non-glucosylated, highly
purified calf thymus DNA is delivered in 100 .mu.l portions to treated
glass tubes in triplicate set. After 15-30 minutes at room temperature,
the solution is removed and the tubes rinsed generously with PBS.Mg.sup.++
buffer [100 mM Na-K-PO.sub.4, pH 6.5, 150 mM NaCl and 10 mM MgCl.sub.2 ].
One set of tubes is checked for the presence of DNA by staining with
ethidium bromide [100 .mu.l of 1 mg/ml solution, 30 minutes in the dark,
at room temperature]. The staining solution is removed and the tubes
rinsed and checked by UV light. Both glucosylated labelled and unlabelled
DNA "probe" bound to the activated glass surface by the observed red
fluorescence characteristic of ethidium bromide.
To another set of tubes is delivered fluorescein-labelled ConA [100 .mu.l
of 0.1 mg/ml in PBS.Mg.sup.++ buffer]. The Concanavalin A [ConA] is
obtained and solubilized in 2.0M NaCl at a concentration of 50 mg/ml, and
fluorescein-labelled by reacting ConA with fluorescein isothiocyanate at
an FITC to protein molar ratio of 3 to 1 in 0.1M sodium borate solution at
a pH of 9.2 and at a temperature of 37.degree. C. for 60 minutes. Any
unreacted FITC is removed by gel filtration on Sephadex G-50. After 60
minutes at room temperature, the solution is removed and the tubes rinsed
and checked under UV light. ConA bound only to glucosylated DNA in tubes
containing T.sub.4 DNA.
To the third set of tubes is delivered 100 .mu.l of unlabeled ConA in
PBS.Mg.sup.++ buffer. After 60 minutes at room temperature, the tubes are
rinsed free of ConA with 0.2M Imidazole buffer pH 6.5.
Acid phosphatase is then added [0.005 units in 100 .mu.l at 0.2 percent
phosphatase-free BSA] and the tubes are incubated at room temperature for
30 minutes. After rinsing with 0.15M NaCl to remove any unbound enzyme,
0.1 mM paranitrophenylphosphate in 0.2M imidazole at pH 6.5 is added and
incubation continued for 60 minutes at 37.degree. C. The enzyme reaction
is terminated by adding 1.0 ml of 0.5 percent sodium bicarbonate and
absorbance is determined at A.sub.300.
The resulting observed test results indicate that acid phosphatase, one
component of the signalling moiety gives a positive visible color
reaction, upon reaction with its chromogen, only in tubes containing
"probe" T.sub.4 DNA and bridging moiety, ConA, but is washed off from the
tubes which contain only ConA or ConA and calf thymus DNA.
EXAMPLE 3
In an example of the method of the present invention, phage lambda DNA was
employed as the analyte, glucosylated DNA as the labelled probe, ConA as
the bridging entity and alkaline phosphatase with paranitrophenylphosphate
as the signalling moiety. Bacteriophage lambda, obtained by heat induction
of E. coli stain W3350 lysogenic for .lambda.C.sub.1 857 phage, was
employed for the preparation of phage lambda DNA. In these tests, the
analyte, phage lambda DNA, was immobilized on an activated glass surface
according to the following procedure. After rinsing with buffer, glass
tubes were coated with 100 .mu.l of coating solution [50 percent
formamide, 5X SSC, 100 .mu.g salmon sperm DNA 0.2 percent polyvinyl
pyrrolidone, 0.1 percent Triton X-100, 0.2 percent BSA and 0.05 percent
SDS] at 42.degree. C. for 90-120 minutes. The coating solution was removed
and the surface was covered with 100 .mu.l of coating solution containing
phage lambda DNA.
Phage lamba DNA employed as the probe is nick translated with
maltose-triose dUTP to introduce glucosyl residues into the DNA. The
glucosylated minutes and rapidly cooled in ice bath immediately before
use. The tubes were then incubated with probe at 42.degree. C. for 24
hours. The solution was removed and tubes were rinsed with PBS.Mg.sup.++
buffer. As described above in example 2, ConA is added to the tubes in
PBS.Mg.sup.++ buffer. After 60 minutes at room temperature the tubes are
rinsed with 0.2M Imidazole buffer.
Also as described in Example 2, the signalling moiety components, acid
phosphatase and paranitrophenyl phosphate, are sequentially introduced
into the tubes, to generate the detectable soluble signal. In these tests,
the glucosyl moiety of the DNA probe is one bridging moiety of the
chemical label, and reacts with and is strongly attracted to the second
bridging moiety, ConA. The results indicated that acid phosphatase was not
washed off from the tubes which contained glucosylated probe, whereas
tubes containing non-labelled probe did not show any enzyme activity.
EXAMPLE 4
As in the above example employing a glucosylated DNA as the labelled probe,
wherein the glucosyl moiety serves as part of the chemical label,
comparable results may also be achieved by employing a biotin-labeled DNA
probe. When biotin is employed as a bridging moiety of the chemical label
of the DNA probe, the presence of the biotin-labeled DNA probe would be
elicited or detected by means of an avidin or streptavidin-linked enzyme,
since avidin is strongly reactive with or strongly bonds to biotin.
For example, a biotin-labeled DNA probe would readily be detected by an
enzyme complex of the character avidin-biotin-alkaline phosphatase. More
specifically, the presence of the biotin-labeled DNA probe would readily
be detected by contacting the hybrid containing the biotin-labeled probe
with the enzyme complex avidin-biotin-alkaline phosphatase, and bringing
the resulting probe and avidin-biotin-alkaline phosphatase complex into
contact with a suitable substrate which, upon contact with the enzyme,
would produce a soluble signal that would be readily noticed or be capable
of being determined, both qualitatively and quantitatively, by photometric
and/or colorimetric means. If desired, instead of an avidin-biotin-enzyme
complex, there could be used an antibody to biotin for attachment to the
biotin moiety of the biotin-labeled DNA probe, followed by a complex
comprising anti-antibody-enzyme in the manner described above.
EXAMPLE 5
The advantages of this invention are also obtainable when the probe is
immobilized on a non-porous plastic surface. When a plastic surface is
employed, it is sometimes desirable to increase the effectiveness or
uniformity of the fixation by pretreating the plastic surface.
Because polystyrene from various batches or sources exhibits different
binding capacities, the adherence or fixing of DNA to a polystyrene
surface is improved by treating the surface with an amino-substituted
hydrophobic polymer or material. Previous experiments demonstrated that
addition of duodecadiamine (DDA) to polystyrene resulted in an uniform
binding coefficient of polystyrene plates of different batches. Another
technique for improving the fixing or uniformity of the plastic surface
for fixing DNA involves treatment of the surface with polylysine (PPL).
In tests involving the fixing of DNA to a plastic surface, biotinylated DNA
(b-DNA) was denatured and aliquoted into Dynatech, Immulon II.TM.
removable wells. Samples were allowed to dry onto the plastic surface at
37.degree. C. The amount of bound b-DNA was determined by sequential
addition of goat anti-biotin antibody and rabbit anti-goat antibody
complexed to the signalling moiety, alkaline phosphatase, followed by
development with p-nitrophenyl phosphate in diethanolamine buffer, pH 9.6.
Enzymatic activity was monitored at 405 nm utilizing the automatic
Dynatech Micro ELISA Scanner. This procedure enables quantitation of the
amount of bound DNA and therefore the degree of biotinylation. To increase
the sensitivity of detection, a fluorogenic substrate such as
4-methylumbelliferyl-phosphate, or its analogues, with companion enzymes,
may be used.
In a further example of the method, denatured adenovirus 2 DNA, the
analyte, was bound to polystyrene plates as described above. After
blocking with Denhardt's formamide blocking buffer, several biotinylated
probes, b-adeno-2-DNA and lambda DNA were hybridized to the immobilized
DNA. To one set of immobilized DNA, no probe was added. The extent of | | |
