Note: Descriptions are shown in the official language in which they were submitted.
1 30993~
8ACKG~OUND
The present invention relates to a nucleic acid
hybridization procedure, and a diagnostic kit for use with
the procedure.
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In traditional microbial diagnostics the presence
of a microbe in a sample is determined by isolatinq the
m~1crobe. After enrichment cultivations, the microbe is
determined either on the basis of its biochemical
properties or its immunological properties. Both methods of
identification require that the microbe in ~he sample be
able to propagate. Such identifica~ion can be laborious and
very time consuming.
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1 30~932
2 5191/98D-106
With the development of genetic research, it was
found that microbes, even ones that are no longer viable, can
be determined by the specific nucleic acids they contain.
The nucleic acid may be deoxyribonucleic acid (DNA), which
is usually double stranded, or ribonucleic acid (RNA), which
is usually single stranded. Organisms such as bacteria,
fungi, viruses, yeasts, etc., can all be thus determined.
Generally the identification procedure includes artificially
induced lysis, whereby the cell wall of the organism is
ruptured to release the nucleic acid, which can then be
determined.
The hydrogen-bonded structure of the double helix
of a nucleic acid molecule (e.g. DNA) can be disrupted by
heating and/or by trea~ment with alkali. Because there are
no covalent bonds connecting the partner strands, the two
polynucleotide chains of a duplex ~ucleic acid molecule
separate entirely when all the hydrogen bonds are broken.
This proce~s of strand separation is called denaturation.
An extremely useful property of denatured nucleic acids is
that, under appropriate conditions, the reaction can be
reversed, so that two separated complementary strands from
the same source can reform into a double helix. This is
called renaturation.~ Renaturation involves the reaction of
two complementary nucleotide sequences that were separated
by denaturation.~ This technique can also be extended to
allow any complementary nucleotide sequences to anneal with
each other~to form a duplex structure This is generally
re~erred to as hybridization when nucleotide sequences from
different nucleic acid moieties are in~olved; e~g., reaction
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3 S191/98D-106
between single-stranded DNA and RNA, or between t~70 single-
stranded ~NA's from different sources.
Nucleic acid hybridization is a well known method
for identifying specific nucleic acids. This ability of two
single-stranded nucleic acid preparations to hybridize forms
the basis of current nucleic acid assaysO The principle of
such assays is to expose two single-stranded nucleic acid
preparations to each other and then to measure the amount of
double-stranded material that is formed.
Hybridization assays usually involve the use of
polynucleotide hybridization probes. A probe generally
comprises a single-stranded fragment of a nucleic acid, or a
double-stranded fragment denatured. It is characterized by
having a specific nucleotide sequence which is complementary
to a corresponding nucleotide sequ~nce on the target nucleic
acid (the nucleic acid to be determined). The probe and the
target polynucleotide (rendered single-stranded), when brought
together, form duplex molecules by base pairing of the
complementary sequences. The probe is generally prepared in
purified reagent form, and a readily detectable label can be
incorporated into the molecular structure of the probe. The
pre~ence of the target polynucleotide can thus be confirmed by
the formation of duplex hybrid molecules carrying the label.
Polynucleotide hybridization probes offer
inexpensive, efficient, and rapid means for detecting,
localizing, and isolating "target'l nucleotide sequences.
KIausner et al., Biotechnoloqf~, August 1983:471~478, provide
interesting background on polynucleotide hybridization
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4 5191/98D-106
probes, including a discussion of their preparation and use.
Known methods for preparing polynucleotide hybridization
probes and for using such probes are well documented in the
literature. See, for example, Southern, J. Mol. Biol.
98:503-517 (1975); Falkow et al., U.S. Patent No. ~,385,535;
Leary et al~, Proc. Natl. Acad. Sci. 80:4045-4049 (1983);
Langer-Safer et al., ~ 79~4381-4385
(1982); and Lanqer et al., Proc. Natl. Acad. Sci~ 78:6633-
6637 (1981). As disclosed by these and other references,
such known methods for preparing probes typically comprise
cloninq a probe region into a double stranded DNA plasmid.
The plasmid carrying the probe region is labeled, typically
by enzymatic polymerization techniques. Such techniques
include, for example, nick translation (Rigby et al., J.
Mol. Biol. 113:237 (1977)); gap-filling (Bourguignon et alO,
J. Virol. 20:290 (1976)); and terminal addition, which
techniques are carried out in the presence of modified
nucleotide triphosphates. The probe nucleic acid can also
be labeled by chemical means with haptens or biotin (Tchen
et al, P.N.A S. 81:3466 (1984), Forster et al., Nucl. Acids
Res. 13:745 (1985), Viscidi et al., J. Clin. Micr~biol.
23~311 (1986).
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There are two principal ways of performin~
hybridization assays: solution (or liquid) hybridization,
and solid carrier hybridization. In solution hybridization,
sinqle-stranded nucleic acid preparations are mixed toqether
;in solution. For larger amounts of materiall the reaction
can be followed by the change in optical density. When
smaller amounts of material are involved, tne probe may
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5191/98D~106
carry 2 readily detectable label, such as a radioactive
label. The unreac~ed single strands are separated from the
double-strands~ and the double-stranded nucleic acid can be
determined by detecting the presence of the label in the
double stranded material.
Hydroxyapatite (HA) has been used as a standard
method for separating hybridized probe from non-hybridized
probe in solution. Vnder the proper conditions, HA can
selectively bind hybridized DNA probe, but does not bind
non-hybridized probe. Other methods are also available for
separating hybridized probe from non-hybridized probe. One
such method involves the use of a specific enzyme, e.g. S
nuclease, to selectively degrade non-hybridized probe to
smaller fragments. The double-stranded molecules are not
affected by the enzyme. The degraded probe can then be
separated by well known size separ~tion techniques, such as
precipitation with polyethylene qlycol, chromatography,
electrophoresis, and ultracentrifugation, etc. Britten et
al, Methods in enzy ___q~, XXIX, page 363, Eds., Grossman &
Moldave, Academic Press, New York, 1974.
Solution hybridization has the advantages of speed
and reaction efficiency. However, it also has serious
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drawbacks. The steps for separating hybridized from non-
hy~ridized probe are generally labor intensive, time
consuming, do not~lend themselves to automation, and may
othe~rwise be limiting. For example, the size separation
techniques described above are relatively non-specific,
inefficient, and show poor reproducibility. Although the HA
separation method is more specific, it is generally useful
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6 5191/98D-106
only if the target polynucleotide is present in larye excess
over the probe, or if the target is RNA, or both. Further,
with unpurified samples, e.g. plant and animal tissue
homogenates, blood, feces, nasal and urethral mucous, etc.,
effective separation can be very difficult.
In solid-carrier hybridization, one of the single-
stranded nucleic acid preparations is immobilized by being
affixed on a solid-carrier. Numerous methods exist for
coupling either terminal end of a polynucleotide to a given
support. See, e.g. Weissback, A. and Poonian, M.,
Methods in Enzymoloqy, vol. XXXIV, Part s, 463-475, 1974.
Also, derivative forms of polynucleotides, for example, one
carrying a terminal aminohexyl nucleotide, can be easily
attached on a variety of supports. Mosbach, K., et zl.,
Methods in ~nzvmolocy, Vol. XLIV, 859 886, 1976. Oligoribo-
nucleotides may be immobilized on b~oronate derivatives of
various supports. Schott, H., et al., ~, 12,
932, 1973.
As an example, nitrocellulose adsorbs single-
stranded DNA, but not RNA. Moreover, further adsorption of
DNA on the nitrocellulose can be prevented by well known
; treatments. Then if a second denatured DNA/ or ~r~A,
preparation i~ added, it will become affixed to the solid-
carrier only if it is able to base pair with the DNA that
was originally adsorbed. Usually, the nucleic acid
preparation which is not originally bound to the solid-
carrier is labeled. After the hybridization reaction is
completed, the solid carrier is separated from the reaction
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1 309932
7 5191/98D-106
mixture, and the degree of hybridization can be determined
by measuring the label affixed to the solid-carrier.
A refinement of solid-carrier hybridization is
sandwich hybridization, such as that discussed in Ranki,
U.S. Patent No. 4,486,539. The sample is subjected to
conditions which render the target polynucleotide (the nucleic
acid to be determined) single-stranded. The sample is then
mixed with two purified nucleic acid reagents. The first
nucleic acid reagent comprises a sinqle stranded fraqment of
nucleic acid, having a nucleotide sequence of at lea~t 10
bases, and being afixed to a solid-carrier. The second
nucleic acid reagent comprises a single stranded fraqment of
nucleic acid, having A nucleotide sequence of at least 10
bases, and being labeled with a radioisotope. The nucleic
acid reagents are capable of forming hybrid molecules by
complementary base pairing with the tarqet polynucleotideO
The two nucleic acid reagents are not capable of hybridizing
with each other. The solid carrier i~ then washed to
substantially remove the label which is not incorporated in
the hybrid molecules. The presence of the target nucleic
acid is then determined by measuring the label on the washed
solid carrier. Sandwich hybridization also provides
improved~specificity over methods using a single nucleic
acid reaqent, because it involves two specific hybridization
processes.
An advantage of solid-carrier hybridization is the
ease with which the hybrid molecule formed from the target
nucleic~acld and the probe~s) can be separated from the
reaction mix~ure. However, prior art solid-carrier
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8 5191/98~-106
hybridization techniques, includin9 the sandwich
hybridization procedure of Ranki discussed above, suffer a
serious drawback. Kinetics dictate much slower reaction
rates in solid-carrier hybridization than in solution
hybridization, as not all the hybridization components are
allowed to diffuse. A reaction that takes minutes in
solution can take hours, even days, to complete when a
solid carrier is involved. ~oreover, the hybridization
efficiency is also much lower, since some nucleic acids are
unavailable for base pairing. Further, the deqree of
preparation required is substantial. Usually several hours
are required to prepare the sample for hybridization, and
one to two hours are required for washing. Relatively large
amounts of probes are also required.
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It is desirable to have a method for assaying
nucleic acid which combines the ad~antages of both solution
hybridization and solid carrier hybridization.
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Genetic disease diagnosis, in the present state of
the art, generally involves time consuming and labor
intensive techniques. A currently accepted diagnostic
method for the determination of genetic disease is a
procedure known as restriction fragment length
poiymorphization (RFLP). RFLP technology qenerally employs
a somewhat tedious separation step based on the size of the
genetic pieces produced pursuant to treat~ent of sample DNA
with a restriction enzyme. The specialized case of sickle-
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cell anemia presents the most easily understood variation of
RFLP technology7~ For example, The New Enqland Journal
~ of Medicine, ~uly, 1982: pp. 30-32, and pp~ 32-36 contains
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1 309932 5191J9~3D-106
two articles which illustrate how the current prenatal test
for sickle-cell anemia is performed. Generally the test
sample DNA is treated with a restriction enzyme (Mst II)
which produces two different sized pieces o~ the qlobin gene
for the normal and sickle alleles. The pieces are separated
by size, for example on an agarose electrophoresis qel. The
pieces are then rendered single-stranded, transferred to a
sheet of filter paper, and the exact pieces determined in
the morass of similarly-sized pieces by a hybridization
probe. Both the gel electrophoresis and transfer steps of
the test require the use of highly skilled personnel, and
are time consuming.
What is needed is a simplified method for diagnosing
the presence of gene mutation due to genetic diseases.
SUMMARY
The present invention satisfies the above needs.
Specifically, it covers a method for detecting, either
qualitatively or quantitatively, a single-stranded target
polynucleotide in a liquid sample. The method comprises the
step~ of:
(a) combining the sample with at least two
different probes, being a first probe and a second probe, to
form a reaction mixture, each probe comprising a single-
; stranded polynucleotide which contains a nucleotide sequence
complementary to a portion of the target polynucleotide, the
probes together forming hybrid molecules by complementary
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1 309932
5191/98D-106
base pairing with the target polynucleotide, neither probe
being affixed to a solid carrier, the probes do not
hybridize with each other, and the second probe havinq a
detectable label;
(b) subsequently contacting the reac~ion mixture
with a solid carrier which binds the first probe but not the
second probe, and not the target polynucleotide; and
(c) subsequently determining if any of the label
is bound on the solid carrier.
The sin~le-stranded target polynucleotide can be
formed by denaturing a double-stranded polynucleotide.
Preferably the two probes bind different portions on the
target polynucleotide to be determined, so that the two
probes do not compete. Preferably the two different
portions are adjacent or close by.
The ~ethod of this invent-~on is suitable for
microbial diagnostics. It is also suitable for genetic
disease diagnosis such as that for sickle cell anemi-a, and
for cancer dia~nosis, amonq other uses. In this latter
aspect, the method of the present invention incorporates a
severing step, such as by the use of a restriction enzyme,
to~ether with a sandwich hybridization procedure. For
exa~ple, the method can be used to detect a target
polynucleotide in a sample which may also contain a standard
polynucleotide, the nucleotide sequences of the two
polynucleotide being substantially similar, the standard
polynucleotide having a portion A and a portion B, the
target polynucleotide having a portion A' and a portion B',
the standard and target polynucleotides differing in at
least one nucleotide~which is located between portions A and
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11- 5191J98D--1~56
B on the standard polynucleotide and between positions A'
and B' on the target polynucleotide, the method compri~ins
the steps of:
- (a) severing both the standard and target
polynucleotides present into single-stranded segments, such
that none of the segments of the standard polynucleotide
contains both portions A and B, while at least one of the
segments of the target polynucleotide contains both portions
A' and B';
i (b) combining the treated sample from step
(a) with at least t-~o probes, being a first probe and a
second probe, to form a reaction mixture, each probe
comprising a-single-stranded polynucleotide, the probes
being unable to hybridize with each other, the first probe
complementary base pairing with portion ~, and with portion
A', the second probe complementary base pairing with portion
BJand with portion B', the two probes together forming
: hybrid ~olecules by complementary base pairing with the
single-stranded segment of the target polynucleotide which
contains both portions A' and B'; and
(c) subsequently determining the presence of
hybrid molecules containing both of the probes.
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Preferably neither probe is affixed to a solid
carrier, the second probe ha~ a detectable label, the step
for determining the persence of hybrid molecules containing
both of the probes further comprising the steps of:
(a) contacting the reaction mixture with a
solid carrier which~binds the first probe but not the second
probe,~and~not segments containing only portion B or only
portion Bi; and
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121 3 Q 9 9 ~ ~ 5191/98D-106
~ b) subsequently determining if any of the
label is bound on the solid carrier.
,
DRAWINGS
These and other features, aspects, and advantaqes
of the present invention will become better understood with
reference to the followinq description, appended claims, and
accompanyinq drawings where:
Fig. 1 is a schematic representation of the steps
of the assay ~ethod o~f the present invention.
Fig. 2 is a schematic representation of a sickle
cell assay using the ~method of the present invention.
Figs. 3 and 4 are plots o f the relative signal of
label detection; vs ~the target DNA concentration.
Fig. 5~is a plot ~of the binding characteristics of~
an avidin-cellulose;solid carrier and a biotin-labelled
pr~obe. ~ ~
DESCRIPTION
A~meth~od and kit~inc1uding features of the present
invention can be used~to detec~t the presence of
polynucleotide~(such~as~DNA or~RNA) containing organisms,
such a~ ~v~ru es~, b-~ ~er~ nq~, yeasts, other-
13 t 3 0 9 9 3 2 5191/98D-106
microorganisms, and other infectious agents. The ~ethod and
the kit can be used, for example, in food hygiene
investigation~, me~ical diagnostic applicationsl and any
microbial diagnostics. Suitable samples include animal and
plant tissue homogenates, blood, serum, feces, nasal and
urethral mucous, water, dust, soil, etc. Solid samples are
first slurried or homogenized in a liquid medium to form the
test sample. The method is also suitable for diagnosing
genetic disease where normal genes have been caused to
mutate. The method is also suitable for cancer diagnosis~
In the method of the present invention, a sample
containing cells suspected to contain the polynucleotide to
be detected (target polynucleotide) is pretreated, as
necessary, to release the target polynucleotide from the
cells of the organism into solution, or to render the cell
wall permeable to the reagents use~ for detecting the target
polynucleotide. The tarqet polynucleotide is ususally a DNA
or a RNA, or derivatives or fragments thereof.
Hybridization takes place between single stranded
poIynucleotides. Thus if the target polunucleotide is
double stranded, the test sample is then subjected to
conditions capable of denaturing the target polynucleotide
present, e.g., heat, or heat plus high pH, such as 100 C
for 5 ~in., or treatment with 0.5 molar NaOH for 5 min. at
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20-40C. Conditions for the denaturation of polynucleotides
are well known.
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The method of the present invention, as illusrated
in Fig. 1, comprises a soIution sandwich hybridi~ation step
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and a harvesting step. In the solution sandwich
hybridization step, the test sample is combined with at
least two different specific nucleotide hybridization probes
to form a reaction mixture. For example, two probes can be
used, being a first probe - a binding probe, and a second
probe - an identification probe. Each probe comprises a
single-stranded polynucleotide which contains a nucleotide
sequence complementary to a portion of the single-~tranded
target polynucleotide. Thus each probe is capable of
complementary base pairing with the corresponding nucleotide
sequence on the target polynucleotide. Preferrably the two
probes bind different portions on the target polynucleotide,
so that the two probes do not compete; preferably the
portions are closely spaced apart (no more than about 300
nucleotides apart~, more preferably the portions are
immediately adjacent to each other (no ~ore than about 10
nucleotides apart). The probes are~incapable of hybridizing
with each other. In the reaction mixture, the two probes
and the sin~le-stranded target polynucleotide together
hybridize to form complex double-stranded hybrid molecules.
o ensure fast and efficient hybridization, neither probe is
af~ixed to a solid carrier. The second probe, the
identification probe, is further characterized by having a
readily detectable labelO The procedure is called sandwich
hybridization because the target polynucleotide is
"sandwiched" between the two probes in the resulting hybrid
molecules.
The probes can be contained in separate reagents.
Because the probes do not hybridize with each other, it is
'also~possible to have all the~probes in a single reaqent.
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1 3 0 9 9 3 2 5191/98D-106
The order of combining the probes with the test sample is
generally not important. The probes can be added to the
test sample in seriatum (one after another), all at the same
time, or in any other combinationO
The reaction mixture is maintained at conditions
conducive to hybridization. The conditions depend on, and
are generally known for, the particular polynucleotide
moieties involved. The hybridization is allowed to go to
substantial completion. For most polynucleotides this takes
no more than about one hour. For example/ at a salt
concentration of about 0.15 molar, a temperature of 65C,
and a plasmid probe concentration at 1.0 microgm/ml, the
time to reach 1/2 completion is less than 20 minutes.
In the harvesting step, the reaction mi~ture is
contacted with a solid carrier capable of binding the first
probe (the binding probe), but not the second probe (the
identification probe), and not the target polynucleotide.
The terms "bind" and "binding" herein shall refer to strong
binding via specific functional groups, in contrast to low
level non-specific binding. The hybrid molecules formed
from the binding of the target polynucleotide to the two
probes are thus bound on to the solid carrier via the
binding probe.
The second probe is chosen such that there is
little non-specific binding of the second probe itself on to
the solid carrier. The presence of the target
polynucleotide in the~test sample is thus confirmed by
~ ~ ~ determlning if any of the label is bound on the solid
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16 1 309932 5191/98D--I06
carrier. Quantitative determination of the amount of the
target polynucleotide in the test sample is also possible,
by measuring the actual amount of label bound on the solid
carrier.
There are at least two basic methods for
determining if any of the label is bound on the solid
carrier, both measurinq the distribution of the second probe
in the solid and lquid phases of the reaction mixture after
the harvesting step.- In the first method, the excess
identification probe not incorporated in the hybrid
molecules is removed by conventional methods such as
washing, aspiration, decantation, etc. The amount of label
bound on the solid carrier is then measured. The presence
of the label on the solid carrier indicates the presence of
the target polynucleo~ide in the ~ample. In the second
method, the solid carrier can be se~arated from the liquid
phase of the reaction mixture, or it can remain mixed with
the~liquid phase. The amount of unbound second probe in
solution in the liquid phase is measured and is compared to
the total amount of second probe added to the reaction
mixture~ A difference in the two amount~ indicates that
some of the second probe is;bound on to the solid carrier,
whi~ch ~in turn indicates~the presence of the target
polynucleotide in the sampIe~
Preferably the first probe (binding probe)
comprises a first binding group and the solid carrier
comprises a second binding qroup, the two bindinq groups
bindinq each other by forming strong bonds with each other
rapidly. The two binding groups together constitute a
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17 1 309~32 Slgl/98D--106
binding pair. One of the binding groups of the binding pair
is linked to the base material of the solid carrier, and the
other binding qroup is part of the first probe. The binding
pair can, for example, be any one of the following pairs:
avidin-biotin, hapten-antibodies, antibodies-antigens,
carbohydrates-lectins, riboflavin~riboflavin binding
protein, metal ions-metal ion binding substances, enzyme-
substrate, boronates-cis dioles, staph A proteins-
antobodies, enzymes-inhibitors, etc. The binding pairs also
include pairs of derivatives of the above moieties. The
criteria for choosing the particular binding pairs include
(1) the speed with which the binding groups of the pair
react to bind each other, (2) the strength of the bond
between ~ha binding groups of the pair, (3) minimal
interference of the binding group on the first probe with
the solution sandwich hybridization step, (4) ease with
which the binding groups can be linked to the base material
of the solid carrier, and be used to form the first probe.
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The base material of the solid carrier can be of a
material selected from, for example, agarose, cellulose,
glass, latex, polyacrylamide, polycarbonate, polyamide (e.g.
~ylon TM)~ polyethylene, polypropylene, polystyrene, silica
qelj silica, and derivative thereof. This list is by no
means exhaustive. Any solid on which one of the binding
groups of the binding pair can be affixed is suitable.
The solid carrier can be in various forms. For
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example, it can be in the form of micro-particulates with
particle sizes less than 100 micron~ (for example,
~ mirocrystaline cellulose), macro particulates with particle
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1 309932
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sizes of 0.1 to 2.0 mm, sheets, tubes, pipet tips, plates,
filters and beads, etc.
Methods for affixing the second binding group to
the solid carrier are well known~ Methods for derivatizing
the polynucleotide moiety of the first probe to contain the
first binding group are also also known. (Tchen et al.,
P.N.A.S. 81:3466 (1084), ~orster et al., Nucl. Acids Res.
13:745 (1985), Viscidi et al., J. Clin. Micr~biol. 23:311
(1986). Biotechnology, August 1983:471-478, J. Mol. Biol.
98:503-517 (1975); Falkow et al., U.S. Patent No. 4,385,535;
L~eary et al., ~r~. N-.l Ac~- 3~i 80:4045-4049 (1983);
Langer-Safer et alO, Pr~ tl. ~:a~. s~l 79:4381-4385
~(1982): and Lanqer et al., Proc. NatlO Acad~ sci. 78:6633
6637 (1981), Rigby et al, J. Mol. Biol. 113:237 1977),
Bourquiqnon et al., J. Virol. 20:290 (1976).
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;~- The second probe (the identification probe)
contains a readily detectable label. Various ~ethods for
labeling specific polynucleotide probes are Xnown. Any label
capable of being readily detected and which does not unduly
interfere with the solution hybridization step, can be used.
Suitabla labels include radioisotopes, light-labels,
enzymes, en~yme cofactors~ haptensr antibodies, avidin,
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biotin, carbohyd~rates, lectins, metal chelators, etc., and
their derivatives. ~Detection can be direct, as with
radioisotopes, or indirect, as with a hapten followed by an
enzyme-labeled antibody.
Radioisotopes such~as 32p, 125I, etc., can be used
to~label the probe. The radio-labeIs can be detected by
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19 1 3 0 9 9 3 2 Sl91/98D-106
well known me~hods such as gamma counting or scintillation
counting. However, the use of radioisotope labels can be
expensive and hazardous. Detection of radioactivity
generally requires expensive equipment. Special training
for personnel and Yafety precautions are required for
handling radioactive material. Moreover, radioisotopes have
finite half-lifes, and thus the labeled polynucleotide probe
usually has a relatively short shelf life (usually in the
order of weeks).
For the above reasons, other label systems were
developed, such as light-labels and enzyme-action based
labels. As discussed in Heller, et al., European Patent
Application No. 82303701.5, light-labels can be
chemiluminescent, bioluminescent, fluorescent, or
phosphorescent, and under the proper conditions can provide
sensitivities comparable to that of~radioisotopes. The
light-label can be attached to any point on the single-
stranded polynucleotide segments of the probe; however,
; termlna~l positions are known to be more desirable.
Some examples of light-labels are as follows: (1)
chemiluminescent: peroxidase and functionalized iron
porphyrin derivativesj (2) bioluminescent: bacterial
luciferase, firefly luciferase, flavin mononucleotide (FMN),
; adenosine ~triphosphate~(ATP), reduced nicotinamide adenine
m~ ~ dinucleotide ~N~DH), reduced nicotinamide adenine
dinucleotide phosphate (NADPH), and various long chain
; aldehydes (decyl~aldehyde, etc.); (3) fluorescent:
luorescent nucleotides such as adenosine nucleotides,
etheno-cytidine nucleotides, etc., or functionalized
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2~ 1 3 0 9 9 3 2 5191/98D-106
nucleotide~ (amino-hexane adenosine nucleotides,
murcurinucleotides, etc.), which can first be fluorescently
labelled, and then covalently attached to the single-
stranded polynucleotide segment of the probe; (4)
phosphorescent: 2-diketones such as 2,3 butadione, 1-
[carboxyphenyl]-1,2-pentanedione, and 1-phenyl-1,2-
propanedione. Means for attaching the light labels to the
probe are well documented in the art. The label is measured
by exciting the label and then measurinq the light response
with photo-detection devices. Fluorescent or phosphorescent
labels can be excited by irradiation with light of the
appropriate wavelength. Chemiluminescent or bioluminescent
labels can be chemically excited, by methods well known in
the art~
The identification probe can also be labeled with
an enzyme. The hybridization product is detected by the
action of the enzyme on a substrate for the enzyme. For
example, an enzyme capable of acting on a chromogenic
substrate can be selected. The conversion ratio of the
substrate can be monitored by optical analysisO The ratio
is then correlated with the presence or absence of the
target polynucleotide~ It is well known that the avidin and
biotin can be used to link an enzyme to a specific nucleic
acid probe. Examples of suitable enzymes include, for
example, beta-galactosidase, alkaline phosphatase,
horseradish peroxidase, and luciferase.
; One of the major advantages of the light-labels
and enzyme action based labels is that their qualitative
detection can be visualized without the need of expensive
:: :
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21 7 3 0 9 9 3 2 5lgl/98D-l06
detection equipment. Of course, quantitative determinations
of such labels can also be made with the use of photometric
equip~ent.
It is preferable that the first and second probes
each be complementary to substantially mutually exclusive
portions of the target polynucleotide. In other words, the
first and second probe~ should not compete for the same base
sequence to the extent that sand~ich hybridization is
prevented.
The probes can be made from appropriate
restriction endonuclease treated polynucleotide from the
organism of interest, or from double-stranded
polynucleotides by enzymatic methods such as Exo III
diqestion or RNA polymerase transcription. In these cases
the probes are RNA or DNA fragments.~ In other cases where
the base sequence of a unique portion is known, the probes
can be synthesized by orqanic synthetic techniques
(Stawinski, J. et al., Nuc. Acids Res. 4, 353, 1977; Gough,
G. R. et al., Nuc. Acids Res. 6, 1557, 1979; Gough, G. R. et
al., Nuc. Acids Res. 7, 1955, 1979; Narang, S. A., Methods
in Enzvmoloay, ~ol. 65, Part I, 610-620, 1980). Also, it is
possible to produce oligodeoxyribuonucleotides of defined
sequence using polynucleotide phosphorylase (~. Coli) under
proper conditions ~Gillam, S., and Smith, M., ethods_in
ol. 65, Part I, pp. 687-701, 1980). It is also
possible to produce large quantities of the probe by
; cloning; e.gO by recombining the particular sequence into a
plasmid or~ M13 bacteriophage vector, and then cloning the
' ::
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::
:. . '. .' : . . '
~` 1 309932
'~19 ~ ~ ar~_ ? ~.
reco~bined moiety using standard methods. The cloning
method i~ preferred.
The size of the probes can be from 10 nucleotides
to 100,000 nucleotides ln length. Below 10 nucleotides,
hybridized systems are not stable and will begin to denature
above 20 degrees C. A complementary polynucleotide sequence
of 12 is about the minimum length required for appropriate
binding specificity. The generally used minimum in practice
is about 15. Above 100i000 nucleotides, hybridization
(renaturation~ becomes a much slower and incomplete process,
see Molecular GenetiCs, Stent, G. S. and Ro Calender, pp.
213-219, 1971. It is not necessary that the entire probe be
complementary to the target polynucleotide on a base by base
scale. Preferably the probes should be from about 15 to
about 50,000 nucleotides long, more preferably from about 15
to about 10,000 nucleotides long. ~he number of
complementary (base pairing) nucleotides on the probe is
;~ preferably between about 200 to abou~ 5,000. Preferably the
j ~ complementary nucleotides are adjacent to each other,
~;~ especially when the number of complementary nucleotides is
low (below 200). But one-to-one complementation of all of
the base pairing nucleotides on the target and the probe is
not necessary. Smaller probes ~15-100) lend themselves to
production by automated organic synthetic techniques.
Probes sized from 100-10,000 nucleotides can be obtained
from appropriate enzymatic methods, or by recombinant DNA
methods.
~:
The labelin~ of smaller polynucleotide segments
with the relatively bulky labeling moieties (e.g.
~ ::
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23 1 ~ O )~5191/98D-106
chemiluminescent label~) may in some cases interfere with
the hybridization process. Therefore the proper choice of
labels i5 important. Some of the criteria for choosing the
labels are: ease of incorporating the label into the
polynucleotide without inhibiting hybridization, ease and
sensitivity o~ detection of the label, low nonspecific
binding of the labeled probe to the solid carrier, and hiqh
stability.
The proper hybridization conditions in the
solution hybridization step are determined by the nature of
the first binding group on the first probe (the binding
probe), and of the label attached to the second probe (the
identification probe), the size of the two probes, the [G] -
~[C] (guanine plus cytosine) content of the probes and the
complementary nucleotide sequences on the target
polynucleotide, and how the test sample is prepared. The
label can affect the temperature and salt concentration used
for carrying out the hybridization reaction. For example,
chemiluminescent catalysts can be sensitive to temperatures
and salt concentrations that absorber/emitter moieties can
tolerate. The size of the probes affect the temperature and
time for the hybridization reaction. Assuming similar salt
and reagent concentrations, hybridizations involving reagent
polynucleotide sequences in the range o 10l000 to 100,000
nucleotides may require from 40 to 80 minutes to occur at 67
degrees C, while hybridizations involving 14 to 100
nucleotides require from 5 to 30 minutes at 25 degrees C.
Similarly, sequences with hi~h [G~ + [C] content will
hybridize at higher temperatures than polynucleotide
sequences with a low [G] + [C] content. Finally, conditions
~: :
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1 30q~32
24 5191/98D-106
used to prepare the test sample and to maintain the tarqet
polynucleotide in the single-stranded form can affect the
temperature, time, and salt concen~ration used in the
hybridization reaction. The conditions for preparing the
test sample are affected by the polynucleotide length
required and the [G] + [C] content. In general, the longer
the sequence or the higher the [G~ f [C] content, the higher
the temperature and/or lower the salt concentration required
for denaturation~ The concen~ration of probe or target in
the mixture also determines the time necessary for
hybridization to occur. The higher the probe or target
concentration the shorter the hybridization incubation time
needed. The basic rate of hybridization is also affected by
the type of salt present in the incubation mix, its
concentration, and the temperature of incubation. Sodium
chloride, sodium phoAphate and sodium citrate are the salts
most frequently used for hybridizat~on and the salt
concentration used can be as high as 1.5 - 2M. The salts
mentioned above yield comparable rates of polynucleotide
hybridization when used at the same concentrations and
temperatures, as dv the comparable potassium, lithium,
rubidium, and cesium salts. Britten et al. (1974)
(Methods in Enzymoloqy~ Volu~me_XXIX, part E., ed. Grossman
and Moldave; Academic Press, New York, pa~e 364) and Wetmur
and Davidson (1968) (J. Molecular Bioloqy, Vo_ 31, page
349) present data which illustrates the standard basic rates
of hybridization attained in commonly used salts. The
hybridization rates of~DNA with RNA vary somewhat from those
of DNA hybridizinq with DNA. The magnitude of the variation
is rarely over tenfold and varies, dependin~ for example, on
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1 3 0 ~ 9 3 2 5191/98D-106
whether an excess of DNA or RNA i9 used. See Galau et al.
(1977) ~ , Vol ?4~ ~6, pg. 2306).
There are preferred general conditions which may
not be optimal, but under which hybridization occurs for
nearly all probe and target combinations of 100 nucleotides
or more. These conditions are about 0.75 molar sodi~m
chloride, 0.0?5 molar sodium citrate, 0.025 molar sodium
phosphate (pH 6.5), 65 degrees C, and polynucleotide
concentrations of 0.001 to 1.0 microg./ml. There are also
known methods for achieving similar conditions at lower
temperatures, for example by including a denaturant such as
formamide in the reaction (Casey et al., Nucl. Acids Res. 4:
1539 (1977)). Addition of other reagents in the
hybridization reaction may also increase the reaction rate,
for example dextran sulfate (Wahl et al., P.N.A.S. 76:3683
(1979), polyethylene glycol (Amasi~o, Anal. ~iochem. 152:304
(1986)) and phenol (Kohne et al., ~ 1605329
(1977)).
A kit suitable for use to detec~ a single stranded
tarqet polynucleotide in a test sample by the assay method
of the present invention can comprise a liquid
:;
polynucleotide reagent or reagents comprising at least two
different probes, being a first probe - a binding probe,
and a second probe~- an identification probe. Each probe
comprises a sinqle-stranded polynucleotide which contains a
nucleotide sequence complementary to a portion of the target
;polynucleotide. Neither probe is affixed to a solid
carrier. The probes cannot hybridize with each other. The
second probe has~a detectable label. The ~it also comprises
:
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i:
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1 3 0 9 9 3 2 5191/98D-106
a solid carrier capable of binding the first probe, but not
the second probe and not the target polynucleotide. The
other characteristics of the probes and of the solid carrier
are generally as described above in the description of the
method of the present invention. The kit can also further
comprise means for qualitative and/or quantitative
determination of the label. There can be more than one
probe in each polynucleotide reagentO For example, because
the probes do not hybridize with each other, they can
conveniently be combined in a single reagent.
It is also preferred that one or more of the
following components be included as part of the kit: (a)
detergents capable of solubilizing the various moieties,
such as sodium dodec~l sulfate, or sodium lauryl
sarcosinate; (b) proteases, such as proteinases K and
pronase; (c) reagents to facilitate the denaturing of the
target polynucleotide, including salts and solvents; (d)
; ~ reagents used in detecting the label on the second probe.
The components of (a), (b), (c3 and the probe reagent can
also be combined into a sisngle reagent or a number of
reagents, as needed.
:~ ,
The above method and kit can be adapted for use for
testing a series of target polynucleotides. A separate set
of probes is provided for each target polynucleotide, each
set being specific for the particular target
polynucleotide. ~ach set of probes comprises at least two
different probes, being the binding and identification
probes (the first and second probes), as characterized
before. ~The solid carrier used has a binding group capable
of binding the binding groups on each of the binding probes
: ~:
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1 309932
27 - 5191/98D-106
specific for the plurality of target polynucleotides, but
not any of the identification probes, and not any of the
target polynucleotides. Therefore a single universal solid
carrier can be used with all of the different binding
probes~ In the k~it, preferably each set of probes is
contained in a separate liquid reagent.
The above method and kit can also be adapted for
simultaneous testinq of several target polynucleotides in a
test sample. A separate set of probes i5 provided for each
target polynucleotide, each set being specific for the
corresponding polynucleotide. All the probes are incapable
of hybridizing with each other. Each set of probes consists
of at least two different probes, being a first probe (a
binding probe) and a second probe (an identification probe),
the probes being as characterized previously~ All the first
probes carry an identical binding group, so that the first
probes can all be harvested on the same solid carrier. The
solid carrier does not bind any of the indentification
probes, and nct any of the tarqet polynucleotides. ~he
second probes comprise different labels, each label
corresponding to a particular target polynucleotide. After
the harvestinq step, detection of the respective labels on
the solid carrier yields information about the presence
and/or quantities of the correspondinq tarqet polynucleotide
in the sample. In the kit, preferably all of the probes are
contained in a single liquid reagent.
The advantages of the assay method and kit of the
present invention are many. The method gives the reaction
speed and reaction efficiency of solution hybridization, and
~ .
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28 1 3 0 9 9 3 2 5191/98D-106
the eaqe o~ separation of the hybridization products from
the test ~ample of solid carrier hybridization. Moreover,
the assay method is highly specific, as it requires
complementation of at least two probes with the target
polynucleotide. The solution sandwich hybridization step
can be completed generally in less than an hour, and the
harvesting step takes no longer than about 5 minutes
(optimally less than about 0.5 minutes). ~hus substantial
time savings can be obtained.
Further, the kit of the present invention is
simple to use. The user need only stock one single solid
carrier reagent, which can be used for assaying all target
polynucleotides. Only one liquid reagent is required for each
target polynucleotide. A third reagent for detecting the
label may be required in some applications.
Another significant advantage of the method of the
subject invention is that no extensive preparation of the
test samples is necessary. Prior art solution hybridization
methods detect target nucleic acids which have been purified
away from other cell components. Nucleic acids in cells and
viruses are normally tightly complexed with other cell
components, usually protein, and, in this form are not
available for hybridization. Simply breaking the cell or
virus open to release the contents does not necessarily
render the~polynucleotides available for hybridization. The
polynucleotides remain complexed to other cell or viral
components even though released from the cell, and may in
` act become extensi~vely degraded by nucleases which also may
be released. In addition a probe added to such a mix may
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2~ 1 3 0 9 9 3 2 5191/98D-106
become complexed to "sticky" cell or viral components and be
rendered unavailable for hybridization, or the probe may be
degraded by nuclease action. A variety of prior art ~ethods
exist for purifying polynucleotides. These methods are all
time consuming - one taking an hour is regarded as very
rapid - and require multiple manipulationsO Surprisingly, it
was discovered that the assay technique of the subject
invention can be used wi~h unpurified samples, thus
obviating the need for laborious and time-consuming
purification steps~ The assay lends itself readily to
automation which could further simplify the user's task.
The assay results can be either qualitative or quantitative.
' .
Another aspect of the method and test kit of the
subject invention is directed to genetic disease diagnosis
and cancer diagno~is.
Genetic disease is the result of mutations in a
polynucleotide's nucleotide sequence. Such mutation is also
a factor in the development of cancer. Therefore, as a
~consequence or such genetic disease or cancer, a sample
taken from the patient, and containing a normal
polynucleotide (standard polynucleotide), can also contain
an abnormal polynucleotide (target polynucleotide~ which is
a variant of the normal polynucleotide. The sample may also
contain only the abnormal polynucleotide. The normal and
abnormal polynucleotides typically have substantially
dentical nucleotide sequences, except at points of mutation
on the polynucleotides. The normal and abnormal
polynucleotides thus react differently to certain reagents.
:~ ;: : :
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1 3 0 9 9 3 2 5191/98D--106
The method of the present invention can be used to
detect such difference between the normal and abnormal
polynucleotides The method combines a severing step with a
solution sandwich hybridization procedure. The severing
step comprises a segmentinq step, and may also comprise a
denaturing step if the target polynucleotide is double
stranded. The order of these two steps is not siqnificant.
Restriction reagents, e.g., restriction enzymeY,
are known. A restriction enzyme can, under the proper
conditions, be used in a segmenting ~tep to divide a
polynucleotide at very specific sites on the nucleotide
backbone. The standard and target polynucleotides are
substantially similar. The standard polynucleotide has a
portion ~ and a portion B. The target polynucleotide has a
portion A' and a portion B'. The tw~o polynucleotides differ
by at least one nucleotide which is located between the
polynucleotides' respective portions A and B, and A' and B'.
The restriction reagent is chosen such that it segments the
standard polynucleotide into segments none of which contains
both~portions A and B; the~ restriction reagent segments the - -
ta`rget polynucleotide into segments at least one of which
contains both portions A' and B'. In the typical case,
w~here the tarqet polynucleotide is a genetic variant of the
standard~polynucleotide, portlons A and A', and portions B
and B'j are ide~ntical.
The target~ and standard polynucleotides presen~,
or their segments (from the segment step), are denatured,
i.e.; rendered single-stranded, l~f necessary, either before
. - . : , :
1 3 0 9 9 3 2 5191/98D--106
or after the segmenting step. The treated sample thus
contains single-stranded versions of the target and/or
standard polynucleotides present, ready for hybridization.
The treated sample is then combined with at least two probes
to form a reaction mixture. The two probes are a ~irst
probe and a second probe. Each probe comprises a single-
stranded polynucleotide. The probes do not hybridize with
each other. The firs~ probe complementary base pairs with
portion A on the standard polynucleotide, and wi~h portion
A' on the ~arget polynucleotide. The second probe
complementary base pairs with portion B on the standard
polynucleotide, and with portion B' on the tarqet
polynucleotide. The two probes together form hybrid
molecules by base pairing with the single-stranded
segment(s) of the target polynucleotide which contains both
portions A' and B'. With respect to the standard
polynucleotide, because portion A and portion B are on
separate segments after sequentation, no hybrid molecules
incorporating both the first and second probes will-form.
The probes are attached to separate segments. Hybrid
molecules havin~ both the first and second probes will form
only if the abnormal (target) polynucleotide is present in
the sample.
:: :
The presence in the reaction mixture of hybrid
molecules incorporating both of the probes would indicate
~; the presence of the target polynucleotide in the sample.
The determination of the hybrid molecule~ formed by this
sandwich hybridization procedure can be performed using
previously known methods. For example, a system similar to
that disclosed in Ranki, U.S. Patent No. 4,486,533, wherein
1 309~32 Sl91/98D-106
one of the probes is already immobilized on a solid carrier,
can be used. That is, not all of the probes ne~d to be in
solution. Other methods are also known. Alternatively,
solu~ion sandwich hybridization, followed by a harvesting
step, as previously discussed can also be used. In the
latter case, the further limitations on the method of this
invention asdescribed above are that: neither probe is
affixed to a solid carrier, and the second probe has a
detectable label. The reaction mixture is contac~ed with a
solid carrier which binds the first probe, but not the
second probe, and not any of the segments containing only
portion B or only portion B'. A subsequent determination
is made to see if any of the label is bound on the solid
carrier, as previously discussed.
For example, the followinq method is suitable for
~;~ sickle cell assays. A test sample~suspected to contain the
mutated gene DNA strands (e.g. in sickle alleles) is traated
with a restriction enzyme, e.g. Mst II. Such enzymes have
been used in qene mapping, in order to detect structural
gene deletions in the DNA strands. Direct identification of
~- mutant qenes in DNA, e.g. hemoglobinopathies due to point
mutation in the DNA, was also possible by virtue of the
specificity of such restriction enzymes. A single
nucleotide change in an enzymeis cleavage site can readily
be detected if the appropriate enzyme is used. It is known
that the restriction enzyme Mst II cleaves DNA on the
average to produce fragments of 1.1 and 1.3 kb from ~ N and
5 genes, respectively. Mst II cleaves a sequence
(CCTNAGG) that is a subset of Ddel sites (CTNAG). The DNA
from the sickle cell gene has a nucleotide variation at-the
' :: : :
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33 1 3 0 9 9 3 2 5l9l~9aD-l06
Mst II site. Therefore, the mutated DNA strand will not be
cleaved a~ that site by the Mst II enzyme. The restriction
en~yme treated test sample is then subject to conditions
which renders the polynucleotides single stranded. A single
target polynucleotide-single sample hybridization assay
procedure as previously described for detecting the presence
of a target polynucleotide would then be carried out, with
the additional limitation that the first and second probes
bind sites on the DNA strand which are on opposite sides of
the Mst II site involved in the mutation. The only way that
a complex hybrid molecule, comprising the target
polynucleotide and both of the probes, will form, is if the
qene is a mutated one - e.g. a sickle cell. The normal gene
is cleaved between the binding sites for the two probes.
The schematic of this sickle cell assay is represented in
Fig. 2. This method eliminates the prior art assay qteps of
qel electrophoresis and transfer to solid support (filter
paper). The simplification is very substantial. This assay
procedure would apply equally well to the diagnosis of other
qenetic diseases in which gene mutations are involved.
The cancer diagnostic aspect would take the form
of a quantitative test for the levels of messenger RNA from
an oncogene, although tests similar to the sickle cell
example would also be useful.
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34 i~ 3 ~ ~ 3 2 5191/98D--106
EXAMPLES
EXAMPLE 1
~ his example correlates the harvested signal to
the target DNA concentration.
- ' .
In this example the target polynucleotide i3
mpB1017: probe 1 is pHBC6 having biotin as the first binding
qroup; probe 2 is M13 10W labeled with 32p; the solid
carrier is avidin-cellulose, which binds biotin on the first
probe.
ABBREVIATIONS. SSC is 0~15 molar sodium chloride and 0.015
molar sodium citrate. SSC is used at various
concentrations, '110X SSC" would be, for example, 1.5 molar
sodium ;chlo~ide, and 0.15 molar sodium citrate. EDTA is
ethylenediamine t~etra-acetate, SDS is sodium dodecyl
sul~ate, ~NA is deoxribopolynucleotide, BSA is bovine serum
albumin, tris:HCL is tris-hydroxymethylaminomethane adjusted
to the appropriate pH~with hydrochloric acid.
; ~ M~-YOO~. Plasmid D~As were obtained by growi-ng
transformed E. coli, qtra~in LE39~, followed by lysis of the
cells with lysozyme,~SDS and NaOH. DNA was further purified
by centrifugation in CsCl in the presense of ethidium
bromide See Mauiatis, T~, Fritsch, E. F., and Sambrook, J.,
1982. ~Molec~1ar ~ A _ Manual. Cold Spring
:
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1 309932
5l9l/98D-l06
Harbor Laboratory, Cold Sprinq Harbor, New Yor~, U.S.A.
Replicative form (RF) DNA from M13 bacteriophage, strain 10w
was obtained in an identical fashion by growing infected E.
coli strain 71018~ Single-s~randed virion DNA from M13
clone mpB1017 was obtained from virions in the growth ~edia
of in~ected E. coli 71.18 by precipitation with polyethylene
glycol, centrifugation in CsCl, and extraction of the
purified viru~ with SDS, phenol, and chlorofor~. Salmon
testes DNA was obtained from Sigma Chemical Company, St.
Louis, MO (USA).
Double-stranded DNAs were labeled by nick-
translation (Rigby, et al., J~ Mol. Biol., 113:237, 1977)
essentially a~ described by Leary et al., Proc. Nat'l. Acad.
Sci. [USA] 80:4045, 1983. Plas~lid pHBC6 contains 5,400 base
pairs of the human beta-globin gene, cloned in the plasmid
pBR322 (Fukumaki et al., Cell 28:5~5, 1982~. M13 clone
mpB1017 contains 1310 bases of the human globin gene,
homolo~ous to 1310 bases from the plasmid pHBC6. M13 strain
10w contains 7,500 bases of bacteriophage genes, homoloqous
to the same number of bases in clone mpB1017. The regions
in mp81017 D~A that are homologous to pHBC6 and M13 10w are
not overlapping.
The solid phase for harvesting the solution-
sandwich product was prepared~by activation of
microcrystalline cellulose with N,N'-carbonyldiimidazole,
followed by coupling to the protein avidin, using methods
similar to those of Paul et al., J. Org. Chem. 27:2094,
i ~
1962. The coupling ratio was 1 qram of activated cellulose
to 1 mg of avidin. A 1:1 slurry of the prepared solid phase
,~ ~
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36 1 3 0 9 9 3 2 5191/98D-106
of avidin:cellulos~ contains about 1 gm of cellulose in 6 ml
of buffer.
Solution hybridizations were preformed in a final
solution containing 5X SSC, 0.02% (w/v) each of
polyvinylpyrolidone-40, ficoll-400, and BSA, as well as 25
mM NaP04 buffer, pH 6.5, and 250 microg./ml sonicated salmon
testes DNA. All DNAs tprobe 1, probe 2, target, and carrier
salmon testes) were denatured at 100 degrees C for 3 to 5
minutes prior to starting the hybridization reaction.
Further details of the example assay are given
below.
The following asqay was.done to examine the
feasibility of the aqsay of the present invention, and to
determine the range of target DNA concentration in which the
assay would be effective. Plasmid pHBC6 was labeled with
the ligand biotin by nick-translation with biotin-ll-
deoxyuridine triphosphate (Bethesda Research Laboratories,
Gaithersburg, MD, USA). This constitutes probe 1 of the
assay. The replicative form DNA of bacteriophage M13 10w
was labeled with a radioactive reporter, 32P, by nick-
translation with alpha-32P-deoxycytidine triphosphate
(Amersham, Chicago, IL, USA). This constitutes probe 2 of
the assay. Probe one contained approximately 6% biotin
nucleotides, and probe 2 had specific radioactivities of 0.7
to 7 X 107 cpm/microg. The target DNA was M13 mpB1017, and
samp1es containing a range of amounts of target DNA from 20
.
37 1 3 ot~q 32 5l9l/98D-l06
picograms to 1.0 microgram were tested. A11 assays
contained 100 ng of probe 1 (biotin) and 50 n~ of probe 2
(32p). Probe and sample DNAs were mixed toqether in a
solution of 10 mM tris:HCl, pH7.5, 1.0 mM EDTA, heat
denatured, and adjusted to the solution hybridization
conditions given in the methods above and incubated in a
total volume of 0.2 ml overnight at 68 degrees C. This
incubation time is excessive, and was used only for
convenience and to assure completeness of the reaction.
Control assays contained probe 1 or probe 2 and 20 ng. of
target, or probe 1 & probe 2 without tarqet. The separation
of the product sandwich from unhybridized probes was
accomplished by adding 0.2 ml of a 1:1 slurry of
avidin:cellulose to the reaction~, incubating at 22 degrees
C for 30 min~, and ~ilterinq the cellulose onto a 13 mm
glass fiber filter. One wash of the reaction vessel with
0~1 ml. of tris:HCl, pH 7O5l 1 mM ED~A was also filtered on
the sa~e filter as the primary reaction. The filtered solid
support was further washed 2 times with 2X SSC and 0.1% (v/v~
SDS, 2 times with 0.2X SSC and 0.1~ SDS, and 2 times with
0.1X SSC and 0.1% SDS. The last two washes were performed
at 50 degrees C, all others at room temperature. All washes
were with 0.5 ml of solution. These washes were adapted
from those used for Southern blot hybridization analysis by
~Leary et al~, P.N.A.S. 80:4045 (1983)) The amount of probe
2 (32p) bound to the solid support was determined by liquid
scin~tillation countingj with the resin in 0.5 ml of solution
in a minivial and 4.5 ml of Beckman Ready Solv TM MP
scintillation fluid. All washes were also collected and
counted similarly.
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1 3 0 9 9 3 2 5l9l/98D-l06
.
The results from one run are given in Table
I, where the amount of probe 2 bound is related directly to
the amount of target DNA in the assay. The data represent
the average of duplicate samples.
; ' .
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,
~: :
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' , ' ' '. .,. ~ ' ' '': ''
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-
~ 3D 9 9 ~:~ slgl/98D-l06
TABLE I
Target DNA in Probe 2 bound
assay (nanogm) to cellulose (nanogm)
0.0 0.2
0.021 0 2
0.062 0 16
0.185 ~.2
0.556 - 0.38
1.67 0.72
5~0 1.67
15.0 ~ 2.26
45 0 2.19
Data from several runs demonstrated that the assay as
performed in this example has a peak of harvested signal
(probe 2) at about 30 ng of target DNA, at amount~ above
this level, the harvested signal decreases in a logarithmic
fashion. Below 30 ng of target DNA, the assay is limited by
background bi~ding of probe 2 to the solid support and the
specific radioactivity of the probe, but increases
exponentially from~about 0~5 ng to 10 ng of targetr The
non-linear nature of the signal as a function of the target
amount: ~i9 graphically depicted in Figure 3. Fig. 4 is an
expanded version of Fig. 3 around the peaked signal~ The
non-linear relation~ship may be a function of the nick-
tran~lation Feaction used~to label the probes, since probes
~ ~ .
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labeled in this fashion are known to show some elements of
cooperative binding (Meinkoth and Wahl, Anal. Biochem
138:267, 1984).
EXAMPLE 2
This example demonstrates the binding
characteristics of an avidin-cellulose solid carrier and a
biotin-labeled probe.
The kinetics and specificity of harvesting a
biotin-labeled probe by binding it out of a hybridization
buffer onto avidin-cellulose were examined using DNA's
labeled with both biotin~ dUTP and 3H-dATP or labeled with
H-dATP alone (control). The DN~ was plasmid pHBC6 labeled
by nick-translation (Rigby et al., J. Md. Biol~, 113:237
(1977)).
Avidin-cellulose (0.05 ml packed volume) 3uspended
in 0.2 ml of 10mM Tris:HC1 plus 1.0 mM EDTA (pH 7.5) was
placed in each of 30 microcentrifuge tubes. A mixture (0 2
ml) containing 100 nanogm. of~the biotin-labeled or control
DNA, 50 microgm. of carrier salmon testes D~A, 20 microgm.
of yeast RNA, 0002~ (W/v) each of bovine serum albumin,
ficoll, and polyvinylpryolidone in 0.075 M sodium citrate,
0.75 M sodium chloridel and 0.025 M sodium phosphate (pH
6.5) was added to the appropriate tubes. The tubes
~;containing the solid phase and hybridization mixture were
incubated at room temperature on a rocking platform for
times ranging from 1 min. to 300 min. After the incubation,
the solid phase was collected by filtration on a teflon
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41 5191/98D-106
membrane filter. The fraction of DNA bound was determined
by subtracting the radioactivity in the filtrate and washes
from the total radioactivi~y added to each sample. The data
are presented in Figure 5. Within 30 minutes, approximately
92~ o~ ~he biotin-labeled DNA became bound to the avidin-
cellulose, while an average of le~s than 2% of the control
DNA bound to the solid carrierO The binding is rapid,
efficient, and specific for the biotin label on the probe.
.
The requirement for the avidin component of the solid
phase for binding to biotin-labeled DNA was examined using the
same DNA's as above, with various pretreatments of the solid
carrier. Free biotin should compete with biotin-DNA for the
avidin sites on the solid phase, and pretreatment of the solid
phase with 20 times the half-capacity o~ free biotin reduced
biotin-DNA bindin~ by 50%. A further pretreatment with 10
fold more free biotin reduced biotin-DNA binding by an
additional 30%. Pretreatment of the solid phase with
pronase, 1% (w/v) sodium dodecyl sulfate and 0.1% (v/v) beta-
mercaptoethanol also reduced biotin-DNA binding by 50~, and
further treatment of this digested resin with free biotin
abolished biotin-DNA binding. A slightly different approach
also indicated that the larqe majority of biotin-DNA bindin~
to the solid carrier was not only biotin-dependent hut also
avidin-dependent. A control solid carrier was prepared by
treatin~ cellulose in the same manner as used for coupling
to avidin, except that the avidin was left out of the
reaction and replaced with Tris:~Cl. This control resin
bound 5 to 6% of control DNA probe and 15 to 15~ of biotin-
labeled DNA probe under the conditions used above for the
kinetics study. Thus, the majority of biotin-labelèd probe
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binding (larger than 85%) to the solid carrier is due to
interaction with the specific ligand avidin.
EXAMPL 3
This example demonstrates the construction of M13
vectors containing Mst II fragments bordering the sickle-
cell mutation.
In order to assay the major genetic for~ of
sickle-cell anemia using the assay method of the present
invention, very specific M13 constructions are required.
Basically, DNA fragments from either side of the Mst II
re~triction site which identifies the 6th codon error in a
sickle-cell individual were inserted into ~he M13 phage. The
two fragments used for this purpose were derived from the
plasmid pHBC6 (Fukumaki et al., Cell 28: 585 (1982)) and
purified by acrylamide gel electropfioresis. Approximately
10 ug of the 200 base pair Sau 1 (Mst II isoschizomer)
located immediately downstream of the diagnostic Mst II site
was treated with Klenow DNA Polymerase in the presence of
250 uM dXTPs to "fill-in" the Mst II site. This "blunt" DNA
fragment was then inserted into the ~c II site of pUC i8.
Analysis of 24 pUC 18-B-200 isolates indicated the presence
of the 200 base insert in 2 isolates, pUC 18-B-200-6 and pUC
18-B-200-9. Following CsCl preparation, approximately 200
ug of pUC lB-B-200-9 was digested with Eco Rl and Hd III
prior to purification by gel electrophoresis~ This Eco Rl,
Hd III ended 200 base pair fragment was then inserted into
M13 mp 18 to obta~in single-stranded DNA. Additional
constructions were made using this 200 base fragment to
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allow simple production of larqe quantities of specific RNA
sequences SpecifiCally, this fragment waq inserted into
the transcription vector pT7-1 and pT7-2 (United States
Biochemical Corporation, P.O. Box 22406, Cleveland, Ohio,
44122). These constructions allow for the in vitro
synthesis of labelled RNA to use as a probe in the sickle-
cell assay of the present invention. The other recombinant
vectors needed for assay of the sickle-cell trait use the
800 base pair Mst II ~ Hpa 1 fragment located îmmediately
upstream of the diagnostic Mst II restriction site. This
fragment was cloned in a similar fashion again usinq Klenow
DNA Polymerase to "round-off" the 5' overhanging ends found
at the Mst IIend. This 800 base pair fragment was inserted
into four vectors in order to obtain single stranded RNA and
DNA. They are:
M13 B-19-1-800 ~ -globin message-like strand
M13 B-18-1~800 ~ -globin non-message-like
pT7-1-800 RNA which is non-message like
pT7-2-800 RNA which is mesaage like
Although the present in~ention has been described
in considerable detail with regard to certain versions
thereof, other versions are possible. Therefore, the spirit
and scope of the appended claims should not necessarily be
limited to the description of the versions contained herein.
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