U.S. patent application number 09/565191 was filed with the patent office on 2003-07-03 for methods, kits and compositions for supressing the binding of detectable probes to non-target sequences in hybridization assays.
Invention is credited to Coull, James M., Fiandaca, Mark J., Godtfredsen, Sven E., Hyldig-Nielsen, Jens J., Stefano, Kyriaki.
Application Number | 20030124521 09/565191 |
Document ID | / |
Family ID | 27364099 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030124521 |
Kind Code |
A1 |
Coull, James M. ; et
al. |
July 3, 2003 |
Methods, kits and compositions for supressing the binding of
detectable probes to non-target sequences in hybridization
assays
Abstract
This invention relates to methods, kits and compositions
suitable for the improved detection, analysis and quantitation of
nucleic acid target sequences using probe based hybridization
assays. The invention is more specifically directed to methods,
kits and compositions suitable for suppressing the binding of
detectable nucleic acid probes or detectable PNA probes to
non-target nucleic acid sequences in an assay for a target nucleic
acid sequence to thereby improve the reliability, sensitivity and
specificity of the assay. The methods, kits and compositions of
this invention are particularly well suited to the detection and
analysis of nucleic acid point mutations.
Inventors: |
Coull, James M.; (Westford,
MA) ; Hyldig-Nielsen, Jens J.; (Holliston, MA)
; Godtfredsen, Sven E.; (Binningen, CH) ;
Fiandaca, Mark J.; (Princeton, MA) ; Stefano,
Kyriaki; (Hopkinton, MA) |
Correspondence
Address: |
BRIAN D. GILDEA
APPLIED BIOSYSTEMS
15 DEANGELO DRIVE
BEDFORD
MA
01730
US
|
Family ID: |
27364099 |
Appl. No.: |
09/565191 |
Filed: |
May 5, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09565191 |
May 5, 2000 |
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08963472 |
Nov 3, 1997 |
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6110676 |
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08963472 |
Nov 3, 1997 |
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08937709 |
Sep 25, 1997 |
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60032349 |
Dec 4, 1996 |
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Current U.S.
Class: |
435/6.18 ;
435/6.1; 530/350 |
Current CPC
Class: |
C12Q 1/6832
20130101 |
Class at
Publication: |
435/6 ;
530/350 |
International
Class: |
C12Q 001/68; C07K
014/00 |
Claims
We claim:
1. A method for suppressing the binding of a detectable probe to a
non-target sequence in an assay of a sample for a target sequence,
the method comprising the steps of: a. contacting the sample with a
set containing two or more probes under conditions suitable for the
probes to hybridize to nucleic acid, wherein, at least one of said
probes is a detectable probe labeled with a detectable moiety and
having a sequence complementary or substantially complementary to
the target sequence, and at least one of the other probes is an
unlabeled or independently detectable probe having a sequence
complementary or substantially complementary to a non-target
sequence which may be present in the sample; provided that at least
one of the detectable probe and the unlabeled or independently
detectable probe is a PNA probe; and b. detecting the presence or
amount of target sequence present in the sample by directly or
indirectly detecting or quantitating the detectable moiety of said
detectable probe which hybridized to the target sequence.
2. The method of claim 1, wherein all probes of the set are PNA
probes.
3. The method of claim 1, wherein the target sequence is DNA or
RNA.
4. The method of claim 1, wherein the target sequence is
immobilized to a surface.
5. The method of claim 4, further comprising the step of: c.
collecting an aliquot, by elution from the surface, containing any
of the one or more detectable probes which hybridized to target
sequence when performing step (a.).
6. The method of claim 5, wherein each of the detectable probes,
having a distinct sequence, comprise independently detectable
moieties which are used to identify or quantitate the presence or
amount of each distinct probe sequence present in the aliquot
collected.
7. The method of claim 6, wherein the detectable moiety of the
detectable probe is a mass marker and the identity of each distinct
probe in the aliquot is determined by mass analysis using
Positive-ion Fast Atom Bombardment Tandem Mass Spectrometry.
8. The method of claim 1, wherein target and non-target sequences
are closely related sequences.
9. The method of claim 8, wherein target sequence and the
non-target sequence are related as a point mutation.
10. The method of claim 1, wherein the assay is used to detect,
identify, or quantitate the presence or amount of an organism or
virus in the sample.
11. The method of claim 1, wherein the assay is used to detect,
identify, or quantitate the presence or amount of one or more
species of an organism in the sample.
12. The method of claim 1, wherein the assay is used to determine
the effect of antimicrobial agents on the growth of one or more
microorganisms in the sample.
13. The method of claim 1, wherein the assay is used to determine
the presence or amount of a taxonomic group of organisms in the
sample.
14. The method of claim 1, wherein each of the different detectable
PNA probes of the set comprise independently detectable
moieties.
15. The method of claim 1, wherein the detectable moiety is
selected from the group consisting of a chromophore, a
fluorochrome, a spin label, a radioisotope, an enzyme, a hapten and
a chemiluminescent compound.
16. The method of claim 15, wherein the enzyme is selected from the
group consisting of alkaline phosphatase, soybean peroxidase and
horseradish peroxidase.
17. The method of claim 15, wherein the hapten is selected from the
group consisting of fluorescein, biotin, 2,4-dinitrophenyl and
digoxigenin.
18. The method of claim 1, wherein step (a.) comprises i.)
incubating the sample with at least one unlabeled or independently
detectable probe having a sequence complementary or substantially
complementary to a non-target sequence which may be present in the
sample for a first period of time under conditions suitable for
probes to hybridize to nucleic acid; and ii.) incubating the sample
with the at least one detectable probe labeled with a detectable
moiety and having a sequence complementary or substantially
complementary to a target sequence for a second period of time,
under conditions suitable for probes to hybridize to nucleic
acid.
19. The method of claim 18, wherein; i.) each of the first period
of time and the second period or time is twenty minutes or less;
ii.) at least one detectable probe is a PNA probe; and iii.) at
least one unlabeled or independently detectable probe is a PNA
probe.
20. The method of claim 1, wherein the ratio of unlabeled or
independently detectable probe to detectable probe is at least two
to one
21. A kit suitable for suppressing the binding of a detectable
probe to a non-target sequence in an assay of a sample for a target
sequence, said kit comprising, a set of two or more probes wherein,
at least one of the probes is a detectable probe labeled with a
detectable moiety and having a sequence complementary or
substantially complementary to a target sequence, and at least one
of the other probes is an unlabeled or independently detectable
probe having a sequence complementary or substantially
complementary to a non-target sequence which may be present in the
sample; provided that at least one of the detectable probe and the
unlabeled or independently detectable probe is a PNA probe.
22. The kit of claim 21, wherein all probes of the set are PNA
probes.
23. The kit of claim 21, wherein each of the different detectable
probes of the set comprise independently detectable moieties.
24. The kit of claim 21, wherein each of the different detectable
probes of the set are nucleic acid probes and all other probes of
the set are unlabeled PNA probes each having a defined sequence
which is complementary to a non-target sequence which may be
present in the sample.
25. The kit of claim 22, wherein the different detectable probes of
the set are PNA probes comprising independently detectable
moieties.
26. The kit of claim 21, wherein the kit is designed to detect,
identify, or quantitate the presence or amount of an organism or
virus in the sample.
27. The kit of claim 21, wherein the kit is designed to detect,
identify, or quantitate the presence or amount of one or more
species of an organism in the sample.
28. The kit of claim 21, wherein the kit is used to determine the
effect of antimicrobial agents on the growth of one or more
microorganisms in the sample.
29. The kit of claim 22 comprising two PNA probes, wherein the
first PNA probe has a sequence complementary to the target sequence
and is labeled with a detectable moiety and the second PNA probe is
unlabeled and has a sequence complementary to a non-target sequence
which may be present in the sample.
30. The kit of claim 29, wherein each of the two PNA probes are
designed to hybridize specifically to complementary target and
non-target sequences wherein the target and non-target sequences
are related as point mutations.
31. The kit of claim 21 comprising a set of four probes, wherein
the probes of the set consist of a single detectable probe labeled
with a detectable moiety and having a sequence complementary to the
target sequence, and three unlabeled probes each having a defined
sequence which is complementary to a non-target sequence which may
be present in the sample.
32. The kit of claim 31, wherein the probes of the kit are PNA
probes which are designed to hybridize to target and non-target
sequences which are related as point mutations.
33. The kit of claim 31, wherein the detectable probe is a nucleic
acid probe and the other probes of the kit are unlabeled PNA probes
each having a defined sequence which is complementary to a
non-target sequence which may be present in the sample.
34. A composition for suppressing the binding of a detectable probe
to a non-target sequence in an assay of a sample for a target
sequence, said composition consisting of a set of two probes,
wherein, one of the probes is a detectable probe labeled with a
detectable moiety and having a sequence complementary to the target
sequence, and the other probe is an unlabeled probe having a
sequence complementary to a non-target sequence which may be
present in the sample, provided that either of the detectable probe
or the unlabeled or independently detectable probe is a PNA
probe.
35. The composition of claim 34, wherein the probes of the
composition are PNA probes which are designed to hybridize
specifically to complementary target and non-target sequences
wherein the target and non-target sequences are related as point
mutations.
36. The composition of claim 34, wherein the detectable probe is a
nucleic acid probe and the unlabeled probe is a PNA probe which is
complementary to a non-target sequence which may be present in the
sample.
37. A composition for suppressing the binding of a detectable probe
to a non-target sequence in an assay of a sample for a target
sequence, said composition consisting of a set of four probes
wherein, one of the probes is a detectable probe labeled with a
detectable moiety and having a sequence complementary to the target
sequence, and the other three probes are unlabeled probes each
having a defined sequence which is complementary to a non-target
sequence which may be present in the sample, provided that at least
one of the detectable probe and the unlabeled or independently
detectable probe is a PNA probe.
38. The composition of claim 37, wherein the probes of the
composition are PNA probes which are designed to hybridize
specifically to complementary target and non-target sequences
wherein the target and non-target sequences are related as point
mutations.
39. The composition of claim 37, wherein the detectable probe is a
nucleic acid probe and the three unlabeled probes are PNA probes
each having a defined sequence which is complementary to a
non-target sequence which may be present in the sample.
40. A method for suppressing the binding of a non-target sequence
to a capture probe immobilized on a surface in a capture assay of a
sample for a target sequence of a nucleic acid target molecule, the
method comprising the steps of: a. contacting the sample with a
solution containing one or more blocking probes under conditions
suitable for the blocking probes to hybridize to nucleic acid,
wherein the blocking probes are complementary or substantially
complementary to one or more non-target sequences which may be
present in the sample; b. contacting the sample with the capture
probe immobilized on a surface under conditions suitable for the
target sequence, if present, to hybridize to the capture probe,
wherein the capture probe is complementary or substantially
complementary to the target sequence and thereby forms a capture
probe/target sequence complex; provided that at least one of a
blocking probe or a capture probe is a PNA probe; and c. detecting
the presence or amount of nucleic acid target molecule immobilized
to the surface.
41. The method of claim 40, wherein the target nucleic acid
molecule is DNA or RNA.
42. The method of claim 40, wherein all probes are PNA probes.
43. The method of claim 40, wherein the target and the non-target
sequences are related as a point mutation.
44. The method of claim 40, wherein the surface comprises an array
of probes, wherein each distinct probe sequence in the array is
designed to capture a specific nucleic acid target molecule, the
presence or quantity of which is indicative of the presence or
quantity of a specific organism virus, fungi or disease state of
interest in the sample
45. The method of claim 40, wherein the presence or amount of
nucleic acid target molecule immobilized to the surface is detected
using a labeled antibody which specifically interacts with the
capture probe/target sequence complex which is formed on the
surface.
46. The method of claim 45, wherein the labeled antibody is a
labeled anti-nucleic acid/nucleic acid antibody which detects the
presence of the capture probe/target sequence complex which is
formed by hybridization of the capture probe to the target
sequence.
47. The method of claim 46, wherein the labeled antibody is labeled
with a detectable moiety selected from the group consisting of a
chromophore, a fluorophore, a spin label, a radioisotope, an
enzyme, a hapten and a chemiluminescent compound.
48. The method of claim 45, wherein the labeled antibody is a
labeled anti-PNA/nucleic acid antibody which detects the presence
of the capture probe/target sequence complex which is formed by
hybridization of the PNA capture probe to the target sequence.
49. The method of claim 48, wherein the labeled antibody is labeled
with a detectable moiety selected from the group consisting of a
chromophore, a fluorophore, a spin label, a radioisotope, an
enzyme, a hapten and a chemiluminescent compound.
50. The method of claim 49, wherein the enzyme is selected from the
group consisting of alkaline phosphatase, soybean peroxidase and
horseradish peroxidase.
51. The method of claim 49, wherein the hapten is selected from the
group consisting of fluorescein, biotin, 2,4-dinitrophenyl and
digoxigenin.
52. The method of claim 40, wherein the presence or amount of
target sequence immobilized to the surface is detected using a
detector probe which hybridizes to a second target sequence of the
nucleic acid target molecule.
53. The method of claim 52, wherein the method of claim 1 is used
to suppress the binding of detector probe to a non-second target
sequence.
54. The method of claim 52, wherein the detector probe is labeled
with a detectable moiety selected from the group consisting of a
chromophore, a fluorophore, a spin label, a radioisotope, an
enzyme, a hapten and a chemiluminescent compound.
55. The method of claim 52, wherein the detector probe is a nucleic
acid probe and the presence or quantity of hybridized detector
probe is detected using a labeled anti-nucleic acid/nucleic acid
antibody.
56. The method of claim 52, wherein the detector probe is a PNA
probe and the presence or quantity of hybridized detector probe is
detected using a labeled anti-PNA/nucleic acid antibody.
57. The method of claim 40, wherein the capture assay is used to
detect, identify, or quantitate the presence or amount of an
organism or virus in the sample.
58. The method of claim 40, wherein the assay is used to detect,
identify, or quantitate the presence or amount of one or more
species of an organism in the sample.
59. The method of claim 40, wherein the assay IS used to determine
the effect of antimicrobial agents on the growth of one or more
microorganisms in the sample.
60. The method of claim 40, wherein the assay is used to determine
the presence or amount of a taxonomic group of organisms in the
sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/032,349, filed on Dec. 4, 1996. This application
is a continuation-in-part of U.S. Ser. No. 08/937,709, filed on
Sep. 25, 1997.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention is related to the field of probe based
nucleic acid sequence detection, quantitation and analysis. More
specifically, this invention relates to methods, kits and
compositions suitable for suppressing the binding of detectable
nucleic acid probes or detectable PNA probes to non-target
sequences in an assay for detecting a target sequence of a nucleic
acid molecule of interest.
[0004] 2. Description of the Related Art
[0005] Probe based assays are useful in the detection, quantitation
and analysis of nucleic acids. Nucleic acid probes have long been
used to analyze samples for the presence of nucleic acid from a
bacteria, fungi, virus or other organism (See for example; U.S.
Pat. Nos. 4,851,330, 5,288,611, 5,567,587, 5,601,984 and
5,612,183). Probe-based assays are also useful in examining
genetically-based clinical conditions of interest. Nonetheless,
probe-based assays have been slow to achieve commercial success.
This lack of commercial success is, at least partially, the result
of difficulties associated with specificity, sensitivity and
reliability
[0006] Nucleic acid hybridization is a fundamental process in
molecular biology Sequence differences as subtle as a single base
(point mutation) in very short oligomers (<10 base pairs "bp")
can be sufficient to enable the discrimination of the hybridization
to complementary nucleic acid target sequences as compared with
non-target sequences. Nonetheless, nucleic acid probes of greater
than 10 bp in length are generally required to obtain the sequence
diversity necessary to correctly identify a unique organism or
clinical condition of interest. However, the ability to
discriminate between closely related sequences is inversely
proportional to the length of the hybridization probe because the
difference in thermal stability decreases between wild type and
mutant complexes as the probe length increases. Consequently, the
power of probe based hybridization to correctly identify the target
sequence of interest from closely related (e.g. point mutations)
non-target sequences can be very limited. A extensive review of the
"Principles and Practices of Nucleic Acid Hybridization" is
available (See: David E Kennell, Principles and Practices of
Nucleic Acid Hybridization, pp. 259-301). In the manuscript, the
author discusses the "Use of Competitor RNA to Estimate
Specificity". This process is based on the principle that two
identical molecules will compete with each other for a common
binding site. This principle is applied to assess similarities
between two RNA populations competing for a common DNA. Typically,
one population of RNA is labeled and the competitor population of
RNA is unlabeled. The competition assay is used to estimate the
degree of relation between the two RNA species. A process called
"presaturation competition", wherein the unlabeled competitor RNA
is hybridized to the DNA before hybridization of the labeled RNA,
has been reported to be useful in improving the results of this
type of assay (See: p 297). However, the author warns that "great
caution should be exercised" in interpreting the data from these
assays (See: p. 291 and p. 298 first full paragraph). No data is
provided which quantitates the benefits associated with the
application of this methodology.
[0007] Gray et al. describe in-situ methods for chromosome-specific
staining wherein the hybridization of labeled nucleic acid
fragments to repetitive sequences of chromosomal DNA is disabled
(See: Gray et al. U.S. Pat. No. 5,447,841). In one embodiment of
the invention, disabling of the hybridization capacity of the
repetitive DNA sequences within nucleic acid fragments involves
blocking the repetitive sequences by pre-reassociation of fragments
with fragments of repetitive-sequence-rich DNA, by
pre-reassociation of target DNA with fragments of
repetitive-sequence-rich DNA, or pre-reassociation of both the
fragments of the heterogeneous mixture and the target DNA with
repetitive-sequence-rich DNA (See: col. 9, Ins. 58-68). The
pre-reassociation procedure may be performed in a number of
differing formats (See: claims 2-5). This method provides blocking
sufficient to permit detection of large labeled nucleic acid
(greater than 1000 bp) hybridized to chromosomal DNA (See: claim
1). No data is provided which quantitates the benefits associated
with the application of this methodology. Moreover, this treatment
merely results in nucleic acid fragments whose repetitive sequences
are blocked by complementary fragments such that sufficient unique
sequence regions remain free for attachment to chromosomal DNA
during the in-situ hybridization step (See: col. 10, Ins.
3-13).
[0008] Hybridization assays hold promise as a means to screen large
numbers of patient samples for a large number of mutations. In
practice, however, it is often difficult to multiplex an assay
given the requirement that each of the many very different probes
in the assay must exhibit a very high degree of specificity for a
specific target nucleic acid under the same or similar conditions
of stringency. Recently however, a probe based assay has been shown
to be effective at selectively detecting up to twelve cystic
fibrosis transmembrane conductance regulator (CFTR) mutations using
pools of allele specific oligonucleotides "ASOs" (See: Shuber et
al., Human Mol. Gen., (1993) 2, 153-158). The authors utilized a
tetramethylammonium chloride (TMAC) buffer to eliminate variability
in the affinity of the nucleic acid probes for their complementary
target nucleic acid sequences. Interestingly, the authors describe
the use of labeled and unlabeled nucleic acid probes in the
hybridization cocktail. However, there is no discussion of the
rational for applying this methodology and there is no data
provided which quantitates the benefits associated with application
of this technology.
[0009] More recently, Shuber and his coworkers introduccd a
technique they coined MASDA (multiplex allele specific diagnostic
assay) See Shuber et al Human Mol Gen (1997) 6, 337-347 In this
assay, a single hybridization is performed with a pool of allele
specific oligonucleotide probes The ASOs are affinity purified from
the pool by hybridization to the target nucleic acid (patient
sample) which has been immobilized to a surface. Probes, which
hybridize to the target nucleic acid, are thereafter eluted from
the surface and analyzed to thereby determine the presence or
absence of one or more clinical conditions of interest. The authors
report that they observe such a high degree of specificity of
hybridization of the component labeled ASOs of the pool that, in a
single assay, the method is capable of analyzing greater than 500
samples for greater than 100 known mutations. As in the prior
Shuber publication, the authors describe the use of a hybridization
cocktail containing both labeled and unlabeled probes. This
cocktail is prepared to achieve uniform hybridization signals in
the assay. However, no data is provided which quantitates the
benefits associated with the application of this methodology.
[0010] The background art thus far discussed does not disclose,
suggest or teach anything about Peptide Nucleic Acids (PNAs).
[0011] Peptide Nucleic Acids (PNAs) are non-naturally occurring
polyamides which can hybridize to nucleic acids (DNA and RNA) with
sequence specificity. (See U.S. Pat. No. 5,539,082 and Egholm et
al., Nature (1993) 365, 566-568). PNA's are candidates for
investigation as alternatives/substitutes to nucleic acid probes in
probe-based hybridization assays because they exhibit several
desirable properties. PNA's are achiral polymers which hybridize to
nucleic acids to form hybrids which are more thermodynamically
stable than a corresponding nucleic acid/nucleic acid complex (See:
Egholm et. al., Nature (1993) 365, 566-568). Being non-naturally
occurring molecules, they are not known to be substrates for the
enzymes which are known to degrade peptides or nucleic acids.
Therefore, PNA's should be stable in biological samples, as well
as, have a long shelf-life. Unlike nucleic acid hybridization which
is very dependent on ionic strength, the hybridization of a PNA
with a nucleic acid is fairly independent of ionic strength and is
favored at low ionic strength under conditions which strongly
disfavor the hybridization of nucleic acid to nucleic acid (See:
Egholm et al, Nature, p 567) The effect of ionic strength on the
stability and conformation of PNA complexes has been extensively
investigated (See: Tomac et at J Am Chem Soc. (1996) 118,
5544-5552). Sequence discrimination is more efficient for PNA
recognizing DNA than for DNA recognizing DNA (See: Egholm et al.,
Nature, p. 566). However, the advantages in point mutation
discrimination with PNA probes, as compared with DNA probes, in a
hybridization assay appears to be somewhat sequence dependent (See;
Nielsen et al. Anti-Cancer Drug Design (1993) 8, 53-65). As an
additional advantage, PNA's hybridize to nucleic acid in both a
parallel and antiparallel orientation, though the antiparallel
orientation is preferred (See: Egholm et al., Nature, p. 566).
[0012] PNAs are synthesized by adaptation of standard peptide
synthesis procedures in a format which is now commercially
available. (For a general review of the preparation of PNA monomers
and oligomers please see: Dueholm et al., New J. Chem. (1997), 21,
19-31 or Hyrup et. al., Bioorganic & Med. Chem. (1996) 4,
5-23). Labeled and unlabeled PNA oligomers can be purchased (See:
PerSeptive Biosystems Promotional Literature: BioConcepts,
Publication No. NL612, Practical PNA, Review and Practical PNA,
Vol. 1, Iss. 2) or prepared using the commercially available
products.
[0013] Labeled PNA probes have been hybridized to target nucleic
acid subsequences of denatured dsDNA as a means to detect the
presence and amount of the DNA of interest in an assay coined
"pre-gel hybridization" (See: O'Keefe et al. Proc. Natl. Acad. Sci.
USA (1996) 93, 14670-14675). This assay relies on the rapid
kinetics of PNA/DNA hybrid formation and the relatively slow rate
of reannealing of the dsDNA. Thus, under conditions of low salt,
the sample is analyzed for the presence of the PNA/DNA hybrid
before the PNA/nucleic acid complex is dissociated by the
reannealing/reformation of the dsDNA. "Pre-gel hybridization" is
reported to provide very good discrimination of point mutations in
a DNA sample (See: FIG. 4 of the O'Keefe manuscript and the
associated description).
[0014] In a similar manner, unlabeled PNAs have been shown to be
effective at blocking the interstrand and intrastrand interactions
of dsDNA to thereby enhance the PCR amplification of variable
numbers of tandem repeat (VNTR) loci (See. Demers et al Nucl Acids
Res., (1995) 23, 3050-3055 and U.S. Pat. No. 5,656,461) For this
application, the unlabeled PNAs need to be designed such that they
form PNA/nucleic acid hybrids which are stable enough to disrupt
the interstrand and intrastrand interactions of dsDNA. However, the
PNA/nucleic acid complex must be susceptible to dissociation by the
operation of the polymerase during primer extension. In still
another related application, a process coined "PCR clamping" can be
used to obtain point mutation discrimination when directing
unlabeled PNAs of defined sequence to interfere with the PCR
process (See: .O slashed.rum et al. Nucl. Acids Res. (1993), 21,
5332-5336). In one embodiment of PCR clamping, an unlabeled PNA,
which is identical in nucleobase composition to the PCR primer,
competes with the PCR primer for binding to the common recognition
site. In another embodiment, the target site for the unlabeled PNA
is located within the PCR amplicon region. In this embodiment,
clamping operates if the PNA/nucleic acid hybrid is stable enough
to prevent read through by the polymerase. In yet another
embodiment, the target site for the unlabeled PNA is located
adjacent to the PCR priming site. In this embodiment, PCR clamping
may operate either by preventing read through of the polymerase or
by preventing (blocking) primer annealing. To obtain point mutation
discrimination using PCR clamping, longer mutant and wild type
nucleic acid PCR primers are designed such that amplification
proceeds only if the longer PCR primer is a perfect complement to
the recognition site and thereby out competes the unlabeled PNA for
binding within that site. PCR clamping has recently been directed
to analysis of the Ki-ras mutations of codon 12 and 13 (See: Thiede
et al. Nucl. Acids Res. (1996) 24, 983-984).
[0015] Very recently, the "Hybridization based screening on peptide
nucleic acid (PNA) oligomer arrays" has been described wherein
arrays of some 1000 PNA oligomers of individual sequence were
synthesized on polymer membranes (See: Weller et al. Nucl. Acids
Res. (1997) 25, 2792-2799). Arrays are generally used, in a single
assay, to generate affinity binding (hybridization) information
about a specific sequence or sample to numerous probes of defined
composition. Thus, PNA arrays may be useful in diagnostic or
antisence applications However, in the present study, the authors
note that the affinity and specificity of DNA hybridization to
immobilized PNA oligomers depended on hybridization conditions more
than was expected. Moreover, there was a tendency toward
non-specific binding at lower ionic strength. Furthermore, certain
very strong binding mismatches were identified which could not be
eliminated by more stringent washing conditions. These results
demonstrate the need for improved methods of suppressing the
binding of nucleic acids to non-complementary PNAs. Moreover, these
unexplained results are also illustrative of the lack of complete
understanding of these newly discovered molecules (i.e. PNA)
[0016] There are indeed many differences between PNA probes and
standard nucleic acid probes. These differences can be conveniently
broken down into biological, structural, and physico-chemical
differences. As discussed above and below, these biological,
structural, and physico-chemical differences may lead to
unpredictable results when attempting to use PNA probes in
applications were nucleic acids have typically been employed. This
non-equivalency of differing compositions is often observed in the
chemical arts.
[0017] With regard to biological differences, nucleic acids, are
biological materials that play a central role in the life of living
species as agents of genetic transmission and expression. Their in
vivo properties are fairly well understood. PNA, on the other hand
is recently developed totally artificial molecule, conceived in the
minds of chemists and made using synthetic organic chemistry. It
has no known biological function.
[0018] Structurally, PNA also differs dramatically from nucleic
acid. Although both can employ common nucleobases (A, C, G, T, and
U), the backbones of these molecules are structurally diverse. The
backbones of RNA and DNA are composed of repeating phosphodiester
ribose and 2-deoxyribose units. In contrast, the backbones of PNA
are composed on N-(2-aminoethyl)glycine units. Additionally, in PNA
the nucleobases are connected to the backbone by an additional
methylene carbonyl unit.
[0019] Despite its name, PNA is not an acid and contains no charged
acidic groups such as those present in DNA and RNA Because they
lack formal charge PNAs are generally more hydrophobic than their
equivalent nucleic acid molecules The hydrophobic character of PNA
allows for the possibility of non-specific (hydrophobic/hydrophobic
interactions) interactions not observed with nucleic acids.
Further, PNA is achiral, providing it with the capability of
adopting structural conformations the equivalent of which do not
exist in the RNA/DNA realm.
[0020] The physico/chemical differences between PNA and DNA or RNA
are also substantial. PNA binds to its complementary nucleic acid
more rapidly than nucleic acid probes bind to the same target
sequence. This behavior is believed to be, at least partially, due
to the fact that PNA lacks charge on its backbone. Additionally,
recent publications demonstrate that the incorporation of
positively charged groups into PNAs will improve the kinetics of
hybridization (See: Iyer et al. J. Biol. Chem. (1995) 270,
14712-14717). Because it lacks charge on the backbone, the
stability of the PNA/nucleic acid complex is higher than that of an
analogous DNA/DNA or RNA/DNA complex. In certain situations, PNA
will form highly stable triple helical complexes or form small
loops through a process called "strand displacement". No equivalent
strand displacement processes or structures are known in the
DNA/RNA world.
[0021] In summary, because PNAs hybridize to nucleic acids with
sequence specificity, PNAs are useful candidates for developing
probe-based assays. However, PNA probes are not the equivalent of
nucleic acid probes. Nonetheless, even under the most stringent
conditions both the exact target sequence and a closely related
sequence (e.g. a non-target sequence having a single point mutation
(a.k.a. single base pair mismatch)) will often exhibit detectable
interaction with a labeled nucleic acid or labeled PNA probe (See:
Nielsen et al. Anti-Cancer Drug Design at p. 56-57 and Weller et
al. at p. 2798, second full paragraph). Any hybridization to a
closely related non-target sequence will result in the generation
of undesired background signal. Because the sequences are so
closely related, point mutations are the some of the most difficult
of all nucleic acid modifications to detect using a probe based
assay Numerous diseases, such as sickle cell anemia and cystic
fibrosis, are caused by a single point mutation of genomic nucleic
acid. Consequently, any method, kits or compositions which could
improve the specificity, sensitivity and reliability of probe-based
assays would be useful in the detection, analysis and quantitation
of nucleic acid containing samples and particularly useful for
nucleic acid point mutation analysis.
OBJECTS OF THE INVENTION
[0022] It is an object of the invention to provide methods, kits
and compositions suitable for the suppression of the binding of
probes to non-target sequences in hybridization assays.
[0023] It is an object of this invention to provide methods, kits
and compositions suitable for improving the specificity sensitivity
and reliability of nucleic acid point mutation detection, analysis
and quantitation.
SUMMARY OF THE INVENTION
[0024] This invention relates to methods, kits and compositions
suitable for the improved detection, quantitation and analysis of
nucleic acid target sequences using probe-based hybridization
assays. The invention is more specifically directed to methods,
kits and compositions suitable for suppressing the binding of
detectable probes to non-target sequences in an assay for a target
sequence of a nucleic acid target molecule. Suppression of the
nonspecific binding of detectable probe directly improves the
sensitivity of the assay thereby improving the signal to noise
ratio of the assay. Suppression of nonspecific binding will also
result in improvements in reliability since the incidence of false
positives and false negative should also be reduced. Because the
methods, kits and compositions of this invention are directed to
the suppression of nonspecific binding of probes to nucleic acids,
they are particularly well suited for the development of sensitive
and reliable probe-based hybridization assays designed to analyze
for point mutations. The methods, kits and compositions of this
invention should also find utility for the detection, quantitation
or analysis of organisms (micro-organisms), viruses, fungi and
genetically based clinical conditions of interest.
[0025] It has been surprisingly observed that the signal caused by
the nonspecific binding of detectable probes to one or more
non-target nucleic acid sequences can be dramatically suppressed by
the addition of one or more unlabeled probes wherein the sequence
of the one or more unlabeled probes is complementary to one or more
non-target sequences to which the detectable probe binds in a
nonspecific manner. For example, it has been observed that the
addition of 25 equivalents of unlabeled PNA probe, having a single
mismatch as compared with the labeled PNA probe, does not
substantially alter the detection limit of the assay. However, the
presence of the unlabeled PNA probe resulted in at least a 10 fold
suppression in the binding of labeled PNA probe to the non-target
sequence (point mutation) and a correlating improvement of
approximately 30 fold, in the signal to noise ratio of the assay
(see Example 4A and FIG. 1)
[0026] When the unlabeled PNA probe Was present at 500 equivalents,
there was very little loss of detectable signal (approximately 3 to
10 fold). However, suppression of binding of the labeled probe to a
non-target sequence (point mutation) is substantially improved as
compared with the experiment wherein only 25 equivalents of
unlabeled PNA probe was present (Compare: Examples 4A and 4B of
this specification). The results demonstrate that point mutation
discrimination improved from approximately 10 fold in the absence
of the unlabeled probe to greater than 1000 fold in the presence of
high levels of unlabeled (blocker) PNA probe. Consequently, when
employing the methods described herein, one can achieve several
logs of improvement in point mutation discrimination and similar
dramatic improvements in the dynamic range of the hybridization
assay.
[0027] The applicants are not aware of any similar method suitable
for obtaining such a dramatic suppression of binding to non-target
sequences and the correlating improvement in signal to noise ratio.
The data presented in Example 6, demonstrates the clear superiority
of PNA probes as compared with DNA probes with regard to
suppression of binding to non-target sequences, improvement in
signal to noise ratios and point mutation discrimination.
[0028] In preferred embodiments of this invention, PNA probes are
used either alone or in combination with nucleic acid probes. When
combined with nucleic acid probes, the preferred combination
involves unlabeled PNA probes used to suppress the binding of
detectable (labeled) nucleic acid probes to non-target sequences.
In the most preferred embodiment of this invention, both the
detectable probes and unlabeled or independently detectable probes
are PNA probes because this embodiment exhibits both the greatest
ability to suppress binding to non-target sequences and the
greatest ability to discriminate point mutations.
[0029] The hybridization assay of this invention can be performed
in solution. Alternatively, one or more assay components may be
immobilized to a surface. Thus, in one embodiment the nucleic acid
target molecule comprising the target sequence is immoblized to a
surface. In this embodiment, the immobilized target sequence is
contacted with a solution containing the detectable and unlabeled
or independently detectable probes (e.g. dot blot format).
Alternatively, one or more probes may be immobilized on a surface
and used, in a capture assay, to capture the nucleic acid target
molecule comprising the target sequence. In a preferred embodiment,
arrays of greater than two probes are used to generate binding
(affinity) or sequence information about one or more nucleic acid
target molecules of interest which may be present in the
sample.
[0030] In one embodiment, the invention is related to a method for
suppressing the binding of detectable probe to a non-target
sequence in an assay of a sample for a target sequence. The method
comprises contacting the sample with a set containing two or more
probes under conditions suitable for the probes to hybridize to
nucleic acid. At least one of the probes is a detectable probe
labeled with a detectable moiety and having a sequence
complementary or substantially complementary to a target sequence.
At least one of the other probes is an unlabeled or independently
detectable probe having a sequence complementary or substantially
complementary to a non-target sequence. The second step comprises
detecting the presence, absence or quantity of a target sequence in
the sample by directly or indirectly detecting or quantitating the
detectable moiety. At least one of either a detectable probe or an
unlabeled or independently detectable probe is a PNA probe.
Preferably, the one or more unlabeled or independently detectable
probes is a PNA probe. Most preferably, all the probes are PNA
probes. In preferred embodiments, the detectable probe is perfectly
complementary to a target sequence and the unlabeled or
independently detectable probe is perfectly complementary to a
non-target sequence which may be present in the sample.
[0031] In another embodiment, the invention relates to a kit
suitable for suppressing the binding of a detectable probe to a
non-target sequence in an assay of a sample for a target sequence.
The kit comprises a set of two or more probes wherein, at least one
of the probes is a detectable probe labeled with a detectable
moiety and having a sequence complementary or substantially
complementary to the target sequence. At least one of the other
probes is an unlabeled or independently detectable probe having a
sequence complementary or substantially complementary to the
non-target sequence. At least one of either a detectable probe or
an unlabeled or independently detectable probe is a PNA probe.
Preferably, the one or more unlabeled or independently detectable
probes is a PNA probe. Most preferably, all the probes are PNA
probes. In preferred embodiments, the detectable probe is perfectly
complementary to a target sequence and the unlabeled or
independently detectable probe is perfectly complementary to a
non-target sequence which may be present in the sample.
[0032] In another embodiment, the invention relates to a
composition for suppressing the binding of a detectable probe to a
non-target sequence in an assay of a sample for a target sequence.
The composition consists of a set of two probes wherein, one of the
probes is a detectable probe labeled with a detectable moiety and
having a sequence complementary to the target sequence. The other
probe is an unlabeled or independently detectable probe having a
sequence complementary to a non-target sequence. Either of the
detectable probe or the unlabeled or independently detectable probe
is a PNA probe. Preferably, the unlabeled or independently
detectable probe is the PNA probe. Most preferably, both probes of
the composition are PNA probes.
[0033] In another embodiment, the invention relates to a
composition for suppressing the binding of a detectable probe to a
non-target sequence in an assay of a sample for a target sequence.
The composition consists of a set of four probes wherein, one of
the probes is a detectable probe labeled with a detectable moiety
and having a sequence complementary to the target sequence. The
other three probes are unlabeled or independently detectable probes
having sequences which hybridize specifically with non-target
sequences which are related to the target sequence as the three
possible single point mutations. Either of the detectable probe or
at least one of the three unlabeled or independently detectable
probes is a PNA probe. Preferably the three unlabeled or
independently detectable probes are PNA probes. Most preferably,
all probes of the composition are PNA probes.
[0034] In yet another embodiment, this invention relates to a
method for suppressing the binding of a non-target sequence to a
capture probe immobilized on a surface in a capture assay of a
sample for a target sequence. The method comprises contacting the
sample with a solution containing one or more blocking probes under
conditions suitable for the blocking probes to hybridize to nucleic
acid. Each of the blocking probes is complementary or substantially
complementary to one or more non-target sequences which may be
present in the sample. The sample is also contacted with at least
one capture probe immobilized on a surface under conditions
suitable for the target sequence, if present, to hybridize to the
capture probe. The immobilized capture probe is complementary or
substantially complementary to the target sequence and thereby
forms a capture probe/target sequence complex. The presence or
amount of nucleic acid target molecule immobilized to the surface
is then detected, identified or quantitated. In preferred
embodiments, an array of two or more capture probes immobilized on
a surface is used to screen one or more samples for one or more
target sequences of interest. In preferred embodiments, the
blocking probe is perfectly complementary to a non-target sequence
and the capture probe is perfectly complementary to a target
sequence.
[0035] In preferred embodiments of this invention, a multiplex
hybridization assay is performed. In a multiplex assay, numerous
conditions of interest are simultaneously examined. Multiplex
analysis relies on the ability to sort sample components or the
data associated therewith, during or after the assay is completed.
In preferred embodiments of the invention, distinct independently
detectable moieties are used to label each of the different labeled
probes of the set. The ability to differentiate between and
quantitate each of the independently detectable moieties provides
the means to multiplex the hybridization assay because the data
which correlates with the hybridization of each of the distinct
independently detectable probes to a target sequence of interest
can be correlated with the data for each of the independently
detectable moieties. Because multiplex hybridization assays involve
numerous probes and target sequences, there is a great potential
for non-specific hybridization to adversely effect the reliability
of the multiplex assay. However, application of the methods of this
invention will substantially improve the sensitivity and
reliability of multiplex probe-based hybridization assays
[0036] In summary, the methods, kits and compositions of this
invention are used to suppress binding of probes to non-target
sequences thereby substantially improving sequence discrimination
and dynamic range of the hybridization assay. These advantages
substantially improve the sensitivity and reliability of
probe-based assays in general and are particularly useful when
employing multiplex methodologies and/or point mutation
analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is an autoradiogram generated by exposure of a film
to enzymatically catalyzed chemiluminescence emanating from a nylon
membrane.
[0038] FIG. 2 is a composite electronic image of autoradiograms
generated by exposure of a film to enzymatically catalyzed
chemiluminescence emanating from a nylon membrane.
[0039] FIG. 3 is a Molecular Diagram of hybridization assay
components.
[0040] FIG. 4A is a schematic of a plate assay.
[0041] FIG. 4B is a graphical illustration of hybridization assay
data.
[0042] FIG. 4C is a graphical illustration of hybridization assay
data.
[0043] FIG. 4D is a tabular illustration of hybridization assay
data.
[0044] FIG. 5A is a schematic of a plate assay.
[0045] FIG. 5B is a tabular illustration of hybridization assay
data.
[0046] FIG. 5C is a graphical illustration of hybridization assay
data.
[0047] FIG. 5D is a tabular illustration of hybridization assay
data.
[0048] FIG. 5E is a graphical illustration of hybridization assay
data.
[0049] FIG. 6A is a schematic of a plate assay.
[0050] FIG. 6B is a tabular illustration of hybridization assay
data.
[0051] FIG. 6C is a tabular illustration of hybridization assay
data.
[0052] FIG. 7 is a sequence schematic of the target and probe
binding sites in Neisseria gonorrhoeae and Neisseria
meningitidis.
[0053] FIG. 8A is a schematic of a plate assay.
[0054] FIG. 8B is a tabular illustration of hybridization assay
data.
[0055] FIG. 8C is a tabular illustration of hybridization assay
data.
[0056] FIG. 8D is a tabular illustration of hybridization assay
data.
[0057] FIG. 8E is a graphical illustration of hybridization assay
data
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0058] 1. Definitions:
[0059] a. As used herein, the term "Peptide Nucleic Acid" or "PNA"
is defined as any of the compounds referred to or claimed as a
Peptide Nucleic Acids in U.S. Pat. No. 5,539,082. The term "Peptide
Nucleic Acid" or "PNA" shall also apply to those compositions
referred to as Peptide Nucleic Acids in the following
publications:
[0060] Diderichsen et al., Tett. Lett. (1996) 37, 475-478;
[0061] Fujii et al., Bioorg. Med. Chem. Lett. (1997) 7,
637-640;
[0062] Jordan et al., Bioorg. Med. Chem. Lett. (1997) 7,
687-690;
[0063] Krotz et al., Tett. Lett. (1995) 36, 6941-6944;
[0064] Lagriffoul et al., Bioorg. Med. Chem. Lett. (1994) 4,
1081-1082;
[0065] Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1,
539-546;
[0066] Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1,
547-554;
[0067] Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1,
555-560;
[0068] Petersen et al., Bioorg. Med. Chem. Lett. (1996) 6, 793-796;
and
[0069] U.S. Pat. No. 5,623,049.
[0070] b. As used herein, the term "complementary sequence" or
"complementary probe" is defined as the subunit sequence of a DNA,
RNA or PNA oligomer designed to hybridize with exact
complementarity to a nucleic acid sequence or subsequence.
[0071] c. As used herein, the term "PNA probe" is defined as any
oligomer, comprising two or more PNA subunits (residues, monomer),
suitable for hybridizing to a nucleic acid (DNA or RNA) sequence.
The PNA probe may be labeled with a detectable moiety or may be
unlabeled.
[0072] d. As used herein, the term "target sequence" is any defined
nucleic acid sequence to be detected in an assay. The "target
sequence" may comprise the entire sequence of interest or may be a
subsequence of the nucleic acid target molecule of interest.
[0073] e. As used herein, the term "non-target sequence" is any
defined nucleic acid sequence which is not a target sequence. The
non-target sequences which generate the most background will be
sequences which are closely related to the target sequence (e.g.
point mutations).
[0074] f. As used herein, the term "single base pair mismatch" or
"point mutation" is defined as the modification of a defined
nucleic acid sequence such that a single nucleotide within the
defined nucleic acid sequence has been substituted.
[0075] g. As used herein, the term "sensitivity" or "assay
sensitivity" is defined as the difference in signal intensity
caused by or attributable to the binding of detectable probe to its
complementary sequence and any background or signal caused or
attributable to any other source.
[0076] h. As used herein, the term "assay limit" or "limit of
detection" is defined as the lower limit of signal intensity caused
by the specific binding of detectable probe which can be detected
above the background (noise).
[0077] i. As used herein, the terms "signal to noise" and "dynamic
range" shall be interchangeable.
[0078] 2. Detailed Description
[0079] General:
[0080] This invention relates to methods, kits and compositions
suitable for the improved detection, quantitation and analysis of
nucleic acid target sequences using probe-based hybridization
assays. The invention is more specifically directed to methods,
kits and compositions suitable for suppressing the binding of
detectable probes to non-target sequences in an assay for a target
sequence of a nucleic acid target molecule. Suppression of the
nonspecific binding of detectable probe directly improves the
sensitivity of the assay thereby improving the signal to noise
ratio of the assay. Suppression of nonspecific binding will also
result in improvements in reliability since the incidence of false
positives and false negative should also be reduced. Because the
methods, kits and compositions of this invention are directed to
the suppression of nonspecific binding of probes to nucleic acids,
they are particularly well suited for the development of sensitive
and reliable probe-based hybridization assays designed to analyze
for point mutations. The methods, kits and compositions of this
invention should also find utility for the detection, quantitation
or analysis of organisms (micro-organisms), viruses, fungi and
genetically based clinical conditions of interest.
[0081] It has been surprisingly observed that the signal caused by
the nonspecific binding of detectable probes to one or more
non-target nucleic acid sequences can be dramatically suppressed by
the addition of one or more unlabeled probes wherein the sequence
of the one or more unlabeled probes is complementary to one or more
non-target sequences to which the detectable probe binds in a
nonspecific manner. For example, it has been observed that the
addition of 25 equivalents of unlabeled PNA probe, having a single
mismatch as compared with the labeled PNA probe, does not
substantially alter the detection limit of the assay. However, the
presence of the unlabeled PNA probe resulted in at least a 10 fold
suppression in the binding of labeled PNA probe to the non-target
sequence (point mutation) and a correlating improvement of
approximately 30 fold, in the signal to noise ratio of the assay
(see Example 4A and FIG. 1).
[0082] When the unlabeled PNA probe was present at 500 equivalents,
there was very little loss of detectable signal (approximately 3 to
10 fold). However, suppression of binding of the labeled probe to a
non-target sequence (point mutation) is substantially improved as
compared with the experiment wherein only 25 equivalents of
unlabeled PNA probe was present (Compare: Examples 4A and 4B of
this specification). The results demonstrate that point mutation
discrimination improved from approximately 10 fold in the absence
of the unlabeled probe to greater than 1000 fold in the presence of
high levels of unlabeled (blocker) PNA probe. Consequently, when
employing the methods described herein, one can achieve several
logs of improvement in point mutation discrimination and similar
dramatic improvements in the dynamic range of the hybridization
assay.
[0083] The applicants are not aware of any similar method suitable
for obtaining such a dramatic suppression of binding to non-target
sequences and the correlating improvement in signal to noise ratio.
The data presented in Example 6, demonstrates the clear superiority
of PNA probes as compared with DNA probes with regard to
suppression of binding to non-target sequences, improvement in
signal to noise ratios and point mutation discrimination
[0084] In preferred embodiments of this invention, PNA probes are
used either alone or in combination with nucleic acid probes. When
combined with nucleic acid probes, the preferred combination
involves unlabeled PNA probes used to suppress the binding of
detectable (labeled) nucleic acid probes to non-target sequences.
In the most preferred embodiment of this invention, both the
detectable probes and unlabeled or independently detectable probes
are PNA probes because this embodiment exhibits both the greatest
ability to suppress binding to non-target sequences and the
greatest ability to discriminate between point mutations.
[0085] Nucleic Acid Synthesis and Labeling
[0086] Those or ordinary skill in the art will recognize that both
labeled, unlabeled or modified oligonucleotides are readily
available. They can be synthesized using commercially available
instrumentation and reagents or they can be purchased from numerous
commercial vendors of custom manufactured oligonucleotides.
[0087] PNA Synthesis:
[0088] Methods for the chemical assembly of PNAs are well known
(See: U.S. Pat. No. 5,539,082, entitled "Peptide Nucleic Acids"
herein incorporated by reference). Chemicals and instrumentation
for the support bound automated chemical assembly of Peptide
Nucleic Acids are now commercially available. Chemical assembly of
a PNA is analogous to solid phase peptide synthesis, wherein at
each cycle of assembly the oligomer possesses a reactive alkyl
amino terminus which is condensed with the next synthon to be added
to the growing polymer. Because standard peptide chemistry is
utilized, natural and non-natural amino acids are routinely
incorporated into a PNA oligomer. Because a PNA is a polyamide, it
has a C-terminus (carboxyl terminus) and an N-terminus (amino
terminus) For the purposes of the design of a hybridization probe
suitable for antiparallel binding to the nucleic acid target
sequence (the preferred orientation), the N-terminus of the PNA
probe is the equivalent of the 5'-hydroxyl terminus of an
equivalent DNA or RNA probe Consequently to design a PNA probe
suitable for parallel binding to a nucleic acid target sequence the
C-terminus of the PNA probe will be the equivalent of the
5'-hydroxyl group of an equivalent DNA or RNA probe.
[0089] PNA Labeling:
[0090] PNA's are labeled using chemical methodologies well known to
those of ordinary skill in the art. Chemical labeling of a PNA is
analogous to peptide labeling. Because the synthetic chemistry of
assembly is essentially the same, any method commonly used to label
a peptide may be used to label the PNA. For example, the polymer
may be labeled by condensation of a suitable detectable moiety to
the amino terminus of the polymer during chemical assembly.
Generally, the amino terminus is labeled by reaction with a
detectable moiety having a carboxylic acid group or activated
carboxylic acid group. Amide formation of this type is a well known
and often utilized chemical reaction. The condensation reaction
forms a very stable amide bond thereby generating the labeled PNA
having a detectable moiety (label).
[0091] Similarly, the PNA can be extended with a linker moiety
before the label (detectable moiety) is attached (e.g. Expedite.TM.
PNA Linker; a.k.a. Fmoc-8-amino-3,6-dioxaoctanoic acid). Generally,
linkers are used to minimize the adverse effects that bulky
labeling reagents might have on hybridization properties of the PNA
oligomers. Specialized reagents can be attached to the PNA terminus
or the linker modified terminus for specific or optimized labeling
reactions. For example, a terminal arylamine moiety can be
generated by condensing a suitably protected 4-aminobenzoic acid
derivative with either of the amino terminus of the PNA oligomer or
the amino terminus of a linker extended PNA oligomer. After
synthesis is complete, the labeled PNA is cleaved, deprotected and
purified using well known methodologies.
[0092] Alternatively, the C-terminal end of the PNA can be labeled
with a detectable moiety. Generally the C-terminal end of the PNA
is labeled by first condensing a labeled moiety with the support
upon which the labeled PNA is to be assembled Next, the first
synthon of the PNA can be condensed with the labeled moiety
Alternatively, one or more linker moieties or amino acids can be
introduced between the labeled moiety and the PNA oligomer using
commercially available reagents (e.g. Expedite.TM. PNA Linker;
a.k.a Fmoc-8-amino-3,6-dioxaoctanoic acid). Thereafter, the PNA is
assembled, cleaved, deprotected and purified using the standard
methodologies.
[0093] For example, the labeled moiety could be a lysine derivative
wherein the .epsilon.-amino group is labeled with a detectable
moiety. For example the moiety could be a fluorochrome such as
5(6)-carboxyfluorescein. Alternatively, the labeled moiety could be
a lysine derivative wherein, the .epsilon.-amino group is
derivatized with a 4-aminobenzoic acid moiety (e.g.
4-(N-(tert-butyloxycarbonyl)-aminobenz- amide). Condensation of the
lysine derivative with the support would be accomplished using
standard condensation (peptide) chemistry. The .alpha.-amino group
of the lysine derivative would then be deprotected and the PNA
assembly initiated by condensation of the first PNA synthon with
the .alpha.-amino group of the lysine amino acid. After complete
assembly, the PNA oligomer is then cleaved from the support,
deprotected and purified using well known methodologies.
[0094] According to another well known method, the label
(detectable moiety) is attached to the PNA after it is fully
assembled and cleaved from the support. This method would be
preferable where the label (detectable moiety) is incompatible with
the cleavage, deprotection or purification regimes commonly used to
manufacture the PNA. For example, this method would be preferred
when the label is an enzyme since the enzyme activity may be
destroyed by any of the commonly utilized cleavage, deprotection or
purification techniques.
[0095] By this method, the PNA will generally be labeled in
solution by the reaction of a functional group on the PNA and a
functional group on the label (detectable moiety). Those of
ordinary skill in the art will recognize that the composition of
the solution will depend on the nature of PNA and the detectable
moiety (label). The solution may comprise organic solvent, water or
any combination thereof. Generally, the organic solvent will be a
polar non-nucleophilic solvent. Non limiting examples of suitable
organic solvents include acetonitrile and N,N'-dimethylformamide.
For labeling reactions involving enzymes, generally the organic
concentration will be less than 50% and preferably less than
20%.
[0096] Generally the functional group on the PNA will be an amine
and the functional group on the label will be a carboxylic acid or
activated carboxylic acid. Non limiting examples of activated
carboxylic acid functional groups include N-hydroxysuccinimidyl
esters. If the label is an enzyme, preferably the amine on the PNA
will be an arylamine. In aqueous solutions, the carboxylic acid
group of either of the PNA or label (depending on the nature of the
components chosen) can be activated with a water soluble
carbodiimide. The reagent, 1-(3-dimethylaminopropyl)-
-3-ethylcarbodiimide hydrochloride (EDC), is a commercially
available reagent sold specifically for aqueous amide forming
condensation reactions.
[0097] Generally, the pH of aqueous solutions will be modulated
with a buffer during the condensation reaction. Preferably the pH
during the condensation is in the range of 4-10. When an arylamine
is condensed with the carboxylic acid, preferably the pH is in the
range of 4-7. When an alkylamine is condensed with a carboxylic
acid, preferably the pH is in the range of 7-10. Generally the
basicity of non-aqueous reactions will be modulated by the addition
of non-nucleophilic organic bases. Non-limiting examples of
suitable bases include N-methylmorpholine, triethylamine and
N,N-diisopropylethylamine.
[0098] Non-limiting examples of detectable moieties (labels)
suitable for labeling nucleic acid or PNA probes used in the
practice of this invention would include chromophores,
fluorochromes, spin labels, radioisotopes, enzymes, haptens and
chemiluminescent compounds. Preferred fluorochromes include
5(6)-carboxyfluorescein, Cyanine 3 (Cy3) Dye and Cyanine 5 (Cy5)
Dye. Preferred haptens include 5(6)-carboxyfluorescein,
2,4-dinitrophenyl, digoxigenin, and biotin. Preferred enzymes
include soybean peroxidase, alkaline phosphatase and horseradish
peroxidase. Other suitable labeling reagents and preferred methods
of attachment would be recognized by those of ordinary skill in the
art of PNA, peptide or nucleic acid synthesis.
[0099] Immobilization of Probes to Surfaces.
[0100] One or more probes will preferably be immobilized to a
surface In one embodiment, the probe can be immobilized to the
surface using the well known process of UV-crosslinking.
Alternatively the probe can be covalently bound to a surface by the
reaction of a suitable functional group on the probe. Methods are
well known in the art for the attachment of oligonucleotide probes
to surfaces. These procedures generally involve the reaction of a
nucleophilic group (e.g. an amine or thiol) on a modified
oligonucleotide with an electrophilic group on the support to be
modified. Thus, one or more suitably prepared PNA probes bearing
nucleophilic moieties can, likewise, be covalently immobilized to a
suitable surface. Because native PNA possesses an amino terminus,
PNA generally will not require modification to thereby immobilize
it to a surface.
[0101] Conditions suitable for the immobilization of a PNA to a
surface will generally be similar to those conditions describe
above for the labeling of a PNA. The immobilization reaction is
essentially the equivalent of labeling the PNA whereby the label is
substituted with the surface to which the PNA probe is to be
covalently immobilized. In preferred embodiments of this invention,
surfaces comprising tresyl groups are reacted with arylamine
modified PNA probes to thereby generate capture surfaces.
[0102] Probes:
[0103] The labeled and unlabeled probes of a set used for the
practice of this invention will generally have a length of between
5 and 100 subunits. Preferably, the PNA probes will be 10 to 20
subunits in length and the nucleic acid probes will be 15-30
subunits is length. The labeled and unlabeled probes of a set may
be nearly the same length or of identical length. When mixing PNA
and nucleic acid probes in a set, typically the DNA probes will be
longer to thereby generate probes which, when hybridized to target
sequences, will have thermal stabilities (Tm values) which are
comparable with the PNA probes (See: Egholm et al. Nature (1993)
365, 566-568).
[0104] In one embodiment, a detectable probe is completely
complementary to the target sequence and an unlabeled or
independently detectable probe, which is complementary to the
non-target sequence, comprises at least one single nucleobase
substitution (mismatch) as compared with the target sequence
Though, both the detectable probe and the unlabeled or
independently detectable probe will hybridize, to some extent, to
the target sequence, hybridization of the perfectly complementary
detectable probe and the target sequence will be thermodynamically
favored as compared with the hybridization of the non-complementary
unlabeled or independently detectable probe and the target
sequence. Similarly, hybridization of the unlabeled or
independently detectable probe to the non-target sequence will be
thermodynamically favored as compared with the hybridization of the
non-complementary detectable probe and the non-target sequence.
[0105] In certain other embodiments, a detectable probe is selected
to be substantially complementary to the target sequence such that
the probe sequence need not comprise a nucleobase sequence which is
exactly complementary to the target sequence to be detected in the
probe-based hybridization assay. However, it is important that an
unlabeled or independently detectable probe comprise a greater
number of non-complementary nucleobases (point mutations) to the
target sequence as compared with the detectable probe.
Additionally, it is important that the detectable probe comprise a
greater number of non-complementary nucleobases (point mutations)
to the non-target sequence as compared with the unlabeled or
independently detectable probe.
[0106] For example, the detectable probe can be designed to
comprise a single nucleobase mismatch as compared with a probe
which is exactly complementary to a target sequence. The unlabeled
or independently detectable probe would be designed to comprise at
least two nucleobase mismatches as compared with a probe which is
exactly complementary to a target sequence to be detected.
Similarly and by design, the detectable probe would then comprise
at least two nucleobase mismatches as compared with a probe which
is exactly complementary to a non-target sequence and the unlabeled
or independently detectable probe would comprise less than two
nucleobase mismatches as compared with a probe which is exactly
complementary to a non-target sequence Thus, when the detectable
and unlabeled or independently detectable probes are present in the
probe-based hybridization assay, hybridization of the more
perfectly complementary detectable probe and target sequence will
be thermodynamically favored as compared with the hybridization of
the less complementary unlabeled or independently detectable probe
and the target sequence. Similarly and by design, hybridization of
the more perfectly complementary unlabeled and independently
detectable probe and non-target sequence will be thermodynamically
favored as compared with the hybridization of the less
complementary detectable probe and the non-target sequence. Thus,
if neither probe is perfectly complementary to the target sequence,
hybridization of the more closely related probes and target or
non-target sequences will be preferred.
[0107] Thus, when a detectable probe is designed to hybridize to a
substantially complementary target sequence, it is a general
requirement that a detectable probe comprise N nucleobase
mismatches as compared with a probe which is exactly complementary
to a target sequence. The one or more unlabeled or independently
detectable probes comprise N+m nucleobase mismatches as compared
with a probe which is exactly complementary to a target sequence.
The integer N is 0, 1 or 2 and the integer m is 1, 2 or 3.
Similarly, the unlabeled or independently detectable probe will
comprise P nucleobase mismatches as compared with a probe which is
exactly complementary to a non-target sequence and the one or more
detectable probes will comprise P+q nucleobase mismatches as
compared with a probe which is exactly complementary to a
non-target sequence. The integer P is 0, 1 or 2 and the integer q
is 1, 2 or 3, provided that N is greater than or equal to P.
[0108] Hybridization Assay:
[0109] In one embodiment, the invention is related to a method for
suppressing the binding of detectable probe to a non-target
sequence in an assay of a sample for a target sequence. The method
comprises contacting the sample with a set containing two or more
probes under conditions suitable for the probes to hybridize to
nucleic acid. At least one of the probes is a detectable probe
labeled with a detectable moiety and having a sequence
complementary or substantially complementary to a target sequence.
At least one of the other probes is an unlabeled or independently
detectable probe having a sequence complementary or substantially
complementary to a non-target sequence. The second step comprises
detecting the presence, absence or quantity of a target sequence
present in the sample by directly or indirectly detecting or
quantitating the detectable moiety. At least one of the detectable
probes or the unlabeled or independently detectable probes is a PNA
probe. Preferably, the one or more unlabeled or independently
detectable probes is a PNA probe. Most preferably, all probes of
the set are PNA probes. In preferred embodiments, the detectable
probe is perfectly complementary to a target sequence and the
unlabeled or independently detectable probe is perfectly
complementary to a non-target sequence which may be present in the
sample.
[0110] The target sequence may comprise the entire sequence of
interest or may be a subsequence of the nucleic acid target
molecule of interest. The target sequence or nucleic acid target
molecule of interest may comprise DNA or RNA.
[0111] According to the method, upon contacting the sample with the
set of probes, a detectable probe will bind (hybridize) to the
complementary or substantially complementary target sequence, if
present in the sample. Thereafter, the presence or amount of target
sequence present in the sample is detected by directly or
indirectly detecting the presence, absence or quantity of
detectable moiety (label). Generally, the one or more detectable
moieties are used to identify or quantitate the probe to which they
are attached. However, the detectable moieties are indirectly used
to identify or quantitate the presence or amount of target sequence
present in the sample. Consequently, the assay must be designed to
correlate the presence of the detectable moiety with the
hybridization of the detectable probe to the target sequence.
[0112] Generally, the unbound or non-specifically bound detectable
probe will be removed so that the presence of the detectable moiety
is a true indicator of the presence, absence or quantity of target
sequence present in the sample. Thus, in certain embodiments, the
probe/target sequence is separated from the detectable probe. For
example, either the complex or the detectable probe may be
immobilized to a support so that the other assays components are
easily washed away. Alternatively, the components are separated by
size using a chromatographic process. In other embodiments, the
detectable moiety of the probe is detectable only when hybridized
to the target sequence and, therefore, the excess detectable probe
need not be removed from the sample to obtain useful hybridization
information (See. Tyangi et al, Nature Biotech (1996), 14,
303-308). In still another embodiment, the detectable moiety may be
cleaved from the probe/target sequence and thereafter detected or
quantitated (See: U.S. Pat. No. 5,410,068 entitled "Succinimidyl
Trityl Compounds and a Process for Preparing Same" which is herein
incorporated by reference).
[0113] Nonetheless, it is the correlation between hybridization and
the presence, absence or amount of detectable moiety which must be
maintained by appropriate design of the hybridization assay.
Consequently, in certain embodiments, the complex formed between
the detectable probe and target sequence may be dissociated to
thereby retrieve the detectable probe for analysis. For example,
this may be preferred where the target sequence has been
immobilized to a surface. The MASDA technique previously described
would be an example of a suitable format for dissociating the
probe/target sequence complex to thereby generate information about
the one or more detectable probes which hybridized to the one or
more immobilized target sequences present in the sample to be
analyzed.
[0114] Generally, a detectable probe will bind most strongly to a
closely related sequence to thereby generate undesirable background
signal. Closely related sequences are sequences having nearly
identical nucleotide composition. Nonspecific binding occurs
because the target and non-target sequences are so closely related
that the detectable probe is very nearly the complement to the
non-target sequence. Point mutations are very closely related
sequences because they differ by the substitution of a single
nucleotide. Consequently, single point mutations are very difficult
to distinguish in a probe-based assay. Because the non-specific
binding of the detectable probe to a non-target point mutation can
be dramatically reduced in the presence of an unlabeled probe which
is complementary to one or more closely related non-target
sequences which may be in the sample of interest, this invention is
very well suited to improving the sensitivity and reliability of
probe-based point mutation analysis.
[0115] According to the method, there is no requirement that only a
single detectable (labeled) probe be used. A mixture of two or more
detectable probes may be preferred when two or more target
sequences are to be identified in the same assay. Moreover, the
suppression of binding to non-target sequences and the associated
improvements in signal to noise ratios make the method of this
invention particularly attractive when applied in a multiplex
analysis of one or more samples for one or more conditions of
interest. A multiplex assay would require numerous detectable
probes wherein each detectable probe was used to detect, identify
or quantitate a individual organism, fungi, virus or clinical
condition of interest. Preferably, two or more detectable probes
will comprise independently detectable moieties to thereby simplify
the analysis of the data.
[0116] According to the method, there is no requirement that only a
single unlabeled (or independently detectable) probe be used. A
mixture of two or more unlabeled probes might be preferred when the
labeled detectable probe binds non-specifically to more than one
non-target sequence. A mixture of probes may also be preferred when
the one or more of the non-target sequence(s) is not known with
certainty. Large mixtures of unlabeled probes will most likely be
preferred in multiplex analysis wherein numerous detectable probes
are used and there are numerous closely related sequences which may
be present in the sample.
[0117] Without intending to be bound to this description, it is
believed that the binding of the detectable probe to the non-target
sequence is suppressed because the unlabeled probe, which is
complementary to the non-target sequence, will preferentially
hybridize to the non-target sequence and thereby form a more
thermodynamically stable complex than is formed by hybridization of
the detectable probe and the non-target sequence. Consequently, the
binding of the detectable probe to the non-target will be
substantially diminished in the presence of the unlabeled probe as
compared to the hybridization which occurs in the absence of the
unlabeled (independently detectable) probe.
[0118] Because the method of this invention may be used in a
hybridization assay, this invention will find utility in improving
assays used to detect, identify of quantitate the presence or
amount of an organism or virus in a sample through the detection of
target nucleic acids associated with the organism or virus. (See
U.S. Pat. No. 5,641,631, entitled "Method for detecting,
identifying and quantitating organisms and viruses" herein
incorporated by reference). Similarly, this invention will also
find utility in an assay used in the detection, identification or
quantitation of one or more species of an organism in a sample (See
U.S. Pat. No. 5,288,611, entitled "Method for detecting,
identifying and quantitating organisms and viruses" herein
incorporated by reference). This invention will also find utility
in an assay used to determine the effect of antimicrobial agents on
the growth of one or more microorganisms in a sample (See: U.S.
Pat. No. 5,612,183, entitled "Method for determining the effect of
antimicrobial agents on growth using ribosomal nucleic acid subunit
subsequence specific probes" herein incorporated by reference).
This invention will also find utility in an assay used to determine
the presence or amount of a taxonomic group of organisms in a
sample (See: U.S. Pat. No. 5,601,984, entitled "Method for
detecting the presence of amount of a taxonomic group of organisms
using specific r-RNA subsequences as probes" herein incorporated by
reference.
[0119] Immobilization of the Target Sequence to a Surface:
[0120] In one embodiment of this invention, the target sequence or
nucleic acid target molecule of interest is immobilized to a
surface. Those of ordinary skill in the art will recognize the
numerous methods suitable for immobilizing a nucleic acid to a
surface. Preferably, the target sequence will be immobilized to a
surface using UV crosslinking. When immobilized to a surface, the
probes of the set will hybridize to the one or more target
sequences of interest, if present. Immobilization to a surface is a
preferred embodiment because non-specifically bound probes of the
set can be easily washed away after hybridization to thereby more
easily detect the presence, absence or quantity of the one or more
detectable moieties.
[0121] The detectable moieties, present as a result of the
formation of detectable probe/target nucleic acid complexes, may be
detected on the support. Alternatively, the detectable probe/target
nucleic acid complexes may be dissociated and the probes eluted
from the support for independent analysis (e.g. the MASDA technique
described in Shuber et al Human Mol. Gen. (1997) 6, 337-347 In
preferred embodiments, probes of distinct composition bearing
independently detectable moieties are used to "probe" for differing
target sequences of interest. Consequently, the presence of a
particular independently detectable moiety in the aliquot eluted
from the support can be used to detect, identify or quantitate the
presence or amount of an organism, virus, fungi or clinical
(disease) state of interest. In a preferred embodiment, each of the
independently detectable moieties is an independently detectable
mass marker and mass spectrometry is used to identify the one or
more probes present in the aliquot. Preferably, positive-ion Fast
Atom Bombardment Tandem Mass Spectrometry is used to identify the
probes present in the aliquot (See: Takao et al., Rapid Comm. Mass.
Spec. (1994), 925-928).
[0122] Detectable and Independently Detectable Moieties:
[0123] In preferred embodiments of this invention, a multiplex
hybridization assay is performed. In a multiplex assay, numerous
conditions of interest are simultaneously examined. Multiplex
analysis relies on the ability to sort sample components or the
data associated therewith, during or after the assay is completed.
In preferred embodiments of the invention, distinct independently
detectable moieties are used to label the different probes of a
set. The ability to differentiate between and/or quantitate each of
the independently detectable moieties provides the means to
multiplex the hybridization assay because the data which correlates
with the hybridization of each of the distinctly (independently)
labeled probes to a target sequence of interest can be correlated
with the data for each of the independently detectable moieties.
Because multiplex hybridization assays involve numerous probes and
target sequences, there is a great potential for non-specific
hybridization to adversely effect the sensitivity and reliability
of the assay. Consequently, the sensitivity and reliability of
multiplex probe-based assays should be improved when applying the
methods, kits and compositions of this invention to multiplex
sample analysis.
[0124] In a simple embodiment of the invention, there is a target
sequence, a non-target sequence, a single detectable probe
complementary to the target sequence and an unlabeled probe
complementary to a non-target sequence. Though both PNA and nucleic
acid probes are inherently detectable because they absorb
ultraviolet light at 260 nanometers, the presence of the labeled
probe (and the target nucleic acid/PNA probe complex) can be
independently detected because of the unique properties of the
detectable moiety (label). For example, the labeled (detectable)
PNA probe could be labeled with a chromophore, a fluorochrome, a
spin label, a radioisotope, an enzyme, a hapten or a
chemiluminescent compound. Non limiting examples of preferred
enzymes include, alkaline phosphatase, soybean peroxidase and
horseradish peroxidase. Non-limiting examples of preferred haptens
include, 5(6)-carboxyfluorescein, biotin, 2,4-dinitrophenyl and
digoxigenin. Non-limiting examples of preferred fluorochromes
include 5(6)-carboxyfluorescein, cyanine 3 dye and cyanine 5 dye.
Non-limiting examples of preferred chemiluminescent compounds
include luminol 1,2-dioxetanes.
[0125] Provided however, that the detectable moieties are
independently detectable, there is no requirement that any of the
probes be unlabeled. Indeed, it may be preferable to perform the
method of this invention with two, or more, labeled PNA probes
wherein the labels (detectable moieties) are independently
detectable provided that the probe suppresses the binding of the
other probe to their respective, non-target sequences.
[0126] Independently detectable moieties are moieties (labels)
which, when both are present in a sample each, can be assayed for
independently whereby the presence of the moiety of interest is not
significantly affected by signal generated for the one or more
other independently detectable moieties in the sample. For example,
two fluorochromes could be used as labels for the PNA probes,
provided the emission wavelengths of each fluorochrome were
independently detectable (e.g. 5(6)-carboxyfluorescein and
cyanine-3-dye(Cy3)). Alternatively two enzymes could be used as
labels for the probes, provided the activity of the enzymes can be
independently detected. For example, the activity of soybean
peroxidase and alkaline phosphatase can be independently
determined. Thus, the detectable probe is the probe which is sought
to be presently detected and the unlabeled or independently
detectable probe is the probe which is subsequently or
independently detected
[0127] Consequently, in still another embodiment of the invention,
a mixture of four labeled, but independently detectable probes,
could be used to analyze for and/or quantitate the relative
abundance of point mutations of a target sequence in a sample. The
set of probes would consist of four independently detectable probes
wherein the sequence of each of the probes differed as a point
mutation by relation to the other three. Thus, each of the probes
is complementary to one of the four possible point mutations of a
target sequence, and by relation, the other three probes are
complementary to a non-target point mutation of that sequence.
According to the method, the detectable probe would be the probe
for which the signal for the detectable moiety of interest was
sought to be presently detected. The assay could then be repeated
three times wherein each time, a different independently detectable
moiety was sought to be detected. In each of the four assays, the
three independently detectable probes would thereby suppress the
binding of the detectable probe to the non-target point mutations
of the target sequence. Thus, the presence or absence of each of
the point mutations could be detected with greater sensitivity and
reliability in each of the four assays because the non-specific
binding of the detectable probe to each of the specific
non-targeted point mutated sequence is suppressed. Moreover, when
compared with a standard curve, the relative abundance of the
target point mutations could be determined by comparison of the
signal intensity obtained for each of the four assays.
Alternatively, the intensity of the point mutation could be
determined by simultaneously detecting the four independently
detectable moieties in a single assay. For example, the assay
described above might be a simple multiplex assay used to analyze
for a single point mutation in a target sequence of interest.
[0128] Order of Probe Addition/Incubation Period:
[0129] In a preferred embodiments, the one or more unlabeled or
independently labeled probes are incubated with the sample for a
first period of time, under conditions suitable for probes to
hybridize to nucleic acid. Then, the one or more detectable probes
are incubated with the sample for a second period of time under
conditions suitable for probes to hybridize to nucleic acid. When
using unlabeled or independently labeled PNA probes, the best
signal to noise ratios are obtained when the first incubation
period is 20 minutes or less. The applicants have determined that
the application of these conditions provide the best results
particularly when using unlabeled or independently detectable PNA
probes to suppress the binding of detectable probes to non-target
sequences
[0130] Equivalents of Unlabeled or Independently Labeled Probe:
[0131] The applicants have surprisingly determined that the
presence of between 2-500 equivalents of unlabeled or independently
detectable probe (as compared with the number of equivalents of
detectable probe) does not dramatically reduce the limit of
detection of signal in the hybridization assay. However, as the
ratio of unlabeled or independently detectable PNA probe to
detectable probe increases the binding of the detectable probe to
non-target sequence is dramatically reduced. Consequently, the
signal to noise ratio dramatically improves as the ratio of
unlabeled or independently detectable PNA probe to detectable probe
is increased. The applicants have not observed any conditions
whereby increasing the ratio of unlabeled probe to detectable probe
is detrimental to the overall sensitivity and reliability of the
hybridization assay. Consequently, the ratio of unlabeled or
independently detectable probe to detectable probe is preferably
greater than or equal to two.
[0132] Kits:
[0133] This invention also relates to kits suitable for suppressing
the binding of a detectable probe to a non-target sequence in an
assay of a sample for a target sequence. In one embodiment, the kit
comprises a set of two or more PNA probes wherein, at least one of
the probes is a detectable probe labeled with a detectable moiety
and having a sequence complementary or substantially complementary
to a target sequence. At least one of the other probes is an
unlabeled or independently detectable probe having a sequence
complementary or substantially complementary to a non-target
sequence. PNA or nucleic acid probes may be used, provided, at
least one of either a detectable probe or an unlabeled or
independently detectable probe is a PNA probe. Preferably, the
detectable probes are nucleic acid probes and the one or more
unlabeled or independently detectable probes are PNA probes. Most
preferably all probes are PNA probes. In preferred embodiments, the
detectable probe is perfectly complementary to a target sequence
and the unlabeled or independently detectable probe is perfectly
complementary to a non-target sequence which may be present in the
sample
[0134] The kits of this invention may optionally contain
instructions, buffers, DNA, RNA, instruments or any other item
desirable for performing the assay.
[0135] There is no requirement that the kit comprise a single
detectable probe because the assay may be designed to detect two or
more target sequences simultaneously. There is no requirement the
kit comprise only one unlabeled or independently label probe
because there may be a need to suppress the binding of the
detectable probe to more than one non-target sequence.
Consequently, in one preferred embodiment, the kit comprises a set
of detectable probes wherein each of the different probes of the
set comprises an independently detectable moiety. In a preferred
embodiment, the detectable probes of the set are nucleic acid
probes and all other probes of the set are unlabeled PNA probes
having a defined sequence and which hybridize to one or more
non-target sequences which may be present in the sample. In another
embodiment, all the probes of the set are PNA probes and each of
the differing detectable PNA probes of the set comprise
independently detectable moieties.
[0136] Kits of this invention will find utility in improving assays
used to detect, identify of quantitate the presence or amount of an
organism or virus in a sample. Similarly, the kits of this
invention will also find utility in an assay used in the detection,
identification or quantitation of one or more species of an
organism in a sample. The kits of this invention will also find
utility in an assay used to determine the effect of antimicrobial
agents on the growth of one or more microorganisms in a sample. The
kits of this invention will also find utility in an assay used to
determine the presence or amount of a taxonomic group of organisms
in a sample.
[0137] In one embodiment, the kit comprises a set of two PNA
probes, wherein the first PNA probe has a sequence complementary to
the target sequence and is labeled with a detectable moiety. The
second PNA probe is unlabeled and has a sequence complementary to a
non-target sequence which may be present in the sample. In a more
preferred embodiment, the target and non-target sequences are
related as point mutations
[0138] In another embodiment, the kit comprises a set of four
probes, wherein the probes of the set consist of a single
detectable probe labeled with a detectable moiety and having a
sequence complementary to the target sequence. The three unlabeled
probes each have a defined sequence which is complementary to a
non-target sequence which may be present in the sample. In a more
preferred embodiment, the target and non-target sequences are
related as point mutations.
[0139] Alternatively, the detectable probe of the set is a nucleic
acid probe and the other probes of the kit are unlabeled PNA probes
each having a defined sequence which is complementary to a
non-target sequence which may be present in the sample. In a more
preferred embodiment, the target and non-target sequences are
related as point mutations.
[0140] Compositions:
[0141] The compositions of this invention may exist as a powder or
they may comprise a solution of one or more components dissolved or
suspended in a solvent. Suitable solvents include water or a
mixture of water and an organic solvent. Non limiting examples of
suitable organic solvents include acetonitrile, formamide,
dimethylformamide, methanol, ethanol, isopropanol, tetrahydrofuran
and 1,4-dioxane. The composition may comprise one or more organic
or inorganic salts to thereby adjust the ionic strength of the
composition. The composition may comprise one or more inorganic or
organic detergents. The composition may comprise one or more
buffers to thereby adjust the pH of the composition.
[0142] In one embodiment, this invention relates to a composition
for suppressing the binding of a detectable probe to a non-target
sequence in an assay of a sample for a target sequence. The
composition consists of a set of two probes wherein, one of the
probes is a detectable probe labeled with a detectable moiety and
having a sequence complementary to the target sequence. The other
probe is an unlabeled or independently detectable probe having a
sequence complementary to a non-target sequence. PNA or nucleic
acid probes may be used, provided, at least one of either a
detectable probe or an unlabeled or independently detectable probe
is a PNA probe. Preferably, the probes are PNA probes The probes of
the composition may be designed to hybridize specifically to
complementary target or non-target sequences wherein the target and
non-target sequences are related as point mutations. In a preferred
embodiment, the detectable probe is a nucleic acid probe and the
other probe is an unlabeled PNA probe.
[0143] In another embodiment, the invention relates to a
composition for suppressing the binding of a detectable probe to a
non-target sequence in an assay of a sample for a target sequence.
The composition consists of a set of four probes wherein, one of
the probes is a detectable probe labeled with a detectable moiety
and having a sequence complementary to the target sequence. The
other three probes are unlabeled or independently detectable probes
having a sequence complementary to a non-target sequence. PNA or
nucleic acid probes may be used, provided, at least one of either a
detectable probe or an unlabeled or independently detectable probe
is a PNA probe. Preferably, the probes are PNA probes. Preferably
the probe set contains probes which hybridize to target and
non-target sequences which are related as the four possible
sequence variations for a single point mutation. In a preferred
embodiment, the detectable probe is a nucleic acid probe and the
other three probes are unlabeled PNA probes.
[0144] The compositions of this invention will find utility in
improving assays used to detect, identify of quantitate the
presence or amount of an organism or virus in a sample. Similarly,
the compositions of this invention will also find utility in an
assay used in the detection, identification or quantitation of one
or more species of an organism in a sample. The compositions of
this invention will also find utility in an assay used to determine
the effect of antimicrobial agents on the growth of one or more
microorganisms in a sample. The compositions of this invention will
also find utility in an assay used to determine the presence or
amount of a taxonomic group of organisms in a sample.
[0145] Capture Assays:
[0146] Still another embodiment of this invention is related to a
method for suppressing the binding of a non-target sequence to a
capture probe immobilized on a surface in a capture assay of a
sample for a target sequence of a nucleic acid target molecule.
Capture assays are often preferred because the surfaces can be
repetitively treated whereby the reagents are easily added and
removed Moreover, arrays of capture probes can be constructed to
thereby enable simultaneous analysis of a sample for the presence,
absence or quantity of numerous nucleic acid target molecules.
Consequently, capture assays are easily adapted for multiplex
sample analysis.
[0147] The method comprises contacting the sample with a solution
containing one or more blocking probes under conditions suitable
for the blocking probes to hybridize to nucleic acid. The blocking
probes are complementary or substantially complementary to one or
more non-target sequences which when present in the sample may bind
non-specifically to the capture probe to generate a detectable
signal in the assay. The sample is also contacted with a capture
probe immobilized on a surface. The conditions are suitable for the
target sequence, if present, to hybridize to the capture probe. The
capture probe is complementary or substantially complementary to
the target sequence and thereby forms a capture probe/target
sequence complex upon hybridization. Finally, the presence or
amount of nucleic acid target molecule which becomes immobilized to
the surface by the formation of the capture probe/target sequence
complex is detected, identified or quantitated. In preferred
embodiments, the blocking probe is perfectly complementary to a
non-target sequence and the capture probe is perfectly
complementary to a target sequence.
[0148] The nucleic acid target molecule which is sought to be
captured by operation of the method of this invention may be DNA or
RNA. Both nucleic acid or PNA blocker probes are suitable for use
in the capture assay of this invention. Preferably, the blocker
probes are PNA probes. Both nucleic acid or PNA capture probes are
suitable for use in the capture assay of this invention, provided
at least one of a capture probe and a blocking probe is a PNA
probe. Preferably, the one or more capture probes are PNA probes.
In one preferred embodiment, the capture probes are nucleic acid
probes and the blocker probes are PNA probes In preferred
embodiments, the capture probes and blocker probes are designed to
hybridize specifically to target and non-target sequences which are
closely related
[0149] In a preferred embodiment of this method, the surface
comprises an array of probes, wherein each distinct capture probe
in the array is designed to capture a specific nucleic acid target
molecule The presence or quantity of a nucleic acid target molecule
is indicative of the presence or quantity of a specific organism,
virus, fungi or clinical (disease) state of interest in the
sample.
[0150] Thus, capture assays of this invention will find utility in
improving the detection, identification or quantitation of the
presence or amount of an organism or virus in a sample. Similarly,
the capture assays of this invention will also find utility in the
detection, identification or quantitation of one or more species of
an organism in a sample. The capture assays of this invention will
also find utility in the determination of the effect of
antimicrobial agents on the growth of one or more microorganisms in
a sample. Finally, the capture assays of this invention will also
find utility in determining the presence or amount of a taxonomic
group of organisms in a sample.
[0151] Because hybridization of the capture probe to the target
sequence results in the formation of a capture probe/target nucleic
acid complex, the presence or amount of nucleic acid target
molecule immobilized to the surface can be detected using a labeled
antibody which specifically interacts with the capture probe/target
sequence complex which is formed on the surface. If the capture
probe is a nucleic acid probe, the complex formed on the surface by
capture is a nucleic acid/nucleic acid complex. Thus, a labeled
anti-nucleic acid/nucleic acid antibody can be used to detect the
presence, absence or amount of the nucleic acid/nucleic acid
complex (See: U.S. Pat. No. 5,200,313 entitled "Nucleic Acid
Hybridization Assay Employing Detectable Anti-Hybrid Antibodies"
herein incorporated by reference). If the capture probe is a PNA
probe, the complex formed on the surface by capture of the target
sequence will be a PNA/nucleic acid complex. Consequently, a
labeled anti-PNA/nucleic acid antibody will be used to detect the
presence, absence or amount of the PNA/nucleic acid complex (See:
U.S. Pat. No. 5,612,458 entitled "Antibody to PNA/nucleic acid
Complexes" herein incorporated by reference). Generally, either of
the antibodies can be labeled with a detectable moiety Non-limiting
examples of such detectable moieties can be selected from the group
consisting of a chromophore, a fluorophore, a spin label, a
radioisotope, an enzyme, a hapten and a chemiluminescent compound.
Non-limiting examples of suitable enzymes include alkaline
phosphatase, soybean peroxidase and horseradish peroxidase.
Non-limiting examples of suitable haptens include fluorescein,
biotin, 2,4-dinitrophenyl and digoxigenin. Detection of capture
probe/target nucleic acid complexes using an antibody directed to
the capture probe/target sequence is particularly well suited for
analysis of arrays because the individual PNA capture probe or
nucleic acid capture probes need not be individually labeled.
Moreover, the blocking probes also need not be labeled. Methods
employing unlabeled probes are preferred because the probes
generally cost less to produce and are easier to synthesize and
purify. Moreover, a single reagent is used to detect and quantitate
the presence or amount of the capture probe/target sequence
complex.
[0152] In another embodiment, the presence or amount of target
sequence immobilized to the surface is detected using a detector
probe which hybridizes to a second target sequence of the nucleic
acid target molecule. The detector probe may be a PNA probe or a
nucleic acid probe. The detector probe may be labeled with a
detectable moiety. Detectable moieties suitable for labeling the
PNA or nucleic acid probes of this invention have been previously
described.
[0153] In certain embodiments, the Hybridization Assay described
above can be used to suppress the binding of one or more detector
probes to the one or more non-second target sequences to thereby
improve the reliability of the assay. According to the method, the
set of probes would comprise at least one detector probe having a
sequence complementary to a second target sequence of interest. The
set of probes would also comprise at least one non-second target
sequence probe complementary to a non-second target sequence which
might be present in the sample.
[0154] Alternatively, the detector probes need not be labeled with
detectable moieties but their presence, absence or quantity can be
determined using labeled antibodies. If the detector probe is a
nucleic acid probe, the complex formed on the surface is a detector
(nucleic acid) probe/second target sequence complex. As previously
described, a labeled anti-nucleic acid/nucleic acid antibody can be
used to detect the presence, absence or amount of the detector
(nucleic acid) probe/second target sequence complex. If the
detector probe is a PNA probe, the complex formed on the surface is
a detector (PNA) probe/second target sequence complex.
Consequently, a labeled anti-PNA/nucleic acid antibody can be used
to detect the presence, absence or amount of the detector (PNA)
probe/second target sequence.
[0155] When using labeled antibodies to detect the detector
probe/second target sequence complex in a capture assay, the nature
of the capture probes and the detector probes should be different.
For example, if the capture probes are nucleic acid and the
detector probes are PNA probes, the hybrids can be independently
detected by using the appropriate labeled antibody. In this example
the labeled anti-PNA/nucleic acid antibody will be used to detect
or quantitate the presence or amount of detector probe present
since no cross reaction should occur with the capture probe/target
sequence complex. Similarly, the capture probe/target sequence
complex can be specifically detected using the labeled anti-nucleic
acid/nucleic acid antibody since no crossreaction with the detector
PNA probe/second target sequence complex should occur.
[0156] Having described the preferred embodiments of the invention,
it will now become apparent to one of skill in the art that other
embodiments incorporating the concepts described herein may be
used. It is felt, therefore, that these embodiments should not be
limited to disclosed embodiments but rather should be limited only
by the spirit and scope of the following claims.
EXAMPLES
Example 1
Synthesis of 4-(N-(tert-butyloxycarbonyl)-aminobenzoic acid
[0157] To 100 mM of methyl-4-amino benzoic acid stirring in 150 mL
of dioxane was added 110 mM of di-tert-butyl-dicarbonate. The
reaction was warmed to 70-80.degree. C. and let stir for about 48
hours. The solvent was then evaporated under reduced pressure and
the residue redissolved in about 300 mL of ethylacetate. The
organic layer was then washed three times with 10% aqueous citric
acid, dried (Na.sub.2SO.sub.4), filtered and evaporated to a solid.
The solid was then suspended in 150 mL of 1N NaOH and 50 mL
acetone. The saponification of the ester was allowed to run
overnight until complete hydrolysis was observed by thin layer
chromatography (TLC). To the solution was added citric acid until
the pH of the solution was approximately 4. The solid was then
collected by vacuum filtration and dried in a vacuum oven at
50.degree. C. Yield 20.3 g, 85%. The product was a single peak when
analyzed by HPLC using 0.1% trifluoroacetic acid (TFA) and a linear
acetonitrile gradient.
[0158] .sup.1H-NMR (d.sub.6-DMSO) .delta.=9.7 (s, 1H), 7.8 (d, 2H),
7.6 (d, 2H), 1.5 (s, 9H).
Example 2
Synthesis of Peptide Nucleic Acids
[0159] Peptide Nucleic Acids (PNAs) were synthesized using
commercially available chemicals and instrumentation from
PerSeptive Biosystems, Inc. Labeling of the amino terminus of the
PNA oligomer with a linker group while the oligomer was still
support bound was accomplished by condensation of two subunits of
Expedite PNA Linker (P/N GEN063032) using one of the auxiliary
positions of the PNA synthesizer and the standard coupling cycle.
To the amino terminus of the elongated polymer was condensed
4-(N-(tert-butyloxycarbonyl)-aminobenzoic acid (see Example 1).
After desired modification of the amino terminus of the polymer,
the oligomers were then cleaved from the support and deprotected
according to the manufactures instructions The crude oligomer
samples were then purified by High Performance Liquid
Chromatography (HPLC) using 0.1% trifluoroacetic acid (TFA) and a
linear acetonitrile gradient This process yielded purified aryl
amine terminating oligomers suitable for either conjugation with
enzyme or suitable for use in the assay as an unlabeled probe (i.e.
not conjugated to SBP).
Example 3
General Procedure for Conjugation of Arylamine Containing Peptide
Nucleic Acids (PNA's) to Soybean Peroxidase or Alkaline
Phosphatase
[0160] Stock Solutions:
[0161] 1. Probe Stock:
[0162] Purified arylamine terminated PNA probe, typically fifteen
residues in length, was dissolved at a concentration of
approximately 0.33 .mu.mol per milliliter in 50% aqueous
dimethylformamide (DMF).
[0163] 2. Enzyme Stock:
[0164] Soybean peroxidase, conjugate grade, obtained from Enzymol
International, Columbus Ohio, was dissolved at a concentration of
2.65 mg per milliliter in an aqueous buffer comprised of 3 M NaCl,
10 mM MgCl.sub.21 0.1 mM ZnCl.sub.2 and 30 mM N-methylmorpholine
adjusted to pH 7.6 with 12 N hydrochloric acid.
[0165] 3. 30% Aqueous DMF:
[0166] An aqueous DMF solution was prepared by combining three
volumes of DMF with 7 volumes of water.
[0167] 4. MES Buffer
[0168] An 0.2 M solution of 4-morpholineethanesulfonic acid (MES)
in water was prepared (not pH adjusted).
[0169] 5. Glycine Solution
[0170] A solution comprised of 0.5 M glycine and 0.25 M sodium
hydroxide in water was prepared.
[0171] 6. Wash Buffer
[0172] An aqueous buffer comprised of 1.5 M NaCl, 5 mM MgCl.sub.2,
0.1 mM ZnCl.sub.2 and 15 mM N-methylmorpholine adjusted to pH 7.6
with hydrochloric acid was prepared.
[0173] 7. Storage Buffer
[0174] An aqueous buffer comprised of 3 M NaCl, 10 mM MgCl.sub.21
0.1 mM ZnCl.sub.2 and 30 mM N-methylmorpholine adjusted to pH 7.6
with 12 N hydrochloric acid was prepared.
[0175] Conjugation Procedure:
[0176] In a small reaction tube was combined 10 .mu.L of Enzyme
Stock, 12.5 .mu.L of 30% Aqueous DMF, and 7 .mu.L of Probe Stock.
In a separate tube was placed 1 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
and 7 .mu.L of MES Buffer. These reagents were mixed until the EDC
had dissolved in the MES Buffer. The EDC/MES Buffer solution was
then added to the tube containing the enzyme and probe (Reaction
Mixture). The contents were mixed, and the tube was placed at
5.degree. C. for 40 min. To the Reaction Mixture was then added 7
.mu.L of Glycine Solution. The contents were again mixed and the
tube was placed at 5.degree. C. for a further 20 minutes. The
contents of the tube were diluted with 50 .mu.L of Wash Buffer and
then transferred to the cup of an Ultrafree microconcentrator
(30,000 molecular weight cut-off, Millipore Corporation, Bedford
Mass.). The concentrator was spun at 5,000.times.g until .about.90%
of the liquid had been removed from the cup. An additional 50 .mu.L
of Wash Buffer was added to the cup and the device spun again to
remove 90% of the liquid. This washing procedure was repeated two
additional times. The contents of the cup were then diluted to a
volume of 1 milliliter in Storage Buffer. The absorbance of this
solution at 260 nM was used to estimate the concentration of the
PNA-enzyme conjugate for subsequent assays (0.05 absorbance units
at 260 nanometers per milliliter was estimated to be 0.33 mmol per
milliliter based on an estimated extinction for a PNA 15-mer of 150
optical density units per .mu.mole of probe).
Example 4
Suppression of the Nonspecific Binding of Detectable Probe in a
Probe Based Assay
[0177] Overview of Experiments A and B:
[0178] Two DNA oligonucleotides which differed in sequence by a
single base.(point mutation) were detected in experimental assays
using labeled (detectable) PNA probes, each of which was
complementary to one of the two target sequences. Experiments A and
B were performed to examine, compare and quantitate the effects
associated with the addition of unlabeled probe on the
hybridization assay performance. Experiments A and B were generally
performed in the same manner except that ratios of labeled to
unlabeled probe were substantially greater in Experiment B. The
results of Experiments A and B demonstrate a dramatic suppression
of binding of labeled probe to non-target nucleic acid which
differs from the target nucleic acid as a point mutation. The
suppression of binding to non-target sequence results in a
correlating dramatic improvement in the signal to noise ratio for
the hybridization assay.
[0179] Probes and Targets (Exp. A & B):
[0180] Target DNA oligonucleotides were obtained from a commercial
vendor of custom synthesized DNA. The biotin was attached by the
commercial vendor using commercially available biotin amidites.
Labeled and unlabeled peptide nucleic acid (PNA) probes were
prepared as described in Examples 2 and 3 above.
[0181] Target DNA Oligomer Sequences:
1 Wild Type: 5' Biotin-GTG GTA GTT GGA GCT GGT GGC SEQ ID NO:1
GTA-OH 3' Mutant: 5' Biotin-GTG GTA GTT GGA GCT TGT GGC SEQ ID NO:2
GTA-OH 3'
[0182] Labeled (Detectable) PNA Probes:
2 PNA-Wild Type: H.sub.2NC(O)-ACC TCG ACC ACC
GCA-(linker).sub.2-P-SBP PNA-Mutant: H.sub.2NC(O)-ACC TCG AAC ACC
GCA-(linker).sub.2-P-SBP
[0183] Unlabeled PNA Probes:
3 PNA-Wild Type: H.sub.2NC(O)-ACC TCG ACC ACC
GCA-(linker).sub.2-P-NH.sub.2 PNA-Mutant: H.sub.2NC(O)-ACC TCG AAC
ACC GCA-(linker).sub.2-P-NH.sub.2
[0184] (Notes For the PNA oligomers, "linker" designates the
Expedite PNA linker attached to the amino terminus of the PNA
(equivalent to the 5' hydroxyl end of a DNA or RNA probe for
hybridization purposes) and the letter "P" designates the 4-amino
benzoic acid moiety attached thereto. SBP designates the Soybean
Peroxidase enzyme).
[0185] Reagents (Exp. A & B):
[0186] Pre-Hybridization and Hybridization Buffer
[0187] 50% Formamide
[0188] 20 mM [Tris[hydroxymethyl]amino]methane (TRIS) pH 9.4
[0189] 1.0% Casein
[0190] 2.3% Polyoxyethylene sorbitan Monolaurate (TWEEN-20)
[0191] Oligo Dilution Buffer
[0192] 100 mM TRIS pH 7.6
[0193] 20 mM Ethylenediaminetetracetic acid (EDTA)
[0194] 20.times.SSC
[0195] 3 M NaCl
[0196] 300 mM C.sub.6H.sub.5Na.sub.3O.sub.7.2H.sub.2O (Sodium
Citrate Trisodium Salt: Dihydrate)
[0197] Wash Buffer
[0198] 2.times.(SSC)
[0199] 0.1% Sodium Dodecyl Sulfate
[0200] 0.5% Casein
[0201] Blocking Buffer
[0202] 500 mM NaCl
[0203] 50 mM TRIS pH 9.0
[0204] 0.5% Casein
[0205] High pH Blocking Buffer
[0206] 100 mM TRIS pH 9.4
[0207] 100 mM NaCl
[0208] 10 mM MgCl.sub.2
[0209] Nylon Membrane: Biodyne A.TM. (Pall, Inc.) was obtained from
Gibco, BRL, Gaithersburg, Md.
[0210] The detection system was a commercially available luminol
based chemiluminescent substrate (SuperSignal NA.TM. from Pierce
Chemical). Signal was enzymatically catalyzed by the enzyme Soybean
Peroxidase which was conjugated to the PNA probe (detectable
moiety). The enzyme catalytically produced chemiluminescent signal
which was detected using an autoradiographic film.
[0211] Experiment A
[0212] Assay Description:
[0213] PNA Probe Solutions:
4 Set 1 No unlabeled PNA probe Set 2 1:5, Labeled (Detectable)
probe:Unlabeled probe Set 3 1:25, Labeled (Detectable)
probe:Unlabeled probe
[0214] Labeled (Detectable) PNA probes were diluted to a final
concentration of 4 pmole/mL using Hybridization Buffer. Set 1 was a
control so no unlabeled PNA probe was added. Unlabeled PNA probes
were diluted to a final concentration of: 20 pmole/mL for Set 2,
and 100 pmole/mL for Set 3, using Hybridization Buffer. Table 1,
summarizes the components of the mixtures used to generate Sets 1,
2 and 3.
5 Wild Type Labeled Probe: 293 pmole/mL Mutant Labeled Probe: 387
pmole/mL Wild Type Unlabeled Probe: 5 pmole/.mu.L (diluted in 50%
formamide) Mutant Unlabeled Probe: 5 pmole/.mu.L (diluted in 50%
formamide)
[0215]
6TABLE 1 Wild Mutant Wild Type Type Mutant unlabled unlabeled Hyb.
Set # Probe Type probe probe probe probe Buffer 1 Wild Type 3.4
.mu.L -- -- -- 46.6 .mu.L 1 Mutant -- 2.6 .mu.L -- -- 47.4 .mu.L 2
Wild Type 3.4 .mu.L -- 1 .mu.L -- 45.6 .mu.L 2 Mutant -- 2.6 .mu.L
-- 1 .mu.L 46.4 .mu.L 3 Wild Type 3.4 .mu.L -- 5 .mu.L -- 41.6
.mu.L 3 Mutant -- 2.6 .mu.L -- 5 .mu.L 42.4 .mu.L
[0216] Method:
[0217] Each Target DNA oligomer was diluted to the following final
concentrations, 100 fmole/.mu.L, 32 fmole/.mu.L, 10 fmole/.mu.L, 3
fmole/.mu.L, 1 fmole/.mu.L, 320 amole/.mu.L, and 100 amole/.mu.L. A
control set of dilutions was also made which consisted of 50% of
each of the two oligomers at each dilution. For example, the
control called 100 fmole/.mu.L was made from equal volumes of the
100 fmole/.mu.L dilution of each target DNA oligomer. The final
concentration of each target DNA oligomer in the 100 fmole/.mu.L
control was 50 fmole/.mu.L. For each hybridization, 0.32 .mu.L of
each of the target DNAs was spotted onto a nylon membrane. Six
membranes containing each of the three dilution series (Mu, WT, and
Control) were made. After spotting, the membranes were dried at
37.degree. C. for 15 minutes, then irradiated with 254 nm@0.033
joules/cm.sup.2 of light to crosslink the DNA to the membrane.
Membranes were individually sealed in plastic bags. Membranes were
then pre-hybridized with 200 .mu.L Hybridization Buffer in a
45.degree. C. water bath for 1 hour. During pre-hybridization,
membranes were periodically shaken. Next, 50 .mu.L of probe
mixtures were added to the bags, the bags were resealed, and
hybridization was allowed to proceed for 30 minutes at 45.degree.
C. with periodic mixing.
[0218] Membranes were then washed together twice at room
temperature in 100 mL of Wash Buffer. A third wash was carried out
at 45.degree. C. while shaking for 15 minutes. Membranes were
blocked for 15 minutes while shaking in 100 mL Blocking Buffer.
Membranes were blocked again for 5 minutes while shaking in 50 mL
High pH Blocking Buffer. Membranes were blotted on Whatman membrane
paper and put collectively into a new plastic pouch. Next, 500
.mu.L of Pierce.TM. "Stable Peroxide NA" and 500 .mu.L of
Pierce.TM. "Luminol/Enhancer NA" were added to the pouch. The pouch
was sealed and shaken at room temperature for 10 minutes. The
membranes were again blotted onto paper then resealed in individual
plastic pouches. The membranes were exposed to autoradiographic
film for 2-5 minutes.
[0219] Results:
[0220] With reference to FIG. 1, the left half of the figure
displays the membranes probed with wild type PNA probe conjugated
to soybean peroxidase as the detectable moiety. With reference to
FIG. 1, the right half displays membrane probed with the mutant PNA
probe conjugated to soybean peroxidase as the detectable moiety.
For convenience of interpreting the Figure, columns have been
designated with the letters A-R and rows have been designated with
the numbers 1-7. In each column of each membrane a dilution series
of target DNA oligomer was applied. For each of the six membranes
there was a column labeled "WT" for the wild type DNA target, "C"
for the control (mixture of wild type and mutant target DNA) and
"M" for the mutant DNA target. All six membranes were identical
with respect to dimensions and DNA target location, type and
concentration. DNA target oligomers were spotted approximately in
the center of 3 mm.times.3 mm squares marked on the membrane.
Dilution series were arranged vertically with the highest
concentration of oligomer at the top and the lowest concentration
at the bottom. The two sets of membranes (wild type-probed, and
mutant-probed) were arranged from left to right as the relative
molar concentration ratio of labeled (detectable) PNA probe to
unlabeled PNA probe in the hybridization was increased from 1:0,
1:5, and 1:25. For the wild type probe, the "WT" dilution series
represented specific signal, and the "M" dilution series represents
non-specific signal. For the mutant probe, the "M" dilution series
represents specific signal, and the "WT" dilution series
represented non-specific signal.
[0221] With reference to FIG. 1, row 5, when either the wild type
or mutant targets are probed with the perfectly complementary wild
type or mutant probes, whether in the presence or absence of
blocker probe, the lowest discernible level of detectable DNA was
320 amole (See: row 5, columns A, D, G, L, O and R) The strength of
the signal at 320 amole of spotted target was slightly reduced, in
the presence of the unlabeled mutant probe (Compare row 5, column A
to row 5, columns D and C) The mutant probe does not display any
visible loss of signal in the presence of unlabeled probe (compare
row 5, column L to row 5, columns O and R).
[0222] The control dilution series contained a 1:1 mixture of wild
type and mutant target DNA. The amount of specific signal seen in
the control lanes should be approximately the same (across rows)
under all conditions tested if the presence of unlabeled probe does
not significantly reduce specific signal, and if the two labeled
probes behave in similar ways under the assay conditions. As can be
seen by comparing the signals in columns B, E, H, K, N, and Q in
FIG. 1, control signal was maintained across the six membranes. On
each membrane, the control signal was approximately 50% of the
specific signal at all levels tested as it should be (for example
compare the signals in column K to those in column L).
[0223] The intensity of the signal caused by non-specific
hybridization of the wild type or mutant labeled probe was,
however, dramatically reduced in the presence of the unlabeled
probe. For example, with reference to FIG. 1, compare the signal
intensity of the labeled wild type probe hybridized to the mutant
target (column C) with that obtained from the hybridization in the
presence of the unlabeled mutant probe (See: columns F and I where
the unlabeled mutant probe was present in the hybridization assay
in 5 and 25 relative molar excess, respectively to the labeled wild
type probe). In the absence of the unlabeled probe the non-specific
signal was present at 3 fmole of target DNA (See: row 3, column C).
Addition of 5 fold molar excess of unlabeled mutant probe reduced
the intensity of the non-specific signal by approximately 10 fold
so that about 32 fmole of mutant DNA is required to observe a
signal (See: row 1, column F). Addition of 25 equivalents of
unlabeled mutant probe reduced the intensity of the signal such
that non-specific binding of the wild type probe to the mutant
target was not detectable within the parameters of this experiment
(See: row 1, column 1).
[0224] Consistent with these results, when the labeled mutant probe
was used for hybridization, the addition of a 25 fold relative
molar excess of unlabeled wild type probe to the hybridization
reaction resulted in approximately a 10 fold reduction of
non-specific binding of the labeled mutant probed to the wild type
DNA target With reference to FIG. 1, compare column J (no unlabeled
wild type probe) with columns M and P (unlabeled wild type probe in
relative molar concentrations of 5 and 25). The mutant probe was
detected at about 3 fmole of wild type target in the absence of any
unlabeled wild type probe (row 3, column J). Addition of a 5 fold
relative molar excess of unlabeled wild type probe reduced the
intensity of the non-specific signal by approximately 10 fold such
that about 32 fmole of wild type target DNA was required to observe
a signal (row 1, column M). Addition of 25 molar equivalents of
unlabeled wild type probe to the hybridization mixture reduced the
intensity of the signal observed such that the lower limit of
detection remained at about 32 fmole of target DNA (row 1, column
P).
[0225] In conclusion, the results illustrated in FIG. 1 demonstrate
that non-specific binding of labeled (detectable) PNA probes to
non-complementary nucleic acid sequences can be suppressed by the
addition of unlabelled PNA probes which are complementary to
non-target sequences to which the labeled (detectable) PNA probe
binds non-specifically. As shown here the ability to discriminate
between the wild type and mutant targets goes up from 10 fold in
the absence of the unlabeled probe to greater than 100 fold in the
presence of unlabeled PNA probe.
[0226] Summary:
[0227] Estimated signal to noise ratio increased from approximately
10 to 320 for the labeled wild type probe in the absence and
presence of unlabeled mutant probe, respectively. For the mutant
oligonucleotide target, the estimated signal to noise ratio
increased from approximately 3 to 100 for the labeled mutant probe
in the presence and absence of unlabeled wild type probe,
respectively. Consequently, point mutation discrimination was
dramatically improved in the presence of the unlabeled (blocker)
PNA probe
[0228] Experiment B
[0229] Assay Description:
[0230] PNA Probe Solutions:
[0231] Table 2 displays the components of Hybridization Sets A
(1-4) and B (1-4). Labeled (detectable) PNA probes were diluted to
a final concentration of 5 pmole/mL with Hybridization Buffer.
Unlabeled probes were aliquoted from a concentrated stock "(conc.)"
of 333 pmole/.mu.L, or a diluted stock "(dil)" of 5 pmole/.mu.L.
The stock which was used to prepare the sample for the
hybridization assay is indicated in Table 2. Because Set 1 was a
control, no unlabeled PNA probe was added. Unlabeled PNA probes
were diluted to a final concentration of: 25 pmole/mL for group 2,
500 pmole/mL for group 3, and 2.5 mmole/mL for group 4. As
indicated in the table, the total volume of probe stock was 50
.mu.L for all Hybridization Sets prepared. The 50 .mu.L of probe
stock was then diluted in 150 .mu.L of Hyb. Buffer so that the
total volume of solution applied to the membrane was 200 .mu.L.
7TABLE 2 Wild Type Mutant Wild Type Mutant Set Labeled labeled
labeled unlabeled unlabeled Hyb. # Probe Type probe probe probe
probe Buffer 1A Wild Type 3.4 .mu.L -- -- -- 46.6 .mu.L 2A Wild
Type 3.4 .mu.L -- -- 1.0 .mu.L (dil.) 45.6 .mu.L 3A Wild Type 3.4
.mu.L -- -- 0.3 .mu.L (conc.) 46.3 .mu.L 4A Wild Type 3.4 .mu.L --
-- 1.5 .mu.L (conc.) 45.1 .mu.L 1B Mutant -- 2.6 .mu.L -- -- 47.4
.mu.L 2B Mutant -- 2.6 .mu.L 1.0 .mu.L (dil.) -- 46.4 .mu.L 3B
Mutant -- 2.6 .mu.L 0.3 .mu.L (conc) -- 47.1 .mu.L 4B Mutant -- 2.6
.mu.L 1.5 .mu.L (conc.) -- 45.9 .mu.L
[0232] Method:
[0233] Dilutions of target oligonucleotides were made in Oligo
Dilution Buffer to final concentrations of 3 pmole/.mu.L, I
pmole/.mu.L, 320 fmote/.mu.L, 100 fmole/.mu.L, 32 fmole/.mu.L, 10
fmole/.mu.L, 3 fmole/.mu.L, 1 fmole/.mu.L, 320 amole/.mu.L, and 100
amole/.mu.L Eight nylon membranes were prepared as described in
Experiment A Each membrane consisted of two columns of 6, 3.times.3
mm squares. Four membranes were designated as Set A, and the other
four were designated as Set B. In the left column of Set A,
aliquots of 0.32 .mu.L of the six consecutive dilutions of the wild
type target were spotted onto the membrane to generate a dot blot
array of target nucleic acid present in half log steps in the range
of 10 fmol to 32 amole (Because there was no observed signal below
320 amole, only the results for the first four targets are shown in
FIG. 2). In the right column of Set A, aliquots of 0.32 .mu.L of
the six consecutive dilutions of the mutant target nucleic acid
were spotted onto the membrane to generate a dot blot array of
nucleic acid present in half log steps in the range of 1 pmole and
3 fmole. In the left column of Set B, aliquots of 0.32 1L of the
six consecutive dilutions of the mutant target were spotted onto
the membrane to generate a dot blot array of nucleic acid present
in half log steps in the range of 10 fmole to 32 amole. In the
right column of Set B, aliquots of 0.32 .mu.L of the six
consecutive dilutions of the wild type target nucleic acid were
spotted onto the membrane to generate a dot blot array of nucleic
acid present in half log steps in the range of 1 pmole and 3 fmole.
Membranes were dried, and crosslinked as described in Experiment
A.
[0234] Materials and methods were identical to Experiment A, except
that the pre-hybridization incubation was extended to 90 minutes,
and the third wash incubation was extended to 30 minutes. As noted
above, the total volume of probe stock applied to each membrane was
200 .mu.L, as compared with Example A wherein the total volume was
250 .mu.L. The membranes of Set A were probed with wild type
labeled PNA probe in the presence of unlabeled mutant PNA probe in
the ratios indicated in FIG. 2. The membranes of Set B were probed
with mutant labeled PNA probe in the presence of unlabeled wild
type PNA probe in the ratios indicated in FIG. 2
[0235] Results
[0236] FIG. 2 is a composite of electronic images which were
obtained by scanning the autoradiographic images (film) obtained as
raw data from analysis of the membrane sets The images have been
arranged to align like target concentrations for easy comparison.
For convenience the columns of the figure have been designated with
the letters A-P and rows have been designated with the numbers 1-8.
Many locations in the figure contain no data as there was either no
data which was acquired for that set of conditions (i.e. rows 1-4
in columns A, C, E, G, I, K, M and 0, and rows 7 and 8 in columns
B, D, F, H, J, L, N, and P) or no detectable signal was observed at
the particular concentration of nucleic acid target (100 amole and
32 amole).
[0237] The left half of the figure displays results for the
membranes of Set A and the right half displays results for the
membranes of Set B. For each membrane there is a column labeled
"WT" for the wild type target, and "M" for the mutant DNA target.
The two sets of membranes (A and B) are arranged from left to right
as the relative molar concentration ratio of labeled (detectable)
PNA probe to unlabeled PNA probe was increased from 1:0, 1:5, 1:100
and 1:500. For the wild type probe, the "WT" dilution series
represents specific signal, and the "M" dilution series represents
non-specific signal. For the mutant probe, the "M" dilution series
represents specific signal, and the "WT" dilution series represents
non-specific signal.
[0238] Signal from non-specific hybridization of the wild type or
mutant labeled probe was greatly reduced in the presence of the
unlabeled (blocker) probes. For example, with reference to FIG. 2,
compare the signal intensity of the labeled wild type probe
hybridized to the mutant target in the absence of blocker probe
(column B) with that obtained from the hybridization in the
presence of various amounts of unlabeled mutant (blocker) probe
(columns D, F and H). In the absence of the unlabeled probe the
non-specific signal was clearly evident at 32 fmole of target DNA
(row 4, column B). Addition of 5 equivalents of unlabeled mutant
probe reduced the intensity of the non-specific signal by
approximately 3 fold, Increasing the lowest detectable level of
non-specific target to 100 fmole (row 3, column D) Addition of 100
equivalents of unlabeled mutant probe reduced the intensity of the
non-specific signal by approximately 32 fold, increasing the lowest
detectable level of non-specific target to 1 pmole (row 1, column
f) Addition of 500 equivalents of unlabeled mutant probe reduced
the intensity of the signal such that non-specific binding of the
wild type probe to the mutant target was not detectable within the
parameters of this experiment (row 1, column H).
[0239] The signal from the labeled mutant probe in the presence of
wild type unlabeled (blocker) probes, and wild type targets was
consistent with these results. For example, with reference to FIG.
2, compare the signal intensity of the labeled mutant probe
hybridized to the wild type target in the absence of blocker probe
(column J) with that obtained from the hybridization in the
presence of various amounts of the unlabeled wild type (blocker)
probe (columns L, N and P). In the absence of the unlabeled probe
the non-specific signal was clearly evident at 10 fmole of target
DNA (row 5, column J). Addition of 5 fold equivalents of unlabeled
wild type probe reduced the intensity of the non-specific signal by
approximately -3 fold, increasing the lowest detectable level of
non-specific target to 32 fmole (row 4, column L). Addition of 100
equivalents of unlabeled wild type probe reduces the intensity of
the non-specific signal by approximately an additional 10 fold
thereby increasing the lowest detectable level of non-specific
target to 320 fmole (row 2, column N). Addition of 500 equivalents
of unlabeled wild type probe reduced the intensity of the of the
non-specific signal by approximately an additional 3 fold so that 1
pmole of wild type target is the lowest detectable level (row 1,
column P). Consequently, the dynamic range (signal to noise ratio
of the assay) for the reactions using the mutant probe has improved
from 10 to I in the absence of unlabeled probe to approximately
1000 to 1 in the presence of 500 equivalents of unlabeled wild type
probe.
[0240] As the ratio of unlabeled probe to labeled probe was
increased there was a slight loss of specific signal. For example,
with reference to FIG. 2, the lowest detectable level of wild type
target was 320 amole (see row 8, columns A and C). In the presence
of 100 fold equivalents of unlabeled mutant (blocker) probe, the
limit of detection was reduced to 1 fmole (row 7, column E), This
was a 3 fold loss of signal. In the presence of 500 fold
equivalents of unlabeled probe (row 6, column G), there is
approximately a 10 fold loss of signal. As discussed above,
however, there was at least a 32 fold reduction in background
associated with non-specific binding to non-target sequence.
Consequently, there was at least a 10 fold improvement in the
signal to noise ratio in the presence of 500 equivalents of
unlabeled mutant probe. Similarly, the mutant target was at
approximately 320 amole (see row 8, column K). The addition of 100
or 500 fold equivalents of unlabeled wild type probe, reduced the
limit of detection to approximately 1 fmole (row 7 columns M and
0). This was approximately a 3 fold loss in detectable signal.
However, there was approximately a 100 fold reduction in background
associated with non-specific binding to non-target sequence.
Consequently, there was at least a 33 fold improvement in the
signal to noise ratio in the presence of 500 equivalents of
unlabeled wild type probe.
[0241] Summary:
[0242] In conclusion, the results illustrated in FIG. 2 further
demonstrate that non-specific binding of labeled PNA probes to
non-complementary nucleic acid sequences can be suppressed by the
addition of unlabelled PNA probes which are complementary to
non-target sequences to which the labeled PNA probes bind in a
non-specific manner. The results demonstrate that point mutation
discrimination improved from approximately 10 fold in the absence
of the unlabeled probe to greater than 1000 fold in the presence of
high levels of unlabeled (blocker) PNA probe. Moreover, there was
very little loss of detectable signal (approximately 3 to 10 fold)
even in the presence of 500 fold equivalents of unlabeled probe.
Consequently, when employing the methods described herein, one can
achieve several logs of improvement in point mutation
discrimination and similar improvements in the dynamic range of the
hybridization assay.
Example 5
Fluorescein labeling of Peptide Nucleic Acids
[0243] Peptide Nucleic Acids (PNAs) were synthesized using
commercially available chemicals and instrumentation from
PerSeptive Biosystems, Inc. The N-terminus of the support bound
oligomer was condensed with Fmoc-1-L-lys-(Fmoc)-OH PerSeptive
Biosystems, Inc. P/N GEN911094) using the automated synthesizer and
standard PNA coupling conditions. Using the standard PNA protocols
and condensation conditions, Fmoc-8-amino-3,6-dioxaoctanoic acid
was condensed with each of the N-.alpha. and N-.epsilon. amino
groups of the deprotected lysine amino acid to thereby branch the
amino terminus of the PNA oligomer. The Fmoc groups of the support
bound oligomer were then removed by treatment with piperidine in
DMF. The resin was then treated with a solution containing 0.076 M
5(6)-carboxyfluorescein-NHS ester (Molecular Probes; P/N C-1311)
0.38 M diisopropyethylamine in DMF at room temperature for 30-60
minutes. The support was then washed to remove excess labeling
reagent. The PNA was then cleaved from the support and purified
using standard methodologies.
Example 6
Comparison Of Blocking Probe Assays Using Combinations of
Detectable Probes (PNA and Nucleic Acid) and Blocker Probes (PNA
and Nucleic Acid)
[0244] Overview of Experiments A through C:
[0245] Experiments A-C were performed to compare combinations of
PNA and nucleic acid probes as both detection and blocking probes
in hybridization assays. The experiments were also performed to
determine the optimal conditions for suppression of the binding of
detectable probes to non-complementary nucleic acid sequences.
[0246] These experiments were directed to point mutation analysis
because point mutations are some of the most difficult of all
nucleic acid modifications to detect using a probe based
hybridization assay. A known point mutation in the codon twelve
region of the human Ki-ras gene is commonly observed in malignant
tissues and immortalized cell lines. This was chosen as a model
system. Two biotinylated 31-mer DNA targets having the subsequence
of the mutant and wild type codon twelve regions of human Ki-ras
were synthesized. These targets differed by one base pair (a base
pair mismatch or point mutation) which occurs in the middle of each
oligomer at position 16 (See below: Probes and Nucleic Acid
Targets). PNA and DNA probes which were complementary to each of
the two target nucleic acid sequences were also synthesized. The
probes were prepared as both labeled with fluorescein (detectable
probes) and unlabeled (blocker probes).
[0247] A set of labeled and unlabeled PNA 15-mers was prepared
since PNAs of this length have demonstrated high specific binding
to nucleic acid sequences with the appropriate affinity and
stability. For comparison, a set of labeled and unlabeled DNA
15-mers was prepared. Additionally, a set of labeled and unlabeled
DNA 25-mers was also prepared. The longer 25-mer DNAs were believed
to be more suitable for comparison to the 15-mer PNAs because they
have a thermal stability (Tm) which was more closely comparable
with the PNA 15-mers.
[0248] In experiments not described herein, blocker probes were
added at various concentrations either before, during or after the
addition of labeled (detectable) probes. The results demonstrated
that incubation of the blocker probe and target nucleic acid prior
to addition of the labeled (detectable) probe resulted in the most
favorable signal to noise ratio.
[0249] Experiment A
[0250] Signal from labeled probes hybridized to complementary or
non-complementary targets over a range of blocker probe
concentrations under conditions optimized for the hybridization of
each probe type (PNA or DNA) was measured. The relative ability of
unlabeled PNA and DNA probes to serve as blocker probes was
compared
[0251] Experiment B
[0252] The sensitivity and specificity of the DNA and PNA probe
sets at various target levels in the presence and absence of
blocking probes was compared.
[0253] Experiment C
[0254] PNA and DNA blocker probes were used in conjunction with
labeled DNA probes in normal and accelerated hybridization
assays
[0255] Materials and Methods
[0256] The PNA and DNA probes and targets used are shown below
8 Biotinylated DNA targets SEQ ID NO:3 Wild type 5'
Biotin-GTGGTAGTTGGAGCTGGTGGCGTAGGC AAGA-OH 3' SEQ ID NO:4 Mutant 5'
Biotin-GTGGTAGTTGGAGCTTGTGGCGTAGGC AAGA-OH 3' PNA Labeled Probes
Wild type N (Flu-linker).sub.2-K-ACGCCACCAGCTCCA-NH.sub.2 C Mutant
N (Flu-linker).sub.2-K-ACGCCACAAGCTCCA-NH.sub.2 C PNA Blocker
probes Wild type N H-ACGCCACCAGCTCCA-NH.sub.2 C Mutant N
H-ACGCCACAAGCTCCA-NH.sub.2 C DNA Labeled Probes SEQ ID NO:5 Wild
type-15 5' Flu-spacer-ACGCCACCAGCTCCA-OH 3' SEQ ID NO:6 Mutant-15
5' Flu-spacer-ACGCCACAAGCTCCA-OH 3' SEQ ID NO:7 Wild type-25 5'
Flu-spacer-TGCCTACGCCACCAGCTCCAACT AC-OH 3' SEQ ID NO:8 Mutant-25
5' Flu-spacer-TGCCTACGCCACAAGCTCCAACT AC-OH 3' DNA Blocker probes
SEQ ID NO:9 Wild type-15 5' HO-ACGCCACCAGCTCC-OH 3' SEQ ID NO:10
Mutant-15 5' HO-ACGCCACAAGCTCCA-OH 3' SEQ ID NO:11 Wild type-25 5
HO-TGCCTACGCCACCAGCTCCAACTAC-OH 3' SEQ ID NO:12 Mutant-25 5
HO-TGCCTACGCCACAAGCTCCAACTAC-OH 3'
[0257] All DNA probes are illustrated from 5' to 3' and all PNA
probes are illustrated from the amino terminus (N) to the carboxyl
terminus (C). Probe and target mismatch sites are underlined and
lie at the centers of the molecules. Probe names indicate the
sequence to which the probe is complementary. For example, the
"wild type PNA" probe is a perfect match to the wild type DNA
target when hybridized in the antiparallel orientation. Nucleic
acid probes were either obtained from commercial vendors of custom
oligonucleotides, or synthesized using commercially available
instrumentation and reagents. For the fluorescein labeled DNA
probes, "spacer" designates a linker incorporated into the
oligonucleotide with the Fluorodite.TM. labeling phosphoramidite
obtained from PerSeptive Biosystems, Inc. (P/N GEN080110). The
biotin labeled DNA targets were prepared by a commercial vendor of
custom oligonucleotides (Genosys) using their standard procedures.
PNA probes were synthesized using commercially available
instrumentation and reagents. PNAs were branched at the N-terminus
by the condensation of a lysine amino acid (illustrated as "K")
during chemical synthesis. PNAs were labeled with fluorescein as
described in Example 5 of this specification. For the PNA
oligomers, "linker" designates the Expedite PNA linker (PIN
GEN063032) attached to the lysine amino acid at the N-terminus of
the PNA oligomer.
[0258] General Assay Procedure:
[0259] Biotinylated target nucleic acids and probes were incubated
under appropriate hybridization conditions, after which the
contents of the hybridization reaction were placed in wells of
Streptavidin coated microtitre plates to thereby capture the
biotinylated target nucleic acids and any probes hybridized
thereto. After removal of the unbound material and subsequent
washing, the wells were contacted with an alkaline phosphates
conjugated to a anti-FITC Fab fragment "Rabbit(Fab) anti-FITC/AP"
The Rabbit(Fab) anti-FITC/AP binds to the fluorescein of the
detectable probe, if present (note, although the PNA probes in this
assay were bis-labeled with fluorescein, prior studies demonstrated
that detection of these probes with the Rabbit(Fab) anti-FITC/AP
conjugate produced similar levels of signal as single-labeled PNA
or DNA probes). Non-specifically bound .alpha.-fluorescein
antibody-alkaline phosphatase enzyme conjugate was then removed by
washing, and an alkaline phosphatase activated chemiluminescent
substrate was then added to the wells. Emitted light was measured
using a suitable plate reader.
[0260] The Molecular Diagram (FIG. 3) illustrates the components of
the hybridization assay as described above. The Streptavidin coated
plate, illustrated by the symbol "X", binds the biotinylated
nucleic acid target illustrated using the symbol "B______".
Fluorescein labeled probe which is hybridized to the target nucleic
acid is illustrated by the symbol "______F". The fluorescein moiety
"F", is used as a hapten to thereby bind an .alpha.-fluorescein
antibody which is illustrated as the upside down "Y" in the figure.
The .alpha.-fluorescein antibody is conjugated to the alkaline
phosphatase enzyme which is illustrated as "AP" in the figure. The
.alpha.-fluorescein antibody-alkaline phosphatase enzyme conjugate
"Rabbit(Fab) anti-FITC/AP" is commercially available from DAKO A/S
(Copenhagen, Denmark) Non-labeled blocker probe is shown as not
binding to any assay component, it is illustrated by the symbol
"______".
[0261] Hybridizations were performed in polystyrene microtiter
plates by mixing stock solutions of probes, nucleic acid targets,
and hybridization buffers. All incubations were performed at room
temperature with agitation. Each assay was performed by first
adding the required amount of target nucleic acid, diluted to 80
.mu.L volume in buffer to the polystyrene microtitre plate. For
reactions containing unlabeled (blocker) probes, a solution
containing the appropriate amount of unlabeled (blocker) probe, in
a 10 .mu.L volume, was then added to the target nucleic acid and
the mixture was incubated at ambient temperature (22.+-.2.degree.
C. for all experiments). For control reactions which contained no
unlabeled (blocker) probe, a 10 .mu.L aliquot of the appropriate
dilution buffer was added. A solution containing the appropriate
amount of labeled (detectable) probe, in 10 .mu.L volume, was then
added to the target/blocker probe mixture and this solution was
again incubated at ambient temperature. Each 100 .mu.L reaction was
transferred to a well of a Streptavidin coated microtitre plate and
allowed to incubate for 30 minutes. The solution in the well was
then removed, and each well was washed six times with 300 .mu.L of
Wash Buffer A. Next, 100 .mu.L of a 1:1000 dilution of Rabbit(Fab)
anti-FITC/AP stock solution in Wash Buffer B was added, and allowed
to incubate for 30 minutes at ambient temperature (the optimal
antibody dilution factor, 1:1000, was determined experimentally,
data not shown). The Anti-FITC-AP solution was then removed and the
wells were washed three times with 150 .mu.L of Wash Buffer B, then
three times with 150 .mu.L of Wash Buffer C. Between washes, the
plates were incubated at ambient temperature with shaking for 1
minute. Finally, 50 .mu.L of Visualization Reagent was added to
each well and the plate was incubated at ambient temperature with
shaking for exactly 4 minutes. The Visualization Reagent was
transferred to an opaque (white) reading plate and read for 1 sec
in a Wallac 1420 Multilabel luminometer. The readings obtained from
the lumminometer in relative light units (RLU) indicated the extent
of binding of the labeled (detectable) probe to the target nucleic
acid. For experiments A-C, each hybridization reaction was
performed at least in triplicate and the resulting measurements
were averaged. No data points were discarded. Background
measurements were performed at least in duplicate and were also
averaged.
[0262] The time required for each step of the assay is shown
below.
[0263] Assay Time Line:
9 Steps: Time Line 1. 1 hour blocker probe hybridization 1.00 hr 2.
1 hour labeled probe hybridization 2.00 hr 3. 30 minute capture of
complexes onto SA coated plate 2.50 hr 4. 6x Wash Buffer A washes
2.60 hr 5. 30 minute Anti-FITC-AP binding 3.10 hr 6. 3x Wash Buffer
B Washes 3.15 hr 7. 3x Wash Buffer C Washes 3.20 hr 8. 4 minute
Visualization Reagent incubation 3.30 hr
[0264] Reagent and Buffer Compositions:
[0265] Wash Buffer A
[0266] 10 mM NaCl
[0267] 5 mM TRIS pH 7.3
[0268] 0.01% TWEEN-20
[0269] Wash Buffer B
[0270] 0.5 M NaCl
[0271] 50 mM TRIS pH 9.0
[0272] Wash Buffer C
[0273] 10 mM TRIS pH 9.4
[0274] 10 mM NaCl
[0275] 1 mM MgCl.sub.2
[0276] Visualization Reagent
[0277] 0.4 mM CDP-Star TM
[0278] 1.times.Sapphire II Enhancer.TM.
[0279] 0.1 M Diethanolamine
[0280] The Visualization Reagent comprises CDP-Star.TM. (Tropix,
Bedford, Mass.), a 1,2-dioxetane, which when dephosphorylated by AP
produces a metastable intermediate, which emits light at 466 nm
upon decay (half life .about.2 min.). Sapphire II.TM. (Tropix,
Bedford, Mass.) is a luminescence enhancer which reduces the
effects of aqueous quenching, producing amplified signals
[0281] Choice of Hybridization Conditions:
[0282] Those of ordinary skill in the art of nucleic acid
hybridization will recognize that factors commonly used to impose
or control stringency of hybridization include formamide
concentration (or other chemical denaturant reagent), salt
concentration (i.e., ionic strength), hybridization temperature,
detergent concentration, pH and the presence or absence of
chaotropes. Optimal stringency for a probe/target combination is
often found by the well known techniques of fixing several of the
aforementioned stringency factors and then determining the effect
of varying a single stringency factor. This was done for the
various probes types and lengths. For the work presented here, the
hybridization conditions for a probe set were defined as optimal
when a further increase in stringency produced large reduction in
signal without a corresponding increase in the ability to
discriminate between perfectly matched and mismatched targets.
[0283] A number of stringency factors were fixed throughout the
experiments for either probe type (PNA or DNA) including pH (7.0),
temperature (ambient), detergent (0.5% v/v, Tween-20) and chaotrope
(none). Ionic strength was fixed at 100 mM NaCl for hybridizations
with PNA detector probes and 250 mM NaCl for hybridizations
involving DNA detector probes. With these stringency factors fixed,
formamide concentration was varied and found to be optimal at 70%,
65% and 35% for the PNA 15-mer, DNA 25-mer and DNA 15-mer probes,
respectively. A summary of buffers which were found to provide
optimal stringency is shown below.
[0284] Hybridization Buffer Table:
10 Probe Buffer Type Formamide NaCl (mM) TWEEN-20 Na.sub.2PO.sub.4
A: PNA 70% 100 0.5% 10 mM B: DNA 25 65% 250 0.5% 10 mM C: DNA 15
35% 250 0.5% 10 mM
[0285] All hybridizations were performed at room temperature when
using the conditions described above.
[0286] Experiment A
[0287] Assay Description:
[0288] FIG. 4A is a plate assay schematic of the configuration of
hybridization reactions performed in the wells of a microtiter
plate for this experiment. Each of the reaction wells in the figure
is given a specific location comprised of an alphanumeric character
(e.g. A) and a number (e.g. 1). For row locations the alphanumeric
character is sequentially incremented and for column locations the
number is sequentially incremented. For each condition tested,
three data points (arranged column wise) were generated to obtain
an average. Dilutions of targets, labeled (detectable) probes and
unlabeled (blocker) probes were prepared as described above.
Complementary, "Match", target sequence was added at 30 fmole to
rows A-C; non-complementary, "Mismatch", target sequence was added
at 300 fmole to the wells in rows D-F, "No Target", control
hybridizations were performed in the wells in rows G and H. Labeled
detector probes were used at 3 pmole/well. The ratio of unlabeled
(blocker) probe to labeled (detectable) probe is indicated at the
top of each column of FIG. 4A. Labeled wild type probes (and mutant
unlabeled (blocker) probes) were used in the wells in columns 1-6,
labeled mutant probes (and wild type unlabeled (blocker) probes)
were used in the wells in columns 7-12.
[0289] The experimental conditions, assay timing and reagent
composition were described in the Materials and Methods section,
above. The assay was performed three times, once with PNA probes,
once with DNA 25-mer probes, and once with DNA 15-mer probes. Wild
type and mutant PNA 15-mer, DNA .sup.15-mer and DNA 25-mer labeled
(detectable) probes were hybridized under conditions optimized for
each probe type (See: Choice of Hybridization Conditions,
above).
[0290] Results and Discussion:
[0291] With reference to FIGS. 4B and 4C, the data displayed are
average RLU (with average backgrounds subtracted) on the ordinate
axis, vs. the relative amount of unlabeled (blocker) probe
("Blocker(X)") along the abscissas. The ordinate axes of FIGS. 4B
and 4C are on the LOG.sub.10 scale. FIG. 4B displays the data for
the labeled (detectable) wild type probes in the presence of
various amounts of unlabeled mutant (blocker) probe. FIG. 4C
displays the data for the labeled (detectable) mutant probes in the
presence of various amounts of unlabeled wild type (blocker) probe.
In both Figures, the solid lines indicate data generated by
hybridization between complementary targets and probes ("Match")
and the dashed lines indicate data generated by hybridization
between non-complementary targets and probes ("Mismatch"). In both
Figures, diamond shaped symbols indicate use of DNA 25-mer probes,
triangle shaped symbols indicate use of PNA probes, and square
shaped symbols indicate use of DNA 15-mer probes. The use of
unlabeled (blocker) probes in conjunction with labeled DNA or
labeled PNA probes suppressed signal from non-complementary targets
(dashed lines). Addition of unlabeled (blocker) probes to
complementary target did not prevent binding (hybridization) of the
complementary probe and target as was indicated by the constant
signal levels maintained (solid lines). Of the three probe sets
tested, the PNA probes provided the greatest level of
discrimination between complementary and non-complementary sets of
probes and targets as indicated by the greatest absolute difference
between the solid and dashed line for each set of probes. These
conclusions are further supported by the data presented in FIG.
4D
[0292] FIG. 4D is a table of Match/Mismatch (Ma/Mi) ratios Column A
displays the relative amount of blocker probe present in each
hybridization reaction Columns B-G display the calculated Ma/Mi
ratio for each probe (defined in row 2) at each relative
concentration of unlabeled (blocker) probe Match refers to signal
from hybridization between perfectly complementary target nucleic
acids and probe sequences, whereas; Mismatch refers to signal from
hybridization between non-complementary target nucleic acids and
probe sequences which are related as point mutations. The Ma/Mi
ratio gives a sense of the relative affinity of a probe for its
matching target as compared to a one base pair mismatch target
(i.e., point mutant). The ratios were generated by application of
the following formula where "avg. bkgd" is the average background
signal. (avg. Match signal-avg. bkgd.)/((avg. Mismatch signal-avg.
bkgd.)/10)=Ma/Mi ratio. The "Mismatch" term (denominator) is
divided by 10 to normalize the different levels of target (30 and
300 fmole) being detected in "Match" and "Mismatch" samples.
Background was calculated by averaging all of the "No Target"
values for each probe tested (wells G1-H6 in FIG. 4A). As an
example, the value of 9 for the wild type PNA probe Ma/Mi ratio
(FIG. 4D, column B, row 3) was derived by applying the equation to
the average of the signals generated in wells A1-C1 (Match), and
D1-F1 (Mismatch).
[0293] The data in FIG. 4D reveals a dramatic increase in the Ma/Mi
ratio upon addition of increasing amounts of unlabeled (blocker)
probe for the two probe types, PNA and DNA (See: rows 3-8 of each
column). In the case of the DNA probe sets (See: columns B, C, F
and G) the Ma/Mi ratios go up almost to 300. For the PNA probe sets
(See: columns D and E) the ratios go well above 1000. The four DNA
probe sets all demonstrate approximately equal Ma/Mi ratios. For
example, at "2X" (row 6) the wild type DNA 25, mutant DNA 25, wild
type DNA 15, and mutant DNA 15 are 110, 173, 105, and 185
respectively. The PNA probes display a greater increase in
discrimination than the DNA probes as the concentration of
unlabeled (blocker) probe was increased. At "2.times." blocker (row
6), the wild type PNA and mutant PNA have Ma/Mi ratios of 1009 and
929, respectively. There is also a substantial difference in Ma/Mi
ratios for the PNA probes present at only 0.5.times.blocker (row 4)
The PNA probes are at 497 and 255, while the DNA probes have values
of only 27, 73, 46, and 83. This observed difference is probably
attributable to the fast rate of hybridization of PNA probes, and
the relative stability difference between perfect match and one
base pair mismatch PNA/DNA hybrids as compared to DNA/DNA
hybrids.
[0294] Summary of Experiment A:
[0295] The data demonstrates that unlabeled PNA and DNA probes can
be effectively used to suppress the binding of detectable probe to
non-target sequence over a wide range of concentrations. At all
concentrations tested, the unlabeled (blocker) PNA probes are
better at enhancing discrimination between complementary and
non-complementary targets than either of the unlabeled (blocker)
DNA probes tested (i.e. 15-mer or 25-mer).
[0296] Experiment B
[0297] Assay Description:
[0298] In this experiment, wild type and mutant PNA 15-mer,
DNA-15-mer and DNA 25-mer labeled (detectable) probes were
hybridized to varying amounts of complementary and
non-complementary nucleic acid targets. Results were obtained both
in the presence and absence of two fold equivalents of unlabeled
blocker probes.
[0299] FIG. 5A is a plate assay schematic of the configuration of
hybridization reactions performed in the wells of a microtiter
plate for this experiment. The well positions are again assigned a
two digit code comprising an alphanumeric character (e.g. A) and a
numeric character (e.g. 1) to designate column and row assignments,
respectively. For each condition tested, four data points (arranged
column wise) were generated to obtain an average (derived data)
useful for comparative analysis. The top half of the plate (rows
1-4) was used to measure signal from labeled wild type sequence
probes. The bottom half of the plate was used to measure signal
from labeled mutant sequence probes.
[0300] Dilutions of target nucleic acids, labeled (detectable)
probes and unlabeled (blocker) probes were performed as described
above. For this experiment, "Match" targets were used in quantities
of 50, 5, and 0.5 femptomoles (fmole) per well (columns 1-6).
"Msmatch" targets were used at high levels, 500 and 50 fmoles per
well (columns 9-12), due to the expected decrease in binding
affinity between non-complementary target and probe. Control
reactions containing no target "NT" were also performed in the
wells in columns 7 and 8. The presence and absence of unlabeled
(blocking) probe is indicated by the (-) or respectively, above the
columns. In this experiment, the wells in the odd numbered columns
(1, 3, 5 etc.) were used to measure signal in the absence of
unlabeled (blocker) probes, and the wells in the even numbered
columns (2, 4, 6 etc.) were used to measure the signal in the
presence of blocker probes. Background for the wild type sequence
probes in the absence of unlabeled (blocker) probe was measured in
wells A7-D7, and background for the mutant sequence probes in the
absence of blocker probe was measured in wells E7-H7. The
concentration of labeled (detectable) probe in each well was 5
pmole/100 .mu.L, the unlabeled (blocker) probe was present at 10
pmole/100 .mu.L, and the target nucleic acids were present at the
concentrations depicted in the figure. The assay was performed
three times, once with PNA probes, once with DNA 25-mer probes, and
once with DNA 15-mer probes, each time under conditions which were
optimized for the particular probe type.
[0301] Results and Discussion:
[0302] With reference to FIG. 5B, the Ma/Mi ratios for 50 fmole of
target nucleic acid are shown for all three probe types, in the
absence and presence of blocker probes. The Ma/Mi ratio is
calculated the same way as in Experiment A except that the
denominator is not divided by 10 because match and mismatch values
were derived from equal amounts of target (50 fmol). Column B
displays Ma/Mi ratios in the absence of blocker probe. Column C
displays Ma/Mi ratios in the presence of two equivalents of
unlabeled (blocker) probe. For example, the Ma/Mi ratio for the
labeled PNA probe in the absence of blocker can be found in FIG. 5B
column B, row 2 (Ma/Mi=2.5). In this example, the values for "Match
signal", "avg. bkgd", and "Mismatch signal" from the above equation
are derived from FIG. 5A, wells A1-D1, A7-D7, and A11-D11,
respectively. With reference to FIG. 5B, column B, all Ma/Mi ratios
are between 11 and 4.1 in the absence of unlabeled (blocker) probe
(whether DNA or PNA). A low value for the Ma/Mi ratio indicates
comparatively poor discrimination between the match and mismatch
targets. Because all values are less than ten in the absence of
unlabeled (blocking) probes these conditions are not be very useful
for point mutation analysis. With reference to column C of FIG. 5B,
there was a dramatic increase in the Ma/Mi ratio for all
experiments where blocker probe was used (compare results with
column B). For the wild type probes, the PNA probe set had more
than twice the power to discriminate between target and non-target
nucleic acid as compared with the DNA probe sets (PNA 15-mers, 78;
DNA 25-mers, 30; DNA 15-mers, 30). Likewise, for the mutant probes,
the PNA probe set had more than twice the power to discriminate
between target and non-target nucleic acid as compared with the DNA
probe sets (PNA 15-mers, 140; DNA 25-mers, 42; DNA 15-mers,
65).
[0303] FIG. 5C is a graphical illustration of the data from FIG.
5B. Note the ordinate axis is on a linear scale. The graphical
illustration is useful as a quick means to visually analyze and
compare the tabular data.
[0304] FIG. 5D shows the "Blocker Effect" (BE) values for all three
probe types at all target amounts tested. The BE value is
determined using the following equation.
(avg. signal w/o blocker-avg. bkgd.)/(avg. signal w/blocker-avg.
bkgd.)=BE
[0305] The BE value measures a fold decrease in signal resulting
from the addition of unlabeled (blocker) probe to a hybridization
reaction. It follows from the equation, that the BE value for an
ideal blocker probe would be approximately equal to 1.0 when the
blocker was added to a hybridization reaction involving a
detectable probe and its matched target (i.e., the blocker probe
would not depress the signal). In the case of a hybridization
between a labeled probe and a mismatched target, an ideal blocker
probe would have a large BE value (i.e., the blocker would suppress
binding of the detectable probe to the mismatch target).
[0306] The calculation of a BE value is illustrated by the
following example. For the wild type PNA probe at 5 fmole of wild
type (Match) target nucleic acid the BE value equals 1.0 (FIG. 5D,
column C, row 2). This value was calculated by using "avg signal
w/o blocker", "avg. bkgd", and "avg signal+blocker" values derived
(as averages) from wells A3-D3, A7-D7, and A4-D4 respectively (FIG.
5A)
[0307] With reference to FIG. 5D, columns B, C, D, there is no
appreciable loss of signal (05.ltoreq.BE.ltoreq.1.5) seen at any
target level, with either the DNA or PNA probes where the labeled
probes and target nucleic acids are perfect complements (i.e.,
match). However for mismatch targets at both the 500 and 50 fmole
levels (See: columns E and F), the BE values were substantially
greater especially for the PNA probes. For example, at 500 fmole of
target (see column E) the wild type and mutant PNA probes have BE
values of 132 and 86, respectively. The wild-type DNA 25-mers and
DNA 15-mers have BE values of 11 and 17, respectively. The mutant
DNA 25-mers and DNA 15-mers have BE values of and 16 and 23,
respectively. This data demonstrates that the use of unlabeled
(blocker) PNA probe, when used with labeled (detectable) PNA probe,
results in a much higher level of discrimination between
complementary and non-complementary target nucleic acid than does
the use of either set of unlabeled (blocker) DNA probes in
conjunction with labeled (detectable) DNA probes.
[0308] Although the data in column F generally exhibit the same
trends as the data in column E, the values are consistently lower
in column F. It is believed that this discrepancy is attributable
to the significantly lower signal at 50 fmole, thereby resulting in
lower signal to noise ratios. Therefore, the data in column E is
believed to be better quantitative data.
[0309] The BE values at 500 and 50 fmole which are listed in FIG.
5D are graphically illustrated in FIG. 5E. Note the ordinate axis
is on the linear scale. The graphical illustration is useful as a
quick means to visually analyze and compare the tabular data.
[0310] Summary of Experiment B:
[0311] The use of unlabeled (blocker) probes significantly
increased the discrimination of point mutations in target nucleic
acids. The improved discrimination was obtained without any
significant loss of signal when using both labeled DNA or PNA
probes, at any target level tested. However, the use of unlabeled
(blocker) PNA probes produced a substantially greater increase in
point mutation discrimination than the use of unlabeled (blocker)
DNA probes
[0312] Experiment C
[0313] Assay Description:
[0314] Experiment C was performed to determine whether the various
probe types could be intermixed (e.g., labeled (detectable)DNA used
with unlabeled (blocker) PNA probes) and if so, how did the results
compare with the all PNA or DNA probe sets. To this end, unlabeled
(blocker) PNA 15-mer probes were used as blockers for labeled
(detectable) DNA 25-mer probes under conditions optimized for the
hybridization of the DNA 25-mers to their targets (Hybridization
Buffer B).
[0315] Hybridization times were also varied to determine whether
there was any benefit to performing the hybridization assay more
rapidly. For this comparison, hybridization time for incubations
with the unlabeled (blocker) probe and labeled (detectable) probe
were shortened from 60 to 20 minutes.
[0316] FIG. 6A is a schematic of the plate set up used in
Experiment C. The well positions are again assigned a two digit
code comprising an alphanumeric character (e.g. A) and a numeric
character (e.g. 1) to designate column and row assignments,
respectively. Hybridization reactions were performed in the wells
in rows A-C for 60 minutes. Hybridization reactions were performed
in the wells in rows D-H for 20 minutes. For comparison, 20 min.
hybridization reactions were also performed using labeled
(detectable) PNA probes with and without unlabeled (blocker) PNA
probes in Hybridization Buffer A (100 mM NaCl and 70 percent
formamide) (See FIG. 6A, rows G and H) which is optimized for the
all PNA system.
[0317] Only the labeled wild type probes, and their corresponding
unlabeled (blocker) mutant probes were used in this experiment.
Hybridization reactions containing the wild type DNA 25-mer probes
were performed in the wells in rows A-F. Hybridization reactions
containing the wild type PNA 15-mer probes were performed in the
wells in rows G and H The unlabeled (blocker) DNA 25-mer probe
(mutant sequence) was used in the hybridization reactions performed
in the wells in rows A and D Unlabeled (blocker) PNA probe (mutant
sequence) was used in the hybridization reactions performed in the
wells in rows B, E and G Control reactions containing no unlabeled
(blocker) probes were performed in rows C, F and H Reactions
performed in columns 1-3 contained 500 fmole of mutant
(non-complementary) target. Reactions performed in the wells in
columns 4-6 contained 50 fmole of mutant target. Control reactions
performed in the wells in columns 7-9 contained no target, and
reactions performed in the wells in columns 10-12 contained 50
fmole of wild type (complementary) target. Data was collected from
three identical hybridization reactions and averaged to generate
the derived data.
[0318] Results and Discussion:
[0319] With reference to FIG. 6B, the table displays derived Ma/Mi
ratios for 50 fmole of target nucleic acid for both probe types in
the presence of unlabeled (blocker) probes. The Ma/Mi and blocker
effect ratios are generated with the same formulas used in
Experiment A, except that the denominator was not divided by 10
since the match and mismatch target amounts were equal (50 fmol).
For example, use of the labeled and unlabeled DNA 25-mer probes and
60 minute hybridization times gave a Ma/Mi value of 125 (FIG. 6B,
column D, row 2). In this example, the terms "Match signal", "avg.
bkgd", and "Mismatch signal" from the above equation are derived
from wells A10-A12, A7-A9, and A4-A6, respectively (FIG. 6A).
[0320] With reference to FIG. 6B, rows 2-4, the presence of
unlabeled (blocker) DNA probe (row 2) and unlabeled (blocker) PNA
probe (row 3) exhibited significant beneficial effects on the Ma/Mi
ratio as compared with hybridizations in which no blocker probes
were present (row 4). When using the conditions optimized for the
DNA 25-mers with 60 minute hybridizations, the results obtained
with the unlabeled (blocker) DNA blocker were somewhat better than
the results obtained with the unlabeled (blocker) PNA probes
(compare rows 2 and 3).
[0321] By comparison however, data obtained for the 20 minute
hybridizations demonstrate that, under these conditions, the
unlabeled (blocker) PNA had a higher Ma/Mi ratio than did the
unlabeled (blocker) DNA probe (compare rows 5 and 6 respectively).
Moreover, the point mutation discrimination was greatly improved In
the presence of unlabeled (blocker) probes (PNA or DNA) (compare
rows 5 and 6 with row 7). With reference to column D, rows 8 and 9,
the presence of the unlabeled (blocker) PNA probes, under
conditions Optimized for PNA hybridizations, resulted in the
highest specificity when used in combination with the labeled
(detectable) PNA probe (Ma/Mi ratio=357). This data demonstrates
that the rapid hybridization assay format when performed in
combination with unlabeled and labeled PNA probes results in the
most dramatic point mutation discrimination.
[0322] With reference to FIG. 6C, the BE values calculated from the
derived data are presented. The BE value was calculated as
described in Experiment B. In all mismatch cases the BE value was
larger when using the unlabeled (blocker) PNA probes than when
using the unlabeled (blocker) DNA 25-mer probes (compare row 3 to
row 2, and row 5 to row 4). Concomitant with the marked increase in
discrimination through the use of PNA blocker probe, there was a
slight cost to signal (1.2.ltoreq.BE.ltoreq.1.6) (see column D,
rows 3, 5, and 6). The BE values for the unlabeled (blocker) DNA
probes are similar to those observed in Experiment B. As observed
in Experiment B, there was no loss of signal associated with the
presence of the unlabeled (blocker) DNA probes (see column D; rows
2, 4). With regard to columns E and F, a comparison of data in rows
1-5 with the data in row 6 demonstrated that although unlabeled
(blocker) PNA probes can increase the discrimination of the labeled
DNA 25-mer probes, the presence of unlabeled (blocker) PNA probes
in conjunction with labeled PNA probes was far better at
suppressing signal from mismatch targets.
[0323] Summary of Experiment C:
[0324] Unlabeled (blocker) PNA probes can be used to improve
discrimination of labeled (detectable) DNA probes with little loss
of sensitivity. However, the presence of unlabeled (blocker) PNA
probes increases the level of point mutation discrimination of
labeled (detectable) DNA probes to a greater extent than the most
nearly equivalent unlabeled (blocker) DNA probes. Rapid
hybridization assay formats are preferred when using the unlabeled
(blocker) PNA probes, particularly when the conditions have been
optimized for hybridization of labeled (detectable) nucleic acid
probes.
Example 7
Synthesis of PNA Capture Probe comprising a C-terminal Arylamine
moiety:
[0325] Experiment A: Synthesis of
N-.alpha.-(Fmoc)-N-.epsilon.-(4-(N-(tert- -butyloxycarbonyl
aminobenzoyl)-L-Lysine-OH
[0326] To 2.6 mmole of N-.alpha.-Fmoc-L-lysine-OH was added 2.7
mmole of trifluoroacetic acid to dissolve the amino acid. Once the
amino acid was completely dissolved this solution was added to the
activated 4-(N-(tert-butyloxycarbonyl)-aminobenzoic acid prepared
as described below.
[0327] To 2.6 mmole of 4-(N-(tert-butyloxycarbonyl)-aminobenzoic
acid (prepared in Example 1) was added 50 mL of
N,N'-dimethylformamide (DMF), 2.7 mmole
[O-(7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate (HATU) and 15 mmole diisopropylethylamine
(DIEA). This solution was allowed to stir for 20 minutes and then
the solution of N-.alpha.-Fmoc-L-lysine-OH was added dropwise.
After reacting for 30 min., the solvent was removed by evaporation
under reduced pressure. The residue was partitioned in 100 mL of
dichloromethane (DCM) and 50 mL of 10% aqueous citric acid. An
attempt was made to wash the organic layer with 50 mL of 5% aqueous
sodium bicarbonate but the product crystallized from the solution.
The white solid was then collected by vacuum filtration. An attempt
was made to dissolve the solid in a mixture of 30 mL of 10% aqueous
citric acid and 70 mL DCM but the product would not dissolve. Thus,
the solid was again collected by vacuum filtration and used as
obtained. Yield 0.924 g (1.57 mmole: 60%).
[0328] Experiment B: Synthesis of
N-.alpha.-(Fmoc)-N-.epsilon.-(4-(N-(tert-
-butyloxycarbonyl)-aminobenzoyl)-L-Lysine-PAL-Peg/PS Synthesis
Support
[0329] The
N-.alpha.-(Fmoc)-N-.alpha.-(4-(N-(tert-butyloxycarbonyl)-aminob-
enzoyl)-L-Lysine-OH prepared as described above was used to prepare
a synthesis support useful for the preparation of C-terminal
arylamine modified PNA probes. The Fmoc group of commercially
available bulk Fmoc-PAL-Peg-PS (PerSeptive Biosystems Inc.)
synthesis support (approx. 1 g) was removed by treatment, in a flow
through vessel with 20% piperidine in DCM for 25 minutes. The
deblocking solution was then removed from the reaction vessel and
the resin was washed with DMF and dried with a flushing stream of
argon.
[0330] A solution containing 0.440 g
N-.alpha.-(Fmoc)-N-.epsilon.-(4-(N-(t-
ert-butyloxycarbonyl)-aminobenzoyl)-L-Lysine-OH, 4.8 mL of DMF,
0.266 g HATU, 0.157 mL DIEA and 0.104 mL 2,6-lutidine was prepared
by sequential addition of the reagents listed. This solution was
added to the washed resin and allowed to react for 2.5 hours. The
solution was then flushed through the vessel with a stream of argon
and the resin washed sequentially with DMF, DCM and DMF. The resin
was then dried with a flushing stream of argon.
[0331] The resin was then treated with 5 mL of standard
commercially available PNA capping reagent (acetic anhydride and
2,6-lutidine in DMF) for three minutes. The capping reagent was
then flushed from the vessel and the resin was washed with DMF and
DCM. The resin was then dried with a stream of argon. Finally, the
resin was dried under high vacuum.
[0332] Final loading of the resin was determined by analysis of
Fmoc loading of three samples of approximately 11-14 mg using well
known methods. Analysis determined the loading to be approximately
0.105 mmol/g.
[0333] Experiment C: Synthesis Of PNA Capture Probe:
[0334] The synthesis support prepared as described in Example B,
above, was then packed into a standard PNA synthesis column. PNA
synthesis was performed using standard commercially available
instrumentation and reagents. The deprotection and purification of
the PNA was also performed using standard methodologies. The PNA
synthesis gave the desired OL6 PNA capture probe suitable for use
in Experiment 8, below
Example 8
Capture Assay in the Presence and Absence of Blocking Probes
Preparation of Nucleic Acid Targets
[0335] Neisseria gonorrhoeae (N g) and Neisseria meningitidis (N m)
16S rDNAs were obtained from Dako (Copenhagen, Denmark) and then
amplified Using the polymerase chain reaction (PCR). The nucleotide
sequence of the forward and reverse primers are reported below.
Pyrococcus furiosus (Pfu) DNA polymerase was used in the PCR
amplification. Each of the two 16S rDNA amplimers were
approximately 1500 base pairs in length. The 16S rDNA amplimers
were then cloned into the transcription vector pGEM-4Z (Promega
Corp., Madison, Wis.) using standard methods such that
transcription from the T7 promotor yields the 16S rRNA sequence.
Regions within the 16S rDNA clones of the N.g. and N.m. clones were
then sequenced using the CircumVent Phototope thermal cycling kit
(New England Biolabs Inc., Beverly, Mass.). The sequence
information obtained was then compared with the data available in
GenBank to confirm that the PCR amplification reactions had not
misincorporated any nucleotides.
[0336] Biotin-labeled 16S rRNA transcripts of N.g. and N.m. were
then prepared in vitro using T7 RNA polymerase and the RiboMax
transcription kit (Promega Corp., Madison, Wis.). A ratio of 3
parts uridine triphosphate (UTP) to 1 part biotin-21-UTP (Clontech
Laboratories, Inc., Palo Alto, Calif.) was used in 18 hr reactions.
The DNA template was then digested with DNase and protein was
removed by LiCl precipitation. The unincorporated nucleotides were
then removed by size exclusion chromatography on a Bio-Spin P30
column (Bio-Rad). The purified transcripts were quantitated by
ultraviolet (UV) absorption at 260 nm. The transcripts were stored
in 10 mM Tris pH 8, 1 mM EDTA at -20.degree. C.
[0337] PCR Primers Used to Prepare the Amplimers:
11 Forward primer SEQ ID NO 13 5'
HO-CCG-AAT-TCG-TCG-ACA-ACA-GAG-TTT-GAT-CMTGGC- TCA-G-OH 3' Reverse
primer SEQ ID NO 14 5'
HO-CCC-GGG-ATC-CAA-GCT-TAA-GGA-GGT-GWT-CCA-RCC- OH 3' M = 1 to 1
ratio of A & C, R = 1 to 1 ratio of A & G, and W = 1 to 1
ratio of A & T
[0338] Capture Section of Target Nucleic Acid Sequences
[0339] Neisseria gonorrhoeae (Ng) 16S rRNA Target Sequence
12 SEQ ID NO:15 5' HO-TGG-CGA-AGG-CAG-CCU-CCU-GGG-AUA-ACA-C-
UG-ACG- UUC-AUG-UC-OH 3' Neisseria meningitidis (N.m.) 16S rRNA
Target Sequence SEQ ID NO:16 5'
HO-TGG-CGA-AGG-CAG-CCU-CCU-GGG-ACA-ACA-CUG-ACG- UUC-AUG-UC-OH
3'
[0340] Capture Probe/Target Site:
[0341] The PNA capture probe, OL6, which is complementary to
positions 740-754 in the N.g. 16S rRNA sequence, was prepared using
commercially available chemistry and reagents, except that the PNA
probe was C-terminally labeled with 4-aminobenzoic acid as
described in Example 7. Within the capture site, the N.g. and N.m.
targets are related as a point mutation wherein the uracil residue
at position 747 in N.g. is replaced by a cytosine residue in N.m.
Consequently, the capture PNA probe, OL6, would comprise a single
base mismatch when hybridizing to the 16S rRNA of N.m. target (FIG.
7).
[0342] PNA Blocker Probes:
[0343] A PNA 15-mer homologous to the capture site of the 16S rRNA
N.m. target was synthesized (FIG. 7) using commercially available
instrumentation and reagents. Similarly, a PNA 15-mer homologous to
the capture site of 16S rRNA N.g. was synthesized (FIG. 7) using
commercially available instrumentation and reagents. Consequently,
these PNA blocker probes are related in that they specifically
hybridize with the N.g. and N.m. capture sites wherein the N.g. and
N.m. capture sites are related as point mutations.
[0344] PNA Probe Sequences:
13 OL6 N Ac-CAG-TGT-TAT-CCC-AGG-(linker).sub.2-- K(P)-NH.sub.2 C N
g. blocker N H-(linker).sub.2-CAG-TGT-TAT-CCC-AGG-NH.sub.2 C N.m
blocker N H-CAG-TGT-TGT-CCC-AGG-NH.sub.2 C
[0345] The PNA probes are illustrated from the amino terminus (N)
to the carboxyl terminus (C) The illustration "Ac" designates that
the amine terminus has been capped with an N-acetyl group. The
illustration "linker" designates the presence of the Expedite PNA
linker (P/N GEN063032). The letter "P" illustrates the
4-aminobenzoic acid moiety which is attached to the .epsilon.-amino
group of the lysine amino acid moiety "K".
[0346] Preparation of Microtitre Plates Comprising Capture
Probe:
[0347] The arylamine (4-aminobenzoic acid) at the 3' terminus of
the PNA capture probe was reacted with the commercially available
tresyl-activated dextran coated AquaBind microtiter plates (M&E
Corp., Copenhagen, Denmark). The covalent immobilization of a PNA
capture probe to the microtiter plate generates a surface suitable
for the specific hybridization of nucleic acid sequences.
[0348] It was experimentally determined that addition of 150 pmole
of PNA capture probe (OL6) to each well in the microtitre plate
produced the optimal specificity and kinetics of capture of the 16S
rRNA targets. Consequently, to each well in the microtitre plate
was added a solution containing 150 pmole of PNA capture probe
(OL6) in 100 .mu.L of 0.1 M carbonate buffer pH 9.62. The reaction
of the capture probe and the tresyl activated Aquabind microtitre
well was allowed to proceed for 2 hours at 25.degree. C., with
shaking. The PNA capture probe solution was then removed and the
wells were washed four times with Wash Buffer 1 (0.01 M phosphate
pH 7.2, 2.7 mM KCl, 0.5M NaCl and 1% (v/v) Triton X-100). Each of
the wells was then washed twice with diethyl pyrocarbonate treated
deionized water (DEPC-dH.sub.2O). Tresyl groups which may not have
reacted during the coupling reaction were quenched by treating each
well with 100 .mu.L of a solution containing 5%
2-(2-Aminoethoxy)ethanol (AEE) pH 10. This quenching reaction was
performed for 15 min. at 25.degree. C., with shaking. The AEE
solution was then removed and each of the wells was washed twice
with DEPC dH.sub.2O. The wells were then treated to block the
surfaces from non-specific interaction, by the addition of 300
.mu.L of a solution containing 1% diethyl pyrocarbonate treated
casein (Boehringer Mannheim, Indianapolis, Ind.) and 0.1 M
carbonate buffer pH 9.62. This reaction was allowed to proceed for
at least 2 hours at 25.degree. C., with shaking. The plates were
then stored at 10.degree. C. without removing the solution
containing 1% diethylpyrocarbonate treated casein (Boehringer
Mannheim, Indianapolis, Ind.) and 0.1 M carbonate buffer pH 9.62.
Prior to use, this solution was removed and each of the wells was
washed twice with Wash Buffer 1 and twice with DEPC dH.sub.2O.
Assay Method:
[0349] Each of the wells in the microtitre plates were equilibrated
by incubation with 100 .mu.L of Hybridization Buffer (100 mM NaCl,
100 mM Tris pH 7.4, 20 mM EDTA, 50% formamide and 0.5% Triton
X-100) for 15 min. at 50.degree. C., immediately before use in the
capture assay. This buffer was then discarded.
[0350] To perform the hybridization, a set of microtubes (supported
in an 8.times.12 rack) containing hybridization buffer and the
various amounts of biotin-labeled N.g. or N.m. target (0.17 pmole
to 0.17 fmole in half log dilutions) were incubated, in the
presence and absence, of 1.7 pmole of either of the N.m. or N.g.
PNA blocker probe. The incubation of each of these hybridization
reactions was allowed to proceed for 30 min. at 50.degree. C.
[0351] After 30 min., the contents of each of the hybridization
reactions was transferred from each of the microtubes to a specific
well in the microtiter plate comprising the OL6 capture probe
immobilized to the well surface. The capture reaction was allowed
to proceed for 30 min. at 50.degree. C., with shaking. The
solutions in each of the wells was then discarded. Any, residual
unhybridized target was then removed by washing once with
hybridization buffer and four times with THT buffer (50 mM Tris pH
7.4, 100 mM NaCl and 0.1% Tween 20) both at 50.degree. C.
[0352] The biotinylated N.g or N.m. target nucleic acid which was
still present in each of the wells of the microtitre plate was
detected using a Streptavidin/HRP conjugate (DAKO Corp. USA). The
Streptavidin/HRP conjugate was diluted 1:5000 in THT buffer and
each well in the microtitre plate was treated with 100 .mu.L of the
diluted stock for 30 minutes at 25.degree. C., with shaking. Each
of the wells was then washed five times with THT to remove any
excess conjugate To each well was then added 100 .mu.L of 3,3',5,5'
tetramethyl benzidine (TMB+) (DAKO Corp, USA) This reagent was
allowed to react for 15 minutes at 37.degree. C. and then the
reaction was terminated by addition of an equal volume of 0 5 M
H.sub.2SO.sub.4 The color which was generated (absorbance at 450
nm) was then measured in a microplate reader (Molecular Devices
Corp., Menlo park, Calif.).
[0353] Assay Design:
[0354] FIG. 8A is a plate schematic of the configuration of
hybridization reactions performed in the wells of the microtiter
plates used for this experiment. For convenience the rows of the
figure have been designated with the letters A-H and the columns
have been designated with the numbers 1-12. As illustrated in the
figure, the reactions performed in the wells in columns 1, 2, 5, 6,
9, and 10 contained various amounts of N. gonorrhoeae target.
Similarly, as illustrated in the figure, the reactions performed in
wells in columns 3, 4, 7, 8, 11 and 12 contained various amounts of
N. meningitidis target. As illustrated in the figure, for both the
N.g. and N.m. containing reactions, the amount of target sequence
present in the wells varied between 170 fmole and 0.17 fmole.
Reactions performed in the wells in row H contained no target and
the values obtained for these reactions were used as the assay
background. The reactions performed in the wells in columns 1
through 4 contained no PNA blocker probe. The reactions performed
in the wells in columns 5 through 8 contained 1.7 pmole of PNA
blocker which was complementary to the N.g. target. The reactions
performed in the wells in columns 9 through 12 contained 1.7 pmole
of PNA blocker which was complementary to the N.m. target. Each of
the conditions examined was performed in duplicate and, unless
otherwise noted, the results were averaged to generate the derived
data.
[0355] Results:
[0356] With reference to FIG. 8B, the derived data for each of the
experimental conditions is presented. Unless otherwise indicated,
the data at each target level represents the average of the two
data points collected after the appropriate averaged background has
been subtracted. (Of 96 data points, 3 data points were not
included in the analysis since these were clearly erroneous).
Though all the derived data which was acquired in the experiment is
presented in the figures essentially all the data for target levels
below 5.4 fmole gave data points which were not significantly above
background to be considered to be reliable or statistically
significant. Consequently, only the data obtained for target levels
greater than 5.4 fmole are discussed in this section.
[0357] With reference to FIG. 8C, a comparison of the percent
decrease in capture of the target which is attributable to the
presence of blocking probe is presented. Thus, all data in FIG. 8C
is derived from data in FIG. 8B. For example, column A represents
the percent decrease in capture of the N.g. target in the presence
of the N.m blocker probe. Consequently, the percent decrease of
38.3 reported in FIG. 8C, column A for the target level of 170
fmole is determined by subtracting the value in FIG. 8B, in columns
A and C and then dividing that difference by the value in FIG. 8B,
column A.
[0358] Columns A and B in FIG. 8B contain data for the signal
(absorbance 450 nm) resulting from capture of the N.g. and N.m.
transcripts in the absence of PNA blocker probe. The data
demonstrates that the signal generated in the presence of 5.4 fmole
of N.g. target is approximately equivalent to signal generated in
the presence of 0.17 pmole of N.m. target. Therefore, the
discrimination between N.g. and N.m., in the absence of blocker
probe, is approximately 1-1.5 logs.
[0359] With reference to FIG. 8B, columns C and D present the data
for the signal (absorbance 450 nm) resulting from capture of the
N.g. and N.m. transcripts in the presence of 1.7 pmole of PNA
blocker probe which is homologous to the N.m. capture site (the
N.m. blocker probe). At all target levels above 5.4 fmole, there is
a measurable decrease in the capture of both the N.m. and N.g.
targets as compared with the absence of the N.m. blocker probe
(Compare: data in FIG. 8B, column A with C and data in FIG. 8B,
column B with column D, respectively) However, as can be seen by
comparison of the relative percent decrease in capture of the
different targets in the presence of the N.m. blocker probe (data
in FIG. 5C) there is a much greater decrease in capture of the N.m
target. For the three highest target levels, the average decrease
in capture of the N.m. target in the presence of the N.m. blocker
probe was .sup.69% while the average decrease in the capture of the
N.g. target was only 31%. Therefore, under the conditions of this
assay, addition of 1.7 pmole of N.m. blocker probe improved
discrimination between N.g. and N.m. by approximately two fold.
[0360] With reference to FIG. 8B, columns E and F present the data
for the signal (absorbance 450 nm) resulting from capture of the
N.g. and N.m. transcripts in the presence of 1.7 pmole of PNA
blocker probe which is homologous to the N.g. capture site (the
N.g. blocker probe). At all target levels above 5.4 fmole, there is
a significant decrease in the capture of the N.g. target as
compared with the absence of the N.g. blocker probe (Compare: data
in FIG. 8B, column A with E) However, as can be seen by comparison
of the relative percent decrease in capture of the different
targets in the presence of the N.g. blocker probe (data in FIG. 8C)
there is a much greater decrease in capture of the N.g. target.
Thus, for the three highest target levels, the average decrease in
capture of the N.g. target in the presence of the N.g. blocker
probe was 86% while the decrease in the capture of the N.m. target
was minimal (only 16% for the highest target level; See: FIG. 8C,
column D).
[0361] In summary, the data in FIGS. 8B and 8C demonstrate that it
is possible to improve the selective inhibition of capture of a
target sequences containing a point mutation on a hybridization
surface composed of PNA capture probe when blocking probes are used
in the hybridization assay.
[0362] The discrimination ratios which are presented in FIG. 8D are
derived from the data in FIG. 8B. When considering the data for
only the three highest target levels examined (170-17 fmole), the
discrimination ratios in the absence of PNA blocker probe are 21,
12 and 8 (See FIG. 8D, column A). In the presence of 1.7 pmole of
N.m. blocker probe, the discrimination ratios for the capture of
the N.m. target were increased from 21 to 40, from 12 to 34 and
from 8 to 18, respectively (Compare: columns A and B).
Consequently, the overall improvement in point mutation
discrimination was two to three fold greater under the conditions
examined.
[0363] In the presence of 1.7 pmole of N.g. blocker probe, the
discrimination ratios for the capture of the N.g. target were 3,
1.6 and 11 at the three highest target levels examined (See column
C). This data demonstrates that, under the conditions examined, it
is necessary to add PNA blocker in at least 30 fold molar excess to
the amount of target present to thereby completely eliminate the
capture of the N.g. target. This is evident since the
discrimination ratio approaches the value of 1 when the signal for
N.g. and N.m. approaches equivalent low levels.
[0364] FIG. 5E is a graphical illustration of the data in FIG. 8D.
The graphical illustration is presented as a useful and quick means
to visually analyze and compare the tabular data.
[0365] Summary:
[0366] A discrimination in a capture assay can be substantially
improved by addition of blocking probes, which are designed to
hybridize to non-target sequences which are closely related to the
target of interest.
Sequence CWU 0
0
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