Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO92/1865~ PC~/~S92/02983
2 ~ 0 ~
DETECTION OF DNA/RNA BY
FLUORESCENCE POLARIZATION
This application is a continuation in-part of our pri~r
co-pending application, serial number 7/430,844 filed
11/1/89.
Field of the Invention
The present invention relates to the field of
detecting target nucleic acid sequences contained in
complex biological mixtures. More particularly, the
invention relates to detection of nucleic acid
sequences originally present in such mixtures n
extremely low concentrations, by first enzymatically
amplifying the specific target sequences, and then
detecting them in a substantially homogene~us assay
utilizing fluorescence polarization. An increase in
fluorescence polarization indicates the extent of
; hybridization of a probe with the amplified target
sequences.
Backaround of the Invention
In he study of cell populations associated with disease
states it is ~requently ~ound that only a subpopulation
of available susceptib~e cells actually exhibit the
morbid phenotype. In infectious diseases, the
proportions of cells which are passively or actively
infected may be very low, and the disease caused by the
infectious agent may go unnoticed clinically even
though an infected individual can transmit the agent to
others. Examples of such infectious agents are clammy,
protozoans, certain bacteria, and many viruses.
Amongst viruses, the human immunodeficiency virus
(HIV) is known to have an extremely long latent period
before the onset of the clinical symptoms known as
AIDS. Latency may extend to several years during which
the infected individual is capable of transmitting the
virus to others through intimate contact, sharing of
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intravenous injection apparatus, or through donation of
blood products.
HIV infection is specific f or thymus-derived
lymphocytes (T cells~, and in particular the subset T
cells having immune helper function. These T cells
possess highly specific HIV receptors on their surfaces
to which the virus attaches to gain entry to the cell.
Monolingual antibodie~, grouped generally in the CD4
cluster, see Leukocyte ~vpina III, Ed. A.J. McMichael,
Oxford University Press, 198~, and specific for the HIV
receptor, have been isolated heretofore (see Xung et
al., U.S. Patent No. 4,381,295~. A signal molecule can
be attached to such antibodies which binds selectively
to those cells expressing the receptor antigen thereby
identifying the helper T cell subpopulation.
Quantitation of cell numbers of such lymphocyte
subpopulations may conveniently be carried out in a
flow cytometer.
In normal individuals approximately So percent of
peripheral T cells are helper cells. In HIV infected
patients, this proportion declines sharply because the
virus is cytotoxic to helper T cells. In latent
infections or early in the course of clinical disease,
the proportion of the helper T cell population actually
containing virus, in either lytic or latent phase, may
be very low, even to the order of l to l000. This
means that in such patients, a 2 ml sample of blood may
contain only one or a few copies of the virus or its
genome. At his stage of infection, no antibodies to
viral proteins can be detected, even with the most
sensitive immunological techniques available. There is
a great danger that individuals at such early stages of
infection may transmit the virus in donated blood
without the virus being detected by conventional
screening methods.
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The most sensitive immunological technique~ arecapable of detecting antibody by HIV at minimally 21
days post-infection. A variety of immunoassays for
detection of HIV have been described including enzyme-
linked immunoassays (ELISA), immunodiffusion assays,radioimmunoassays (RIA~, and the classical Western
blot. Also a number of distinct assay strategies have
been developed. One group of assays utilizes ~IV viral
antigens, particularly viral protein containing
epitopes in conserved domains, bound covalently to a
solid matrix. The matrix-bound enzyme is contacted
- with a serum sample, and any anti~antigen antibodies
contained therein bind to antigen. In the typical
sandwich assay, antiserum raised in a heterologous
species against human antibody antigens conjugated to
an enzyme (ELISA), fluorescent molecule, or other
signal generating substance, is then reacted with the
washed matrix-bound antigen-anti-antigen complex. The
signal emitted by the signal generating substance is
typically a chromophor, fluorescent signal, beta or
gamma radiation, or other such measurable emission.
Alternatively, analysis of serum antibodies maybe
obtained by Western blot consisting of gel
electrophoresis of viral proteins, electrotransfer of
the proteins to blotting paper, followed by reaction
with antisera, and color development of the individual
protein ~ands. The Western blot analysis is employed
on a confirming test by blood banks in blood screening
procedures. For a ~eneral review of the various
immunological methods available, see Stites et al.,
Basic & Clinical Immunology, Appleton & Lange, 1987.
There have been many attempts in the prior art to
make detection of serum antibodies to HIV or other 1GW
concentration targets more sensitive and selective.
~ 35 The two major approaches have been target amplification
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2~8~40~ 4
and signal amplification. In signal amplification, the
object is to couple a very low level signal ~vent to a
large number of subsequent secondary signals which can
be detected a~d quantif ied . It is apparent that this
coupling must be highly specific so that background
secondary signals do not proportionally incrsase. One
such signal amplification system takes advantage of the
extremely high affinity of avidin for biotin. A large
number of biotin molecules can be covalently coupled to
an antibody specific for viral antigens. When reacted
with f luorochome-coupled a~idin a large complex is
formed havin~ unusually brilliant fluorescence.
Another system utilizes a mixture of monoclonal
antibodies conjugated to a signal generating substance,
each individual antibody type being specific for a
different structurally distinct epitope. The t~eory is
that a greater number of signal generating antibody
molecules will ~ind to antigen i~ there are no
overlapping specificities.
Another approach is to target nucleic acid
sequences of the virus with a homologous nucleic acid
probe coupled to a signal amplification system. Under
renaturing conditions the viral RNA (or denatured DNA)
anneals to the complementary sequence of an
oligonucleotide probe. Detection of the hybrid is
afforded by signal generating substances covalently
conjugated to th~ probe. Applicable to hi~ approach
are the enzyme proteolyzes a zymogen which then acts
upon a substrate to generate a ~easurable signal. Many
of the variations in such techniques are described in
Lelie et al., Detection of HIV Infection Usinq Second-
Generation HIV Assay, IV International AIDS Symposium,
Stockholm, 1988.
The second major approach involvés target
amplification in which the target interacting with the
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signal-generating entity is itself multiplied in
number. Since proteins cannot replicate, target
amplification inherently requires a nucleic acid
sequence, and an enzymatic system which can replicate
the target sequence in vitro. One such target
amplification technique is disclosed in U.S. Patent
Nos. 4,683,195 (Mullis et al.~ and 4,683,202 ~Mullis),
and is called polymerase chain reaction (PCR)
amplification.
In PCR, a mixture of nucleic acids containing a
DNA sequence in a small quantity is heated to denature
double stranded DNA. Primers consisting of a
oligonucleotide capable of mediating DNA synthesis from
a single stranded template is added under conditions
which favor annealinq of the primers to their specific
complementary sequences. A thermostable DNA polymerase
is added, and an extension reaction proceeds at 72C in
the presence of deoxynucleotide triphosphates,
adenosine triphosphate and cofactors. The reaction is
run at high temperatures to avoid non-specific binding
of primer to non-homologous sequences. ~nder these
strinqen~ conditions fidelity of pol~merization to t~e
desired sequences is very high.
After polymerization is complete, the mixture is
again heated to denature the double stranded DNAs, and
the extension reaction is repeated. Such repetition of
extension polymerization may occur several times until
the target sequence is amplified in numbers sufficient
to detect by any of the signal conjugated probe assays
described hereinabove.
In a second target amplification scheme called
TAS, a first primer oligonucleotide or oligonucleotide
containing an RNA transcriptase promoter-~inding
sequence is annealed to the target sequence and
extended by DNA polymerase or reverse transcriptase.
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2a~4~ 6
Following melting, a second primer complementary to the
newly formed oligomer in a region distal to the first
primer binding sequence is added, annealed, and
extended. The resultant duplex DNA oligomer thus has a
sequence flanking the target region and containing a
transcriptional promoter. Addition of RNA
transcriptase, in the presence of oligonucleotide
triphosphates, adenosine triphosphate, and cofactors
institutes transcription in vitro yielding up to looo
copies of the target sequence. The TAS methods have
been-disclosed in wo 88/01050 (Berg et al.).
In a variation of TAS, RNAse H is added to the
reaction mix. RNAse H specifically catalyzes the step-
wise hydrolysis of RNA bases in an RNA-DNA duplex, so
that after a cDNA strand has been synthesized with
reverse transcriptase the RNAse digests the RNA strand
of the duplex to permit synthesis of the second
complementary DNA strand by DNA polymerase. It will be
apparent that since a heating step to melt the DNA-RNA
duplex is unnecessary for cyclization of the reaction,
the entire amplification can be performed in a single
incubation. The disadvantages of the TAS and 3SR
methods, compared to PCR is the lesser degree of
stringency because of non-specific primer ~onding at
the lower temperature.
Finally, target amplification can be carried out
in a ligase-mediated procedure. In this procedure,
complementary primer sets form adjacent hybrids on ~oth
complementary strands of the target. Ligase then joins
the primers together at the nick separation, after
h~bridization. The ligated double primer can then act
as a template for further ligation of primers in a
~ subsequent melting and rehybridization step.
; After the target amplification, the nucleic acids
are ordinarily extracted and the amplified se~uences
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are detected by the procedures set forth hereinabove.
It should be emphasized that the known procedures o~
the prior art ar~ heter~geneous, that is, they require
multiple steps in which the DNA is first hybridized to
a signal generating probe, followed by a step in which
the unhybridized probe is separated from hybridized
probe. Ordinarily different sets of reagents are
required for signal generation than for probe
hybridization and separation. A completely homogeneous
method for detection sf amplified nucleic acids without
a separation step is unknown in the prior art.
Summary of the Invention
In accordance with the present invention, nucleic
acids amplified by any method are detected in a
15 convenient homogeneous one-step method utilizing
fluorescence polarization. The method of this
invention comprises incubation of denatured amplified
nucleic acids under hybridizing conditions with a
nucleic acid probe of homologous sequence covalently
coupled to a fluorophor. The fluorophor-conjugated
probe is added at a concentratiDn sufficient t~ produce
a measurable increase in fluorescence polarization when
hybridized to the amplified nucleic acid sequences, but
not in an excess quantity so as to appreciable quench
the increase in fluorescence polarization attributable
to hybridization.
It will be apparent that the method of the present
inve~tion is adaptable to a simple automated format for
convenient processing of a large number of samples.
This is because there is no nucleic acid extraction
` step, and it is unnecessary to separate unhybridized
probe from the mixture. Further, the increase in
fluorescence polarization upon hybridization is
virtually instantaneous, and no repetitious shifts in
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W092/~86s0 2a~4~ PcT/uss~/02s~3
temperature are required in practicing the present
~ethod.
More particularly, the method of the invention
comprises: a) Heating or otherwise denaturing a liquid
mixture and for a length of tim~ sufficient to separate
duplex nucleic acids into single strands, b) adding a
fluorochrome-conjugated nucleic acid probe having a
base sequence complementary to a target sequence
contained in the amplified nucleic mixture, c~ lowering
the temperatura of th2 mixture for adjusting the pH to
permit hybridization of probe sequences to amplified
nucleic acids target sequences; d) incubating the
mixture for a time sufficient for substantially
complete hybridization to occur; and e) measuring the
degree of fluorescence polarization.
In a preferred method, target nucleic acid
sequences are incubated under hybridization conditions
with a fluorescein-conjugated probe comprising an
oligonucleotide having substantial complementarity to
the target nucleic acid sequences, the fluorescein
being amino-linked to the oligonucleotide probe through
an aminochlorotriazinylaminoalkylphosphoryl group,
incubating for a time sufficient to obtain
substantially complete hybridization, and measuring the
fluorescence polarization. Probes for detecting a
target ~ucleic acid sequence are also disclosed which
comprise a fluorescein conjugated oligonucleotide
~equence of substantial complementarity to the target
nucleic acid sequence, the fluorescein molecule being
amino-linked to the oligonucleotide portion through an
aminochlorotriazinylaminoalkylphosphoryl group. Most
preferred probes are selected from the group. The
method of claim 4 wherein the said probes are selected
~rom the group consisting of oligonucleotides
substantially homologous to target nucleic acids
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WO9~J186so PCT/US92/02983
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containing a guanosine ~r cytosine base in the position
immediately 5' of the base annealing to the
fluorescein-labelled terminal nucleotide of the said
probe.
A kit is also contemplated by this inventi~n,
comprising a vessel containing a concentrated buffer
solution of a composition optimizing hybridization of
nucleic acids but without i~terfering with the
measurement of fluorescence polarization, and a second
vessel containing a solution of one or a plurality of
nucleic acid probes conjugated to one or a plurality of
assays. The solutions of the kit of this invention are
readily deliverable step-wise to a multiplicity of
sample eontainers. The samples can be processed
through each of the method steps without transferring
to another container, so that the incubations and
fluorescence polarization determination can be
accomplished in the same vessel and same machine under
automation.
Detailed Descriptic~L~ he Preferred Em~odim~ent
In the technique of fluorescence pol~rization,
light from a source is utilized to excite fluorescence
emission from a fluorochrome molecule. A fluorophor or
flùorochrome is a molecular entity, usually of
molecular weight less than lO,OOo daltons which emits
fluorescent light at a characterictic wavelength when
impacted with a rad~ant energy source. The
fluorochrome is covalently coupled to a nucleic acid
probe hybridizable with a complementary target
3 0 amplif ied nucleic acid sequence. A variety of
fluoroc~romes are known in the art which are adaptable
to the present invention, including fluorescein
derivatives, acidine orange (Van Bertalanffy et al., J.
~istochem. Cytochem., 4:481 t1g56)), benzimi~azole
derivatives (Hilwig et al., Exp. Cell Res., 75:1~2,
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W092/l86~0 PCT/US92/029~3
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1972)), and a series of fluorescen~ nu~leic acid
stains, as disclosed in U.S. Pa~ent NQ. 4,544, 5~6. In
addition, oligonucleotides bonded to DNA intercalating
agents have been utilized as nucleic acid probes, as
discl~sed in U.S. Patent N~. 4,83S,263.
In the preferred embodiment, fluorescein is
covalently attached to an oligonucleotide probe through
an aminochlorotriazinylaminoalkyphosphoryl group ha~ing
the g~neralized structure:
Dligonu~leotlùe ~ ` ~ o
wherein n is an integer from 2 to about 12 (herein~fter
referred to as DTAF). Most preferred is the
fluorescein conjugted oligonucleotide amino-lin~ed
t~rough an aminochlorotriazinylaminoethylphosphoryl
group. It has been determined empirically that
compounds of the preferred generalized structure are
superior in generating signal than other amino-linker
configurations, such as carboxy-fluorescein
succinimidyl ester or the fluorescein isothiocyan~te
derivative (FITC) having the structure:
~lLgorll~c~.uot~.du
o
Applicants have further discovered that detection
of hybridization by fluorescence polarization is
substantially enhanced in the preferred DTAF conjugated
probe when the target sequence selected contains a
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WO 92/18650 PCI'/US92/02983
11 2`~g~
guanDsine or a cytosine base in the position
immediately 5' of the base annealing to the
fluorescein-labelled terminal nucleotide of the probe.
The oligonucleotide portion of the probe has a seque~ce
of substantial complementarity to the amplified target
nucleic acids, so that a duplex between probe and
target is formed. It is apparent that perfect
complementarity is not necessary so long as a stable
duplex is formed under the hybridization conditions
lo utilized, and that some base ~ismatching may be
tolerated. It is further apparent that these probes
may be used in the detection of any substantially
homologous target nucleic acid sequence, and is not
limited to detection of only amplified nucleic acid.
The principle of this technique is based upon the
characteristic rotational properties of molecules in
solution. Molecules have a tendency to tumbe about
their various axes of rotation. In general, larger
molecules tumbe more slowly than smaller ones.
Compared to the amplified DNA target molecules, the
oligonucleotide fluorochrome-conjugated probes tumbe
` very rapidly and along several axes of rotation.
fluorescent light emitted from such spinning molecules
is diffuse and characteristically multiplanarn
~owever, when ~he probe molecule has annealed to the
target sequence forming a substantially rigid structure
of h~gh molecular weig~, it loses much of its spin.
As a result fluorescence emission becomes relatively
polarized. The emission fluorescence polarization of
solution in which intercalating fluorochrome binds iwth
DNA was described by Arschine et al., The EMBO J., 3,
795 ~1984). This polarization effect can be ~easured -
conveniently in a ~luorometer and a polarizatin value
is calculated accordingly to the following eguation:
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WC~ 92/18650 PCI/USg2/0~!9~3
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P = IPA -- IPE
IPA I IPE
wherein P is polarization units, IpA is the parallel
S intensity and IpE is the perp~ndicular intensity.
The oligonucleotide portions of the probe may have
a variable number of bases, preferably about 10-~0
which are substantially homologous with the target
sequence. For generic identifications, it is important
to select highly conserved regions of the genome s~ as
to ensure substantial homology between different
species, or genetic variants of the same infectious
organism or other nucleic acid tarqet. Further
increased sensitivity of the present method may be
attained by s~e of more than one probe specific for
different amplified sequences. In the case of ~IV,
selection of conserved sequences is an important
consideration because the envelope proteins are
genetically labile and mutant variations occur
extremely rapidly. This has been found to result from
replication errors. Reverse transcriptase is a highly
imprecise replicator. On the other hand, since this
property of the enzyme is particularly functional, the
sequence encoding the en2yme itself is highly
conserved. Accordingly, the sequences encoding the
reverse transcriptase make excellent oliqonucleotide
probes which can be expected to hybridize to virtually
- any HIV variant. Conversely, selection of probes of
short hybridizable sequences; preferable of 6 to 12
bases, from highly mutogeneic genomic regions may ~e
utilized diagnostically to assess the degree o~ drift
in homology over a period of time, or between different
patient hosts.
;` As indicated hereinabove, there are now a number
of different methods for amplifying nucleic acids.
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WO 92/18650 P~/US92/02983
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Once amplification has occurred, whether by PCR. TAS,
3SR, or LAS, the meth~d of the present invention is
equally applicable t detection of the nucleic acid so
amplified, without regard to the amplificati~n method
used. It will be apparent to those skilled in the art
that the inventive method of detection will be
applicable as well to any future improvement or new
technology in the nucleic acid amplification art.
The buffer utilized during hybridization may be
selected from the group of buffers having buffering
agents with a pH in the range of 6.5 to 8, having an
ionic strength of 100 500 mM, and having 10 to 100 mM
of a chelating substance. A typical buffer (20x
concentration) is described in Maniatis et al.,
Molecular_Cloninq, 1982 and contains 200 mM sodium
monobasic phosphate, 3 M sodium chloride, 20 mM
ethylene diamnetetracetic acid, at pH 7.4 It was found
empirically that this buffer provides good conditions
for hybridization without appreciable quenching or
other deleterous effect on the fluorescence
polarization detection step.
The kit of the present invention provides a first
vessel in which amplification of specific nucleic acid
is done in a small volume. The amplified nucleic acid
is then denatured by boiling or by the addition of
sodium hydroxide. A second vessel o~ the ~it contains
the probe in a reconstitutable, lyopholozed, or
concentrated liquid form which also contains
hybridization buffer. The second v~ssel of the kit is
made of a substance which does not readily absorb
oligonucleotide molecules to its surfaces. Examples of
such materials are polyethylene, polypropylene, teflon,
silanized or siliconized glass. Finally, the opening
of the vessels may have flange or valve means whereby
to facilitate drawing of liquids contained therein into
W092/18650 PST/US92/029.8~
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lines conveying the liquids to an automated machine for
processing of a large number of amplified nucleic ac~d
samples. Other advantages of the present invention
will be apparent from the Examples which follow.
`5 ~xamples
In initial experiments, the targPt sequence
selected for study was a section of the HIVPV22
sequence partially encoding reverse transcriptase of
HIV. A section sf thi~ sequence (bases 1195-2690) was
cloned into plasmid p24L utilizing conventional
techniques. The PCR reactions on both the plasmid and
DNA from infected cells, termed HIV-DASH amplification
region. The oligonucleotide primers at the 3' and 5'
termini are shown on the figure. Upon dPnaturation of
HIV~DASH and annealing o~ the primers t~ their target
sequences, DNA polymerase, preferably a highly purified
form of recombinant enzyme from which the endonuclease
encoding sequences have been deleted, is added to
obtain chain extension. This procedure was repeated
several times. This method of PCR amplification does
not depart appreciably from the method disclosed in
U.S. Patent Nos. 4,683,145 and 4,683,202, and
variations reported in the literature.
Upon completion of amplification, he nucleic acids
are once again denatured. Denaturation can be effected
thermally by ~oiling or, preferably, by addition of
NaO~ at 55C resulting in a pH of approximately 13.
T~e fluorochrome-conjugated oligonucleotide probes are
added. If denaturation was effected by boiling,
renaturation will proceed by simply lowering the
temperature to 37 - 48C. If denaturation was
`~ effected at high pH, then a sufficient amount of Tris
pH 5.5 buffer is added to reduce the pH to 8, so that
hybridization of probe proceeds.
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Empirically, it was discovered t~at signal
detection is enhanced when complementary probe pairs
are employed, and also when more than one target
sequence is used. Also, it was found that surprisingly
a much better detection signal was generated in
relatively large volumes of low target DNA
concentration. Accordingly, the experiments of these
e~amples were conducted in 1.5 - 2.0 ml volumes instead
of 50 microliter microassays.
In the experiment of Example l, reagents comprised
a probe designated DASH3-AMI-FITC which has the
oligonucleotide sequence shown in Figure 2, covalently
attached through an amide linkage t~ fluorescein
isothiocyanate designated DASH3, and a second
unconjugated oligonucleotide sequence complementary to
DASH3-AMI-F~TC, designated DASHIC, and a third
unconjugated oligonucleotide sequence, designated
DASH#, as a noncomplementary control. These reagents
were added together as follows: with 1.9 ml 5x SSPE
buffer (750 mM NaCl, 10 mM sodium phosphate monobasic,
1 mM EDTA), lO0 microliters 120 mM DASH3-AMI-FITC in 5x
SSPE buffer, 5, 20, 50, 100 microliters respectively,
in separate tubes; 100 mM DASH3c in 5x SSPE buffer, or
alternatively, 5, 20, 50, lO0 microliters 100 ~M DASH3
; 25 in 5x SSPE, respectively in separate tubes. Background
fluorescence polarization values were obtained for each
tube immediately after addition of the DAS~3-AMI-FITC
and before addition of the other probes. After the
hybridization reactions were begun, fluorescence
polarization was monitored over a 35 minute period.
The results are shown in Figure 3 of the drawings.
It is readily apparent that hybridization of the
fluorochrome-conjugated probe with its complementary
probe sequence produces substantial polarization of
3 5 f luorescence, and that the amount of polarization is
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W092/18650 PCT/US92tO29~
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substantially quantitative. This is essentially a
control experiment demonstrating that hybridization to
the relativ~ly short complementary sequence brings
about a significant degree of molecular spin
inhibition.
Example 2
In another experiment, of substantially identical
format, the capture target sequence for labelled probe
is a PCR amplified HIV target derived from
amplification of the p24L plasmid known to contain the
- DASH sequence. The results are shown in Figure 4 of
the drawings. Hybridization was monitored over 55
minutes and carried not in a total reaction volume of
1.6 ml. The glossary of graph p~ints on the right side
of the figure expresses the DNA content of the reaction
mix as the number of tipomoles of pre-PCR DNA present
prior to the amplification ~tep and, correspond to 0,
1o2, 103, 104, and 105 pre-amplification molecules of
p24L plasmid DNA. Denaturation was carried out by
incubating 20 microliters PCR reaction mix with 25
microliters water and 5 microliters NaOH at 55c for 15
minutes. Tubes containing 1.5 ml 5x SSPE buffer, 7
microliters of 3.2 M Tris-Hcl and-100 microliters
DASH3-AMI-FITC ~10 mM in 5x SSPE buffer) were prepared
and background fluorescence polarization measured.
Fifty microliter aliquots of the PCR-DNAs were added
and fluorescence polarization measured at the times
indicated.
The results graphed in Figure 4 of the drawings
: 30 clearly show an increase in fluorescence polarization
which is generally dose-dependent with increasing
amounts of added PCR-DNA. This is an especially
significant finding becauce of the large excess of DNA
present which is noncomplementary to the DASH sequence
in this homogeneous assay.
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Example 3
In a still further experiment the RNA fraction ofH-9 cells infected with HIV virus was extracted by
conventional methods. A portion containing 0.1
attomoles of ~IV ~NA was the~ amplified utilizing the
3SR amplification techniques described hereinabove.
Test tubes for assay of probe-RNA hybrids were prepared
by adding 1.~ ml 5x SSPE buffer to 100 microliters of a
10 mM DASH3-AMI-FITc solution. 3SR RNA was alkalie
denatured. Sixty microliters of the denatured 3SR
amplified ~NA was added to the test tubes, and
fluorescence polarization was monitored over a 45
minute time period. The r~sults, shown in Figure 5,
indicate that 3SR amplified RNA derived from the RNA
fraction of HIV infected cells gives a strong,
unequivocal positive signal compared to an unamplified
control. This example illustrates t~at the detection
method of the present invention is a versatile assay of
nucleic acids containing target sequences amplified by
any method.
Exam~le 4
In the experiment of this example, the resolving
power of the present method was evaluated. The
supernatant from a growing culture of H-9 cells
infected with HIV was obtained and the DNA putatively
contained therein was subjected to 60 cycles of PRC.
The a~ounts of ~upernatant amplified corresponded to a
volume of cultured cells csntaining 102, 103, 104
cells. Similarly, identical control supernatants were
prepared from growing cultures of non-infected H-9
cells. Test tubes were prepared containing 1.5 ml 5x
SSPE buffer, 5 microliters lN NaOH, 7 microliters 3~2 M
Tris pH 5.5, and 100 microliters of 10 mM DASH3-AMI-
FITC. Target DNA was denatured by adding 5 microliters
lN NaOH and, 25 microliters water to 20 microliters PCR
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2a ~ 18
reaction mix followed by incubation at 55C ~or 15
minutes. Fifty microliters of the denatured PCR
reaction solution was added to the test tubes
containing the DASH3-AMI-FITC probe and hybridization
5 proceeded with monitoring by fluorescence polarization.
Figure 6, shows that a~f irmative RIV sequence detection
can be obtai~ed of the PCR a~plification of the
supernatant containing as few as 200 cells. This means
that the method of the present invention has sufficient
sensitivity and resolving power to be useful as a
routine serum screening-assay for individuals with very
early pre-clinical HIV infection.
In practicing the present invention, it must be
emphasized that the probe technology and hybridization
conditions are critical. The target sequences must
have high binding affinity to probe and be highly
specific for host sequences. Purity is also an
important factor. Also, the fluorochrome-conjugated
probe sequence length must be short enough that the
rotational and relaxational properties show contrast in
fluoroscence polarization values when hybridized.
Specific probe se~uence may influence the rate at which
annealing of probe to target sequence occurs, compared
to the rate at which reannealing of native
complementary target regions oc~urs. While is does
appear that the amino-linker probe coupling strategy
utili2ed in these examples is particularly efficacious,
other linkage modes may theoretically be substituted by
trial and error.
; 30 Example 5
In these experiments the probe and targ~t reayen~s
were as follows: a probe designated DASH3-15-AMI-FITC
which has the oligonucleotide sequence shown in Figure
7 covalently attached through an amino-linker arm to
fluorescein isothiocyanate, a second probe designated
~ ~ .
.
., ~ ~ . . .
W092/l8650 PCT/US921029~3
19 2~4~
DASH3-15-AMI-DTAF whch has an idPntical sequence as the
above probe but covalently attached through an amino-
; linker arm to dichlorotrianzinylaminofluorescein, and
an unconjugated oligonucleotide target sPquence
designated T23-DASHIC which has the sequence shown in
Figure 7 and which is complementary to bo~h of the
ab~e probes. In separate tubes each probe was added
to the target sequence and fluorescence polarization
was monitor as in Example 2. The results tabulated in
Table 1 clearly shown that upon hybridizatio~ to a
complementary tarqet sequence, a probe labelled with
DTAF gives higher changes in fluorescence polarization
(delta mP) than a probe labelled with FITC, upon
hybridization to a complementary target seguence.
Table 1
FLUORESCENCE POLAR~ZATION ~mP)
Pre- Post-
` PROBE ~ybridiz~tion hy~ridization Delta
DASH-15-AMI-FITC93.0 169.8 76.8
DASH-15-AMI-D~AF111.~ 240.1 128.3
In further experiments the probe and target were
as follows: a probe designated DASH3-15-AMI-DTAF which
has the oligonucleotide sequence shown in Figure 8
covalently attached through an a~ino-linker arm to
dichlorotrianzinylaminofluorescein, and five
unconjugated oligonucleotide target sequence designated
-~; T23-DASHIC-G, T23-DAS~IC-A, T23-DASHIC-T, and T23-
DASHIC-C which have the sequences shown in Figure 8 and
are complementary to the above pro~e. In separate
tubes the probe w c added to each of target sequences
and f luorescence polarization was monitored as in
Example 2. The results, graphed in Figure 9 clearly,
shows that the nucleotide of target sequence adjacent
to the probes' linker arm after the hybridization
affects the degree of fluorescence polarization.
-
~ .
.- ~ ,
W092/18650 PCT/US92/02983
2 ~ '
Greater changes (delta mP) in fluorescence polarizatio~
are obtained if the said nucleotide is a deoxycytidine
(C) or a deoxyguanidine (G).
