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Description  |
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The invention relates generally to the field of nucleic acid amplification,
and more particularly, to methods of monitoring the progress of nucleic
acid amplification reactions, especially polymerase chain reactions (PCR).
BACKGROUND
Nucleic acid amplification techniques have opened broad new approaches to
genetic testing and DNA analysis, e.g. Arnheim and Erlich, Ann. Rev.
Biochem., 61: 131-156 (1992). PCR in particular has become a research tool
of major importance with applications in cloning, analysis of genetic
expression, DNA sequencing, genetic mapping, drug discovery, and the like,
e.g. Arnheim et al (cited above); Gilliland et at, Proc. Natl. Acad. Sci.,
87:2725-2729 (1990); Bevan et at, PCR Methods and Applications, 1:222-228
(1992); Green et at, PCR Methods and Applications, 1: 77-90(1991);
Blackwell et al, Science, 250:1104-1110 (1990). The widespread
applications of such nucleic acid amplification techniques has driven the
development of instrumentation for carrying out the amplification
reactions under a variety of circumstances. Important design goals for
such instrument development have included fine temperature control,
minimization of sample-to-sample variability in multi-sample thermal
cycling, automation of pre- and post-reaction processing steps, high speed
temperature cycling, minimization of sample volumes, real time measurement
of amplification products, minimization of cross-contamination, or "sample
carryover," and the like. In particular, the design of instruments
permitting amplification to be carried out in closed reaction chambers and
monitored in real time would be highly desirable for preventing
cross-contamination, e.g. Higuchi et al, Biotechnology, 10:413-417 (1992)
and 11: 1026-1030(1993); and Holland et al, Proc. Natl. Acad. Sci., 88:
7276-7280 (1991). Clearly, the successful realization of such a design
goal would be especially desirable in the analysis of diagnostic samples,
where a high frequency of false positives and false negatives--caused by
"sample carryover"--would severely reduce the value of an amplification
procedure. Moreover, real time monitoring of an amplification reaction
permits far more accurate quantitation of starting target DNA
concentrations in multiple-target amplifications, as the relative values
of close concentrations can be resolved by taking into account the history
of the relative concentration values during the reaction. Real time
monitoring also permits the efficiency of the amplification reaction to be
evaluated, which can indicate whether reaction inhibitors are present in a
sample.
Holland et al (cited above) and others have proposed fluorescence-based
approaches to provide real time measurements of amplification products
during a PCR. Such approaches have either employed intercalating dyes
(such as ethidium bromide) to indicate the amount of double stranded DNA
present, or they have employed probes containing fluorester-quencher pairs
(the so-called "Tac-Man" approach) that are cleaved during amplification
to release a fluorescent product whose concentration is proportional to
the amount of double stranded DNA present.
The latter approach, illustrated in FIG. 1, involves the use of an
oligonucleotide probe that specifically anneals to a region of the target
polynucleotide "downstream," i.e. in the direction of extension, of primer
binding sites. The probe contains a fluorescent "reporter" molecule and a
"quencher" molecule such that the whenever the reporter molecule is
excited, the energy of the excited state nonradiatively transfers to the
quencher molecule where it either dissipates nonradiatively or is emitted
at a different emission frequency than that of the reporter molecule.
During strand extension by a DNA polymerase, the probe anneals to the
template where it is digested by the 5'->3' exonuclease activity of the
polymerase. Upon digestion, the quencher molecule is no longer close
enough to the reporter molecule to quench emissions by energy transfer.
Thus, as more and more probe gets digested during amplification, a
stronger and stronger fluorescent signal is generated.
Three main factors determine the performance of such a doubly labeled
fluorescent probe: First is the degree of quenching observed in the intact
unbound probe. This can be characterized by the ratio, designated herein
as "RQ.sup.- ", of fluorescent emissions of the reporter molecule and the
quencher molecule absent hybridization to a complementary polynucleotide.
That is, RQ.sup.- is the ratio of fluorescent emissions of the reporter
molecule and the quencher molecule when the S oligonucleotide probe is in
a single stranded state. Influences on the value of RQ.sup.- include the
particular reporter and quencher molecules used, the spacing between the
reporter and quencher molecules, nucleotide sequence-specific effects, the
degree of flexibility of structures, e.g. linkers, to which the reporter
and quencher molecules are attached, the presence of impurities, and the
like, e.g. Wu et at, Anal. Biochem., 218: 1-13 (1994); and Clegg, Meth.
Enzymol., 211:353-388 (1992). (A related quantity, RQ.sup.+, is the ratio
of fluorescent emissions of the reporter molecule and the quencher
molecule when the oligonucleotide probe is in a double stranded state with
a complementary polynucleotide). A second factor is the efficiency of
hybridization, which depends on probe melting temperature, T.sub.m, the
presence of secondary structure in the probe or target polynucleotide,
annealing temperature, and other reaction conditions. Finally, a third
factor is the efficiency at which the DNA polymerase 5'.fwdarw.3'
exonuclease activity cleaves the bound probe between the reporter molecule
and quencher molecule. Such efficiency depends on the proximity of the
reporter or quencher to the 5' end of the probe, the "bulkiness" of the
reporter or quencher, the degree of complementarity between the probe and
target polynucleotide, and like factors, e.g. Lee et al, Nucleic Acids
Research, 21:3761-3766 (1993).
As quenching is completely dependent on the physical proximity of the
reporter molecule and quencher molecule, it has been assumed that the
quencher and reporter molecules must be attached to the probe within a few
nucleotides of one another, usually with a separation of about 6-16
nucleotides, e.g. Lee et al (cited above); Mergny et at, Nucleic Acids
Research, 22:920-928 (1994); Cardullo et at, Proc. Natl. Acad. Sci.,
85:8790-8794 (1988); Clegg et at, Proc. Natl. Acad. Sci., 90:2994-2998
(1993); Ozaki et at, Nucleic Acids Research, 20:5205-5214 (1992); and the
like. Typically, this separation is achieved by attaching one member of a
reporter-quencher pair to the 5' end of the probe and the other member to
a base 6-16 nucleotides away. Unfortunately, there are at least two
significant drawbacks to this arrangement. First, attaching reporter or
quencher molecules typically involves more difficult chemistry than, for
example, that used to attach moieties to an end. And second, attachment of
reporter or quencher molecules to internal nucleotides adversely affects
hybridization efficiency, e.g. Ward et at, U.S. Pat. No. 5,328,824; Ozaki
et al (cited above); and the like.
In view of the above, the application of techniques for real-time
monitoring of nucleic acid amplification would be facilitated by the
availability of a conveniently synthesized probe having efficient
hybridization characteristics and distinct fluorescent characteristic in a
bound double stranded state and an unbound single stranded state.
SUMMARY OF THE INVENTION
A broad object of our invention is to provide a method for real-time
quantitation of nucleic amplification that employs a probe which is easier
to synthesize than currently used probes and which has superior
hybridization efficiency than those of currently used probes.
Another object of our invention is to provide a probe for use in the above
method that has different fluorescent characteristics depending on whether
it is in a double stranded state hybridized to a complementary
polynucleotide or whether it is in a single stranded state.
Still another object of our invention is to provide a conveniently
synthesized probe for use in the above method that has a reporter molecule
attached to one end and a quencher molecule attached to the other end.
Another object of our invention is to provide a probe for use in the above
method that does not require reporter or quencher moieties to be attached
to internal bases or internucleotide linkages.
These and other objects of the invention are achieved in the method
described below.
Generally the method of our invention relates to monitoring the progress of
a nucleic acid amplification reaction that employs a nucleic acid
polymerase having 5'.fwdarw.3' exonuclease activity. More particularly,
our invention relates to a method of monitoring the amplification of a
target polynucleotide by (1) providing an oligonucleotide probe capable of
annealing to the target polynucleotide, the oligonucleotide probe having a
reporter molecule capable of fluorescing attached to a first end and a
quencher molecule attached to a second end such that the quencher molecule
substantially quenches any fluorescence of the reporter molecule whenever
the oligonucleotide probe is in a single-stranded state and such that the
reporter is substantially unquenched whenever the oligonucleotide probe is
in a double-stranded state; and (2) extending a primer annealed to the
target polynucleotide with a nucleic acid polymerase having 5'.fwdarw.3'
exonuclease activity such that the oligonucleotide probe is degraded by
the 5'.fwdarw.3' exonuclease activity of the nucleic acid polymerase as it
extends the primer.
Preferably, reporter and quencher molecules are attached to the terminal 5'
carbon and terminal 3' carbon of the oligonucleotide probe by way of 5'
and 3' linking moieties, respectively, such that either the reporter
molecule is on the 5' end of the probe and the quencher molecule is on the
3' end of the probe, or the reporter molecule is on the 3' end of the
probe and the quencher molecule is on the 5' end of the probe.
Our invention advantageously overcomes several deficiencies of currently
used probes for monitoring the amplification of nucleic acids. In
particular, probes provided in the method of our invention are readily
synthesized and ameliorate inefficiencies in hybridization and exonuclease
cleavage due to groups which are attached to internal bases or
internucleotide linkages.
An important aspect of the invention is the discovery that in probe a
quencher molecule need not be attached to a nucleotide adjacent to a
reporter molecule to successfully quench fluorescence produced by the
reporter when the probe is in a single stranded state. Thus, facilely
synthesized end-labeled probes may be used in place of
more-difficult-to-synthesize and less efficient internally labeled probes
for monitoring nucleic acid amplification.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a method for real-time monitoring nucleic acid
amplification utilizing a probe which is degraded by the 5'.fwdarw.3'
exonuclease activity of a nucleic acid polymerase.
DETAILED DESCRIPTION OF THE INVENTION
Preferably, the method of the invention is used in conjunction with the
amplification of a target polynucleotide by PCR, e.g. as described in many
references, such as Innis et at, editors, PCR Protocols (Academic Press,
New York, 1989); Sambrook et at, Molecular Cloning, Second Edition (Cold
Spring Harbor Laboratory, New York, 1989); and the like. The binding site
of the oligonucleotide probe is located between the PCR primers used to
amplify the target polynucleotide. Preferably, PCR is carried out using
Taq DNA polymerase, e.g. Amplitaq.TM. (Perkin-Elmer, Norwalk, Conn.), or
an equivalent thermostable DNA polymerase, and the annealing temperature
of the PCR is about 5.degree.-10.degree. C. below the melting temperature
of the oligonucleotide probes employed.
The term "oligonucleotide" as used herein includes linear oligomers of
natural or modified monomers or linkages, including deoxyribonucleosides,
ribonucleosides, and the like, capable of specifically binding to a target
polynucleotide by way of a regular pattern of monomer-to-monomer
interactions, such as Watson-Crick type of base pairing, or the like.
Usually monomers are linked by phosphodiester bonds or analogs thereof to
form oligonucleotides ranging in size from a few monomeric units, e.g.
3-4, to several tens of monomeric units. Whenever an oligonucleotide is
represented by a sequence of letters, such as "ATGCCTG," it will be
understood that the nucleotides are in 5'.fwdarw.3' order from left to
right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G"
denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoranilidate, phosphoramidate, and the like.
Generally, oligonucleotide probes of the invention will have a sufficient
number of phosphodiester linkages adjacent to its 5' end so that the
5'.fwdarw.3' exonuclease activity employed can efficiently degrade the
bound probe to separate the reporter and quencher molecules.
"Perfectly matched" in reference to a duplex means that the poly- or
oligonucleotide strands making up the duplex form a double stranded
structure with one other such that every nucleotide in each strand
undergoes Watson-Crick basepairing with a nucleotide in the other strand.
The term also comprehends the pairing of nucleoside analogs, such as
deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may
be employed. Conversely, a "mismatch" in a duplex between a target
polynucleotide and an oligonucleotide probe or primer means that a pair of
nucleotides in the duplex fails to undergo Watson-Crick bonding.
As used herein, "nucleoside" includes the natural nucleosides, including
2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker,
DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in
reference to nucleosides includes synthetic nucleosides having modified
base moieties and/or modified sugar moieties, e.g. described by Scheit,
Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,
Chemical Reviews, 90:543-584 (1990), or the like, with the only proviso
that they are capable of specific hybridization. Such analogs include
synthetic nucleosides designed to enhance binding properties, reduce
degeneracy, increase specificity, and the like.
Oligonucleotide probes of the invention can be synthesized by a number of
approaches, e.g. Ozaki et at, Nucleic Acids Research, 20:5205-5214 (1992);
Agrawal et at, Nucleic Acids Research, 18:5419-5423 (1990); or the like.
The oligonucleotide probes of the invention are conveniently synthesized
on an automated DNA synthesizer, e.g. an Applied Biosystems, Inc. Foster
City, Calif.) model 392 or 394 DNA/RNA Synthesizer, using standard
chemistries, such as phosphoramidite chemistry, e.g. disclosed in the
following references: Beaucage and Iyer, Tetrahedron, 48:2223-2311 (1992);
Molko et al, U.S. Pat. Nos. 4,980,460; Koster et al, U.S. Pat. No.
4,725,677; Caruthers et al, U.S. Pat. Nos. 4,415,732; 4,458,066; and
4,973,679; and the like. Alternative chemistries, e.g. resulting in
non-natural backbone groups, such as phosphorothioate, phosphoramidate,
and the like, may also be employed provided that the hybridization
efficiencies of the resulting oligonucleotides and/or cleavage efficiency
of the exonuclease employed are not adversely affected. Preferably, the
oligonucleotide probe is in the range of 15-60 nucleotides in length. More
preferably, the oligonucleotide probe is in the range of 18-30 nucleotides
in length. The precise sequence and length of an oligonucleotide probe of
the invention depends in part on the nature of the target polynucleotide
to which it binds. The binding location and length may be varied to
achieve appropriate annealing and melting properties for a particular
embodiment. Guidance for making such design choices can be found in many
of the above-cited references describing the "tatman" type of assays.
Preferably, the 3' terminal nucleotide of the oligonucleotide probe is
blocked or rendered incapable of extension by a nucleic acid polymerase.
Such blocking is conveniently carried out by the attachment of a reporter
or quencher molecule to the terminal 3' carbon of the oligonucleotide
probe by a linking moiety.
Preferably, reporter molecules are fluorescent organic dyes derivatized for
attachment to the terminal 3' carbon or terminal 5' carbon of the probe
via a linking moiety. Preferably, quencher molecules are also organic
dyes, which may or may not be fluorescent, depending on the embodiment of
the invention. For example, in a preferred embodiment of the invention,
the quencher molecule is fluorescent. Generally, whether the quencher
molecule is fluorescent or simply releases the transferred energy from the
reporter by non-radiative decay, the absorption band of the quencher
should substantially overlap the fluorescent emission band of the reporter
molecule. Non-fluorescent quencher molecules that absorb energy from
excited reporter molecules, but which do not release the energy
radiatively, are referred to herein as chromogenic molecules.
There is a great deal of practical guidance available in the literature for
selecting appropriate reporter-quencher pairs for particular probes, as
exemplified by the following references: Clegg (cited above); Wu et al
(cited above); Pesce et at, editors, Fluorescence Spectroscopy (Marcel
Dekker, New York, 1971); White et at, Fluorescence Analysis: A Practical
Approach (Marcel Dekker, New York, 1970); and the like. The literature
also includes references providing exhaustive lists of fluorescent and
chromogenic molecules and their relevant optical properties for choosing
reporter-quencher pairs, e.g. Berlman, Handbook of Fluorescence Sprectra
of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971 );
Griffiths, Colour and Consitution of Organic Molecules (Academic Press,
New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford,
1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals
(Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and
Phosphorescence (Interscience Publishers, New York, 1949); and the like.
Further, there is extensive guidance in the literature for derivatizing
reporter and quencher molecules for covalent attachment via common
reactive groups that can be added to an oligonucleotide, as exemplified by
the following references: Haugland (cited above); Ullman et al, U.S. Pat.
No. 3,996,345; Khanna et al, U.S. Pat. No. 4,351,760; and the like.
Exemplary reporter-quencher pairs may be selected from xanthene dyes,
including fluoresceins, and rhodamine dyes. Many suitable forms of these
compounds are widely available commercially with substituents on their
phenyl moieties which can be used as the site for bonding or as the
bonding functionality for attachment to an oligonucleotide. Another group
of fluorescent compounds are the naphthylamines, having an amino group in
the alpha or beta position. Included among such naphthylamino compounds
are 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate
and 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include
3-phenyl-7-isocyanatocoumarin, acridines, such as 9-isothiocyanatoacridine
and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide;
benzoxadiazoles, stilbenes, pyrenes, and the like.
Preferably, reporter and quencher molecules are selected from fluorescein
and rhodamine dyes. These dyes and appropriate linking methodologies for
attachment to oligonucleotides are described in many references, e.g.
Khanna et al (cited above); Marshall, Histochemical J., 7:299-303 (1975);
Mechnen et at, U.S. Pat. No. 5,188,934; Menchen et al, European pat. No.
application 87310256.0; and Bergot et al, International application
PCT/US90/05565. The latter four documents are hereby incorporated by
reference.
There are many linking moieties and methodologies for attaching reporter or
quencher molecules to the 5' or 3' termini of oligonucleotides, as
exemplified by the following references: Eckstein, editor,
Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford,
1991 ); Zuckerman et al, Nucleic Acids Research, 15: 5305-5321 (1987)(3'
thiol group on oligonucleotide); Sharma et al, Nucleic Acids Research,
19:3019 (1991 )(3' sulfhydryl); Giusti et al, PCR Methods and
Applications, 2:223-227 (1993) and Fung et al, U.S. Pat. No. 4,757,141 (5'
phosphoamino group via Aminolink.TM. II available from Applied Biosystems,
Foster City, Calif.); Stabinsky, U.S. Pat. No. 4,739,044 (3'
aminoalkylphosphoryl group); Agrawal et al, Tetrahedron Letters,
31:1543-1546 (1990)(attachment via phosphoramidate linkages); Sproat et
al, Nucleic Acids Research, 15:4837 (1987)(5' mercapto group); Nelson et
al, Nucleic Acids Research, 17:7187-7194 (1989)(3' amino group); and the
like.
Preferably, commercially available linking moieties are employed that can
be attached to an oligonucleotide during synthesis, e.g. available from
Clontech Laboratories (Palo Alto, Calif.).
Rhodamine and fluorescein dyes are also conveniently attached to the 5'
hydroxyl of an oligonucleotide at the conclusion of solid phase synthesis
by way of dyes derivatized with a phosphoramidite moiety, e.g. Woo et al,
U.S. Pat. No. 5,231,191; and Hobbs, Jr. U.S. Pat. No. 4,997,928.
EXAMPLE
The method of the invention was carded out using the oligonucleotides shown
in Table 1. Linker arm nucleotide ("LAN") phosphoramidite was obtained
from Glen Research. Standard DNA phosphoramidites, 6-carboxyfluorescein
("6-FAM") phosphoramidite, 6-carboxytetramethylrhodamine succinimidyl
ester ("TAMRA NHS ester"), and Phosphalink.TM. for attaching a 3' blocking
phosphate were obtained from Perkin-Elmer, Applied Biosystems Division.
Oligonucleotide synthesis was performed on a model 394 DNA Synthesizer
(Applied Biosystems). Primer and complement oligonucleotides were purified
using Oligo Purification Cartridges (Applied Biosystems). Doubly labeled
probes were synthesized with 6-FAM-labeled phosphoramidite at the 5' end,
LAN replacing one of the T's in the oligonucleotide sequence, and
Phosphalink.TM. at the 3' end. Following deprotection and ethanol
precipitation, TAMRA NHS ester was coupled to the LAN-containing
oligonucleotide in 250 mM Na-bicarbonate buffer (pH 9.0) at room
temperature. Unreacted dye was removed by passage over a PD-10 Sephadex
column. Finally, the doubly labeled probe was purified by preparative HPLC
using standard protocols. Below, probes are named by designating the
sequence from Table 1 and the position of the LAN-TAMRA moiety. For
example, probe Al-7 has sequence of Al with LAN-TAMRA at nucleoside
position 7 from the 5' end.
All PCR amplifications were performed in a Perkin-Elmer Thermocycler 9600
using 50 .mu.l reactions that contained 10 mM Tris-HCl (pH 8.3 ), 50 mM
KCl, 200 .mu.M dATP, 200 .mu.M dCTP, 200 .mu.M dGTP, 400 .mu.M dUTP, 0.5
units AmpErase.TM. uracil N-glycolyase (Perkin-Elmer), and 1.25 units
AmpliTaq.TM. (Perkin-Elmer). A 295 basepair segment of exon 3 of the
.beta.-human 13-actin gene (nucleotides 2141-2435 disclosed by
Nakajima-Iijima) was amplified using the AFP and ARP primers listed below.
The amplification reactions contained 4 .mu.M MgCl.sub.2, 20 ng human
genomic DNA, 50 nM Al or A3 probe, and 300 nM of each primer. Thermal
regimen was 50.degree. C. (2 min); 95.degree. C. (10 min); 40 cycles of
95.degree. C. (20 sec.), 60.degree. C. (1 min); and hold at 72.degree. C.
A 515 basepair segment was amplified from a plasmid that consists of a
segment of .lambda. DNA (nucleotides 32,220-32,747) inserted into the Sma
I site of vector pUC 119. These reactions contained 3.5 mM MgCl.sub.2, 1
ng plasmid DNA, 50 nM P2 or P5 probe, 200 nM primer F119, and 200 nM
primer R119. The thermal regimen was 50.degree. C. (2 min); 95.degree. C.
(10 min); 25 cycles of 95.degree. C. (20 sec.), 57.degree. C. (1 min); and
hold at 72.degree. C.
For each amplification reaction, 40 .mu.l was transferred to an individual
well of a white 96-well microtiter plate (Perkin-Elmer). Fluorescence was
measured on a Perkin-Elmer TaqMan.TM. LS-50B System, which consists of a
luminescence spectrometer with a plate reader assembly, a 485 nm
excitation filter, and a 515 nm emission filter. Excitation was carried
out at 488 nm using a 5 nm slit width. Emission was measured at 518 nm for
6-FAM (the reporter, or R value) and 582 nm for TAMRA (the quencher, or Q
value) using a 10 nm slit width. In order to determine the increase in
reporter emission that is due to cleavage of the probe during PCR, three
normalizations are applied to the raw emission data. First, emission
intensity of a buffer blank is subtracted for each wavelength. Second,
emission intensity of the reporter is divided by the emission intensity of
the quencher to give an RQ ratio for each reaction tube. This normalizes
for well-to-well variation in probe concentration and fluorescence
measurement. Finally, .DELTA.RQ is calculated by subtracting the RQ value
of the no template control (RQ.sup.-) from the RQ value for the complete
reaction including a template (RQ.sup.+).
Three pairs of probes were tested in PCR assays. For each pair, one probe
has TAMRA attached to an internal nucleotide and the other has TAMRA
attached to the 3' end nucleotide. Results are shown in Table 2. For all
three sets, the probe with the 3' quencher exhibits a .DELTA.RQ value that
is considerably higher than for the probe with the internal quencher.
Table 3 gives the results of fluorescence measurements of the indicated
probes in single and double stranded states. For probes having reporter
and quencher at opposite ends of the oligonucleotide, hybridization caused
a dramatic increase in RQ.
TABLE 1
__________________________________________________________________________
Sequences of oligonucleotides.
Name Type Sequence
__________________________________________________________________________
F119
SEQ ID NO: 1
primer ACCCACAGGAACTGATCACCACTC
R119
SEQ ID NO: 2
primer ATGTCGCGTTCCGGCTGACGTTCTGC
P2 SEQ ID NO: 3
probe TCGCAT TACTGATCGTTGCCAACCAG Tp
P2C
SEQ ID NO: 4
complement
GTACTGGTTGGCAACGATCAGTAATGCGATG
P5 SEQ ID NO: 5
probe CGGATTTGC TGGTATCTATGACAAGGA Tp
P5C
SEQ ID NO: 6
complement
TTCATCCTTGTCATAGATACCAGCAAATCCG
AFP
SEQ ID NO: 7
primer TCACCCACACTGTGCCCATCTACGA
ARP
SEQ ID NO: 8
primer CAGCGGAACCGCTCATTGCCAATGG
A1 SEQ ID NO: 9
probe A TGCCC TCCCCCA TGCCA TCC TGCG Tp
A1C
SEQ ID NO: 10
complement
AGACGCAGGATGGCATGGGGGAGGGCATAC
A3 SEQ ID NO: 11
probe CGCCC TGGACTTCGAGCAAGAGA Tp
A3C
SEQ ID NO: 12
complement
CCATCTCTTGCTCGAAGTCCAGGGCGAC
__________________________________________________________________________
TABLE 2
__________________________________________________________________________
518 582
Probe
no temp.
+temp.
no temp.
+temp.
RQ.sup.-
RQ.sup.+
.DELTA.RQ
__________________________________________________________________________
A3-6
34.06
50.12 73.78
70.83 0.46
0.71 0.25
A3-24
58.85
202.27
69.66
78.81 0.84
2.57 1.72
P2-7
67.58
341.15
85.78
87.87 0.79
3.89 3.10
P2-27
124.57
722.22
152.58
118.42
0.82
6.10 5.28
P5-10
77.32
156.10
75.41
67.01 1.02
2.33 1.30
P5-28
73.23
507.28
106.64
96.28 0.69
5.28 4.59
__________________________________________________________________________
TABLE 3
______________________________________
518 582 RQ
Probe ss ds ss ds ss ds
______________________________________
P2-7 63.81 84.07 96.52 142.97 0.66 0.59
P2-27 92.31 557.53 165.13
89.47 0.56 6.23
P5-10 266.30 366.37 437.97
491.00 0.61 0.75
P5-28 51.91 782.80 141.20
154.07 0.37 5.08
A1-7 18.40 60.45 105.53
218.83 0.17 0.28
A1-26 87.75 734.37 90.91 118.57 0.97 6.19
A3-6 44.77 104.80 90.80 177.87 0.49 0.59
A3-24 45.57 857.57 100.15
191.43 0.46 3.47
______________________________________
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 12
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ACCCACAGGAACTGATCACCACTC24
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
ATGTCGCGTTCCGGCTGACGTTCTGC26
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TCGCATTACTGATCGTTGCCAACCAGT27
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GTACTGGTTGGCAACGATCAGTAATGCGATG31
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CGGATTTGCTGGTATCTATGACAAGGAT28
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
TTCATCCTTGTCATAGATACCAGCAAATCCG31
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
TCACCCACACTGTGCCCATCTACGA25
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CAGCGGAACCGCTCATTGCCAATGG25
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATGCCCTCCCCCATGCCATCCTGCGT26
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
AGACGCAGGATGGCATGGGGGAGGGCATAC30
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CGCCCTGGACTTCGAGCAAGAGAT24
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CCATCTCTTGCTCGAAGTCCAGGGCGAC28
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