U.S. patent application number 12/646514 was filed with the patent office on 2010-09-23 for oligonucleotides labeled with a plurality of fluorophores.
Invention is credited to Fei Mao, Xing Xin.
Application Number | 20100240103 12/646514 |
Document ID | / |
Family ID | 34632765 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240103 |
Kind Code |
A1 |
Mao; Fei ; et al. |
September 23, 2010 |
OLIGONUCLEOTIDES LABELED WITH A PLURALITY OF FLUOROPHORES
Abstract
An embodiment of the invention discloses new methods for
designing labeled nucleic acid probes and primers by labeling
oligonucleotides with a plurality of spectrally identical or
similar dyes and optionally with one or more quencher dyes.
Oligonucleotides labeled in accordance with some embodiments of the
invention exhibit a detectable increase in signal, for example,
fluorescent signal when the labeling dyes are separated from one
another. Methods for separating the dye include cleaving the
labeled oligonucleotides include using enzymes that have
5'-exonuclease activity. In one embodiment nucleic acid primers of
the present invention may fluoresce upon hybridization to a target
sequence and incorporation into the amplification product. Nucleic
acid probes and primers of the present invention have wide
applications ranging from general detection of a target nucleic
acid sequence to clinical diagnostics. Major advantages of the
oligonucleotides including nucleic acid probes and primers of many
embodiments of the present invention are their synthetic
simplicity, spectral versatility and superior fluorescent
signal.
Inventors: |
Mao; Fei; (Fremont, CA)
; Xin; Xing; (Foster City, CA) |
Correspondence
Address: |
WILSON, SONSINI, GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Family ID: |
34632765 |
Appl. No.: |
12/646514 |
Filed: |
December 23, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10993625 |
Nov 19, 2004 |
7667024 |
|
|
12646514 |
|
|
|
|
60523263 |
Nov 19, 2003 |
|
|
|
Current U.S.
Class: |
435/91.2 ;
536/22.1; 536/24.3; 536/24.33; 536/25.32 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 1/6818 20130101; Y10S 435/975 20130101; C12Q 1/6818 20130101;
C12Q 1/6851 20130101; C12Q 2561/101 20130101; C12Q 2565/1015
20130101; C12Q 2565/1025 20130101; C12Q 2565/1015 20130101 |
Class at
Publication: |
435/91.2 ;
536/22.1; 536/24.3; 536/24.33; 536/25.32 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C07H 21/00 20060101 C07H021/00 |
Claims
1. A labeled oligonucleotide, comprising: an oligonucleotide
sequence that hybridizes to a target polynucleotide sequence; and
at least two photometric labeling molecules attached to said
oligonucleotide; wherein said at least two photometric labeling
molecules have excitation wavelengths that are within 15 nm of one
another.
2. The labeled oligonucleotide according to claim 1, wherein at
least two of said photometric molecules produce a detectable change
in signal when at least two of said at least two photometric
molecules are separated from one another.
3. The labeled oligonucleotide according to claim 2, wherein said
at least two photometric molecules are separated from one another
when said labeled oligonucleotide is cleaved at a position between
said at least two photometric molecules.
4. The labeled oligonucleotide according to claim 2, wherein said
at least two photometric molecules are separated from one another
when said labeled oligonucleotide hybridizes to the target
sequence.
5. The labeled oligonucleotide according to claim 2, wherein said
at least two photometric molecules are separated from one another
when said labeled oligonucleotide is incorporated into a
polynucleotide.
6. The labeled oligonucleotide according to claim 2, wherein said
two photometric molecules is separated from one another when said
two molecules are cleaved from said labeled oligonucleotide.
7. The labeled oligonucleotide according to claim 1, wherein said
at least two photometric labeling molecules are fluorescent
molecules.
8. The labeled oligonucleotide according to claim 1, wherein any
pair of said photometric labeling molecules on said oligonucleotide
is separated from one another by about 3 to about 60
nucleotides.
9. The labeled oligonucleotide according to claim 1, wherein any
pair of said photometric labeling molecules on said oligonucleotide
is separated from one another by about 12 to about 35
nucleotides.
10. The labeled oligonucleotide according to claim 1, wherein any
pair of said photometric labeling molecules on said oligonucleotide
is separated from one another by about 15 to about 25
nucleotides.
11. The labeled oligonucleotide according to claim 1, further
including: at least one quenching molecule attached to said
oligonucleotide, wherein said quenching molecule quenches a signal
produced by said at least two photometric labeling molecules when
said at least two photometric labeling molecules are attached to
said oligonucleotide.
12. The labeled according to claim 1, wherein at least one of said
at least two photometric labeling molecules is a fluorescent dye,
selected from the group consisting of a dye that has delocalized
positive charge, a dye that has a delocalized negative charge, and
a dye that has an equal number of positive and negative
charges.
13. The labeled oligonucleotide according to claim 1, wherein at
least one of said at least two photometric labeling molecules is a
dye selected from the group consisting of BODIPY, a cyanine dye
with delocalized positive charge and a zwiterionic cyanine dye.
14. The labeled oligonucleotide according to claim 1, wherein at
least one of said at least two labeling molecules is xanthene dye
molecule.
15. The labeled oligonucleotide according to claim 1, wherein at
least one of said at least two photometric labeling molecules is a
non-sulfonated cyanine dye molecule.
16. The labeled oligonucleotides according to claim 1, wherein at
least one of said at least two photometric labeling molecules is a
dye selected from the group consisting of CR110, CR6G, TAMRA and
ROX.
17. The labeled oligonucleotide according to claim 1, wherein said
oligonucleotide further includes at least one minor groove binder
(MGB).
18. The labeled oligonucleotide according to claim 1, including: at
least one Guanidine nucleotide in said oligonucleotide, wherein at
least one of said at least two photometric labeling molecule is
positioned near enough to said Guanidine nucleotide such that a
signal produced by said at least one labeling molecule is quenched
when said oligonucleotide is not hybridized to said target
oligonucleotide.
19. The labeled oligonucleotide according to claim 1, wherein said
oligonucleotide has a 5'-end and a 3'-end; wherein one of said at
least two photometric labeling molecules is attached to the said
5'-end of said oligonucleotide; wherein another of said at least
two photometric labeling molecules is attached to the 3'-end of
said oligonucleotide.
20. The labeled oligonucleotide according to claim 16, wherein said
photometric labeling molecules attached to said 5'-end and said
3'-end of said oligonucleotide molecule are attached to said
oligonucleotide by flexible aliphatic linkers.
21. The oligonucleotide according to claim 1, wherein said
oligonucleotide sequence is substantially devoid of internal
secondary structure.
22. The oligonucleotide according to claim 1, wherein said
oligonucleotide is suitable for use as a probe.
23. The oligonucleotide according to claim 1, wherein said
oligonucleotide is suitable for use as a primer in a polynucleotide
amplification reaction.
24. The oligonucleotide according to claim 23, wherein said
polynucleotide amplification reaction is selected from the group
consisting of PCR, multiplex PCR; real time PCR, quantitative PCR
and real time quantitative PCR.
25. A method of labeling an oligonucleotide, comprising: providing
an oligonucleotide sequence that hybridizes to a target
polynucleotide sequence; and attaching at least two photometric
labeling molecules to said oligonucleotide, wherein said at least
two photometric labeling molecules have excitation wavelengths that
are within 15 nm of one another.
26. The method of labeling an oligonucleotide according to claim
25, wherein said at least two photometric labeling molecules are
fluorescent molecules.
27. The method of labeling an oligonucleotide according to claim
25, wherein any pair of said photometric labeling molecules on said
oligonucleotide is separated from one another by about 3 to about
60 nucleotides.
28. The method of labeling an oligonucleotide according to claim
25, wherein any pair of said photometric labeling molecules on said
oligonucleotide is separated from one another by about 12 to about
35 nucleotides.
29. The method of labeling an oligonucleotide according to claim
25, wherein any pair of said photometric labeling molecules on said
oligonucleotide is separated from one another by about 15 to about
25 nucleotides.
30. The method of labeling an oligonucleotide according to claim
25, further including: attaching at least one quenching molecule to
said oligonucleotide, wherein said quenching molecule quenches a
signal produced by said at least two photometric labeling molecules
when said at least two photometric labeling molecules are attached
to said oligonucleotide.
31. The method of labeling an oligonucleotide according to claim
25, wherein at least one of said at least two photometric labeling
molecules is a fluorescent dye, selected from the group consisting
of a dye that has delocalized positive charge, a dye that has a
delocalized negative charge, and a dye that has an equal number of
positive and negative charges.
32. The method of labeling an oligonucleotide according to claim
25, wherein at least one of said at least two photometric labeling
molecules is a dye selected from the group consisting of BODIPY, a
cyanine dye with delocalized positive charge and a zwiterionic
cyanine dye.
33. The method of labeling an oligonucleotide according to claim
25, wherein at least one of said at least two labeling molecules is
xanthene dye molecule.
34. The method of labeling an oligonucleotide according to claim
25, wherein at least one of said at least two photometric labeling
molecules is a non-sulfonated cyanine dye molecule.
35. The method of labeling an oligonucleotide according to claim
25, wherein at least one of said at least two photometric labeling
molecules is a dye selected from the group consisting of CR110,
CR6G, TAMRA and ROX.
36. The method of labeling an oligonucleotide according to claim
25, wherein said method further includes attaching at least one
minor groove binder (MGB) to said oligonucleotide.
37. The method of labeling an oligonucleotide according to claim
25, including providing at least one Guanidine nucleotide in said
oligonucleotide, wherein at least one of said at least two
photometric labeling molecule is positioned near enough to said
Guanidine nucleotide such that a signal produced by said at least
one labeling molecule is quenched when said oligonucleotide is not
hybridized to said target oligonucleotide.
38. The method of labeling an oligonucleotide according to claim
25, wherein said oligonucleotide has a 5'-end and a 3'-end further
comprising: attaching one of said at least two photometric labeling
molecules to the said 5'-end of said oligonucleotide; and attaching
another of said at least two photometric labeling molecules to the
3'-end of said oligonucleotide.
39. The method of labeling an oligonucleotide according to claim
25, wherein said photometric labeling molecules attached to said
5'-end and said 3'-end of said oligonucleotide molecule are
attached to said oligonucleotide by flexible aliphatic linkers.
40. The method of labeling an oligonucleotide according to claim
25, wherein said oligonucleotide sequence is substantially devoid
of internal secondary structure.
41. The method of labeling an oligonucleotide according to claim
25, further comprising using said oligonucleotide as a primer
probe.
42. The method of labeling an oligonucleotide according to claim
25, further comprising using said oligonucleotide as a primer in a
polynucleotide amplification reaction.
43. A kit for labeling an oligonucleotide, comprising: an
oligonucleotide sequence suitable for hybridizing to a target
polynucleotide sequence; and at least two photometric labeling
molecules suitable for attachment to said oligonucleotide; wherein
said at least two photometric labeling molecules have excitation
wavelengths that are within 15 nm of one another.
44. The kit for labeling an oligonucleotide according to claim 43,
wherein at least one of said at least two photometric labeling
molecule are selected from the group consisting of a dye that has
delocalized positive charge, a dye that has a delocalized negative
charge, and a dye that has an equal number of positive and negative
charges.
45. The kit for labeling an oligonucleotide according to claim 43,
wherein at least one of said at least two photometric labeling
molecule are selected from the group consisting of BODIPY, a
cyanine dye with delocalized positive charge and a zwiterionic
cyanine dye.
46. The kit for labeling an oligonucleotide according to claim 43,
wherein at least one of said at least two photometric labeling
molecules is a xanthene dye molecule.
47. The kit for labeling an oligonucleotide according to claim 43,
wherein at least one of said at least two photometric labeling
molecule is a non-sulfonated cyanine dye molecule.
48. The kit for labeling an oligonucleotide according to claim 43,
wherein at least one of said at least two photometric labeling
molecules is a dye selected from the group consisting of CR110,
CR6G, TAMRA and ROX.
49. The kit for labeling an oligonucleotide according to claim 43,
wherein said kit further includes a minor groove binder (MOB).
50. A method of using a labeled oligonucleotide comprising the
steps of: providing a labeled oligonucleotide, wherein said labeled
oligonucleotide includes an oligonucleotide sequence suitable for
hybridizing to a target polynucleotide sequence; at least two
photometric labeling molecules, wherein said at least two
photometric labeling molecules have excitation wavelengths that are
within 15 nm of one another and, wherein said photometric labeling
molecules are attached to said oligonucleotide; contacting said
labeled oligonucleotide with a sample.
51. The method of using a labeled oligonucleotide according to
claim 50, wherein said sample is selected from the group consisting
of tissue extract, cell extract, bodily fluid, in vitro nucleic
acid synthesis reaction, and PCR reaction mixture.
52. A method of using a labeled oligonucleotide according to claim
50, wherein said labeled oligonucleotide is used in a nucleic acid
amplification reactions wherein the reaction is selected from the
group consisting of PCR, Multiplex PCR, Real Time PCR, Quantitative
PCR, and Real Time Quantitative PCR.
53. A method of using a labeled oligonucleotide according to claim
50, wherein said oligonucleotide is a probe for identifying the
presence of a target sequence of nucleic acid polymer in the
sample.
54. A method of using a labeled oligonucleotide according to claim
50, wherein said sample is applied to a nucleic acid chip.
Description
PRIORITY CLAIM
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/523,263 filed on Nov. 19, 2003,
which is incorporated herein in its entirety.
BACKGROUND
[0002] The present invention relates in general to oligonucleotides
labeled with a plurality of spectrally identical or similar dyes
and optionally one or more quencher dyes, as well as methods of
creating labeled oligonucleotides and various uses of the labeled
oligonucleotides as primers or probes for highly sensitive nucleic
acid detection, including real-time polymerase chain reaction
(PCR).
[0003] Nucleic acid polymers such as DNA and RNA are essential to
the transmission of genetic information from one generation to the
next and in the routine functioning of all living organisms.
Accordingly these molecules are the object of intense study and a
number of techniques have been developed to study of these
molecules. These methods include but are not limited to methods for
identifying the presence of a specific polynucleotide sequences in
a given sample and methods designed to measure the number of
specific nucleic acid molecules originally present in a given
sample.
[0004] Practical uses for these techniques include identifying
specific species and relationships between various species based
upon similarities in oligonucleotide sequences. Other uses include
diagnosing disease by identifying specific sequences in a given
sample as indicative of a given pathology. Still other uses, too
numerous to mention, include identifying individuals with a
predisposition for developing a specific pathology as well as
assessing the efficacy of proposed treatment regimes based on the
presence of specific polynucleotides in a given patient's
genome.
[0005] One of the most widely used and powerful techniques for the
study and manipulation of oligonucleotides is the polymerase chain
reaction (PCR). PCR is a primer extension reaction that provides a
method for amplifying specific nucleic acids in vitro. This
technique was first described in 1987. PCR can produce million fold
copies of a DNA template in a single enzymatic reaction mixture
within a matter of hours, enabling researchers to determine the
size and sequence of target DNA. This DNA amplification technique
has been widely used for cloning and other molecular biological
manipulations. Further discussion of PCR is provided in Mullis et
al., Methods Enzymol. (1987); and Saiki et al, Science (1985).
[0006] In PCR the particular stretch of DNA to be amplified is
referred to as the `target sequence`. The target sequence is
replicated by first binding a complimentary `primer` to a single
stranded portion of the target polynucleotide. One PCR based
technique that is particularly useful is Quantitative PCR (qPCR).
Briefly, the mechanism of qPCR is based on the fact that PCR
amplifies a target DNA in an exponential manner. By running a PCR
reaction and measuring the total number of DNA copies at given
points during the course of the amplification reaction, one can
retroactively calculate the amount of starting DNA material.
[0007] Various methods have been developed for determining the
amount of PCR product made without having to stop the PCR run or
even to sample the reaction during a given PCR run. One such method
follows the course of the PCR run in real time by measuring the
amount of product at each cycle of DNA synthesis. This process is
referred to as real time PCR(RT-PCR). Because of its great
sensitivity and because measurements can be made with the sample
still in the PCR thermocylcer, various fluorescence-based assays
that monitor the formation of PCR products have been developed. A
number of instruments and methods have been developed for real-time
PCR(RT-PCR). A real-time PCR instrument is typically a fluorometer
built upon a thermocycler. Commercially available real-time PCR
instruments include Prism7700 by ABI, LightCycler by Roche, Opticon
by MJ Research, iCycler IQ by BioRad, and MX4000 by Stratagene.
[0008] An oligonucleotide used to identify a given sequence of
nucleic acid by hybridizing to it, but that does not serve to
amplify the sequence may be referred to as a `probe`. Probes also
find utility in PCR reactions where they are used to signal
polynucleotide amplification.
[0009] Given the importance of oligonucleotide and the myriad of
ways in which these molecules can impact human, animal and plant
life there is a need for ever more efficient methods for the study
and manipulation of oligonucleotide. Including new techniques for
efficiently producing labeled oligonucleotide. One object of the
present invention is to provide labeled oligonucleotide and
efficient methods for making and using the same.
SUMMARY
[0010] The present invention provides a methods for labeling
oligonucleotides that find utility in assays such as those designed
to identify the presence of a given polynucleotide in a sample or
to amplify the amount of a given oligonucleotide or polynucleotide
in a given sample. It provides methods for using these types of
labeled oligonucleotides.
[0011] One embodiment includes oligonucleotides labeled with at
least two photometric molecules that have excitation wavelengths
that are within 15 nm of one another. In some embodiments the
labeled oligonucleotides produce relatively little spectral signal
until at least two of the photometric molecules are permanently
separated from each other as a result of oligonucleotide
cleavage.
[0012] One embodiment is an oligonucleotide labeled with two
spectrally similar or identical photometric molecules that are
fluorescent. The detectable emission from the fluorescent molecules
increases when at least two of the molecules are permanently
separated from one another as a result of -oligonucleotide
cleavage. In another embodiment the florescence signal produced by
the at least two fluorescent molecules attached to the
oligonucleotide increases when the oligonucleotide hybridizes to a
target oligonucleotide sequence.
[0013] One embodiment is an oligonucleotide labeled with at least
two photometric molecules in which the oligonucleotide sequence is
substantially devoid of secondary structure, such as hairpin loops
and stem-loop structures.
[0014] One embodiment includes an oligonucleotide labeled with
photometric molecules that have identical or similar spectral
properties and are attached at the 5' and 3' ends, respectively of
the oligonucleotide. In one variation of this embodiment one dye is
attached at the 5' terminal backbone phosphate and the other dye
attached at the 3' terminal backbone phosphate.
[0015] In one embodiment the oligonucleotide is suitable for use as
primer comprising at least two spectrally identical or similar
fluorescent dyes and optionally one or more fluorescence quencher
dyes. In one embodiment the oligonucleotide is suitable for use as
a labeled primer. In another embodiment the fluorescent molecules
may be attached to the bases of nucleosides, or to a combination of
the 5' terminal backbone phosphate and the bases.
[0016] In one embodiment an oligonucleotide is labeled with at
least two spectrally identical or similar fluorescent dyes. The
oligonucleotide may be a primer for use in nucleic acid
amplification reactions including, for example, spectrally similar
or identical fluorescent dyes attached to the 5' terminal phosphate
backbone and the base of a nucleoside, for example, a thymidine
nucleotide.
[0017] One embodiment includes methods for producing
oligonucleotides that are labeled with at least two photometric
molecules in which the photometric molecules are spectrally similar
or identical. Labeled oligonucleotides produced using some of these
methods produce more detectable signal when at least two of the at
least two photometric molecules are permanently separated from one
another.
[0018] Yet another embodiment includes methods for utilizing
oligonucleotides labeled with at least two photometric molecules
that have excitation wavelengths that are within 15 nm or one
another's.
[0019] Still other embodiments includes uses for oligonucleotides
labeled according to embodiments of the invention. These uses
include, but are not limited to, assays to analyze biological
samples comprising nucleic acid sequence in a variety of contexts.
One exemplary application includes, fluorescence in situ
hybridization (FISH), wherein oligonucleotide labeled in accordance
with the invention can be used for localizing and determining the
relative abundance of a target nucleic acid sequences with
biological importance in, for examples, live cells, fixed tissue or
a chromosome sample.
[0020] Other embodiments include using oligonucleotides labeled
with a plurality of dyes in solution-based or chip-based array
detection systems and quantification of differential expression of
genes linked with disease in basic research and the diagnosis of
disease.
[0021] Still other embodiments includes methods and kits suitable
for making oligonucleotides labeled according to embodiments of the
invention. These kits may be used in the detection of amplified
oligonucleotide sequences, which includes iso-thermo
amplifications, ligase chain reactions and the like.
[0022] Various embodiments are suitable for use with PCR to detect
biomolecules other than nucleic acids by using an
oligonucleotide-antibody conjugate wherein the antibody is specific
for the biomolecules to be detected.
[0023] Further forms, embodiments, objects, functions and aspects
from the present invention shall become apparent from the
description contained herein.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
[0024] FIG. 1 is a schematic amplification plot for a typical real
time PCR run that illustrates some of the parameters associated
with real time PCR.
[0025] FIG. 2A shows plots of data collected when the MCG gene was
amplified using PCR. These data were collected using a
6-ROX-labeled probe various at starting concentrations of plasmid
DNA. (Example 2)
[0026] FIG. 2B shows plots of data collected when the MCG gene was
amplified using PCR. These data were collected at various
concentrations of human genomic DNA using a 6CR110-labeled probe.
(Example 3)
[0027] FIG. 2C shows plots of data collected with the MCG gene
being amplified using PCR. These data were collected from various
starting concentrations of human cDNA using a 6CR110-labeled probe.
(Example 3)
[0028] FIG. 2D shows the amplification plots of MCG gene amplified
from 100,000 copies of plasmid DNA using a non-sulfonated
cyanine-labeled probe. (Example 2)
[0029] FIG. 3A illustrates data collected by comparing a TaqMan
probe with a probe made in conformity with one embodiment of the
invention. The TaqMan probe was labeled with JOE at 5' terminus and
TAMRA at 3' terminus; the probe of the present invention has the
same oligonucleotide sequences as the TaqMan probe but it was
labeled with R6G at both 5' and 3' termini. (Example 4)
[0030] FIG. 3B shows the result of a comparison of a TaqMan probe
with a probe made in conformity with one embodiment of the present
invention. The TaqMan probe was labeled with FAM at 5' terminus and
TAMRA at 3' terminus; the probe of the present invention has the
same oligonucleotide sequences as the TaqMan probe but it was
labeled with 6-CR110 at both the 5' and 3' termini. (Example 4)
[0031] FIG. 3C shows the result of a comparison of a TaqMan probe
with a probe made in conformity with one embodiment of the present
invention. The TaqMan probe from ABI with an undisclosed sequence
had a FAM at 5' end, a MGB and a quencher at the 3' end; the probe
of the present invention was labeled with 6CR110 at both the 5' and
3' termini. (Example 4)
[0032] FIG. 4A shows the absorption spectra of a GAPDH probe
labeled with two molecules of 5-CR110 at the termini both before
and after S1 digestion (SEQ ID No. 3, Example 5).
[0033] FIG. 4B shows the absorption spectra of a GAPDH probe
labeled with one 5-CR110 at the 5' terminus (SEQ ID No. 10) both
before and after S1 digestion. (Example 5)
[0034] FIG. 4C shows the absorption spectra of a GAPDH probe
labeled with one 5-CR110 at the 3' terminus (SEQ ID No. 11) both
before and after S1 digestion. (Example 5)
[0035] FIG. 4D shows the amplification plots collected using the
three probes used in 4A, 4B and 4C. (Example 6)
[0036] FIG. 5A shows the absorption spectra of linear and
stem-looped GAPDH probes labeled with two 6-CR110. This figure
illustrates that stem-loop structures facilitate 6-CR110-dimer
formation. (Example 7)
[0037] FIG. 5B shows the absorption spectra of linear and
stem-looped GAPDH probes labeled with two molecules of 6-TAMRA.
These data illustrate that a stem-loop structure strongly promotes
6-TAMRA-dimer formation. (Example 7)
[0038] FIG. 5C shows absorption spectra of a linear probe and
stem-loop probe for GAPDH probes labeled with two molecules of
6-ROX. The data depicted in this figure illustrates that a
stem-loop structure strongly promotes 6-ROX-dimer formation.
(Example 7)
[0039] FIG. 6A depicts the result of comparing the performance of a
doubly 6-CR110-labeled linear probe and a stem-loop probe in real
time PCR at various probe concentrations. This figure illustrates
that the linear probe generates a significantly stronger signal
than the probe that forms a stem-loop structure. (Example 8)
[0040] FIG. 6B illustrates a comparison of the performance of a
doubly 6-TAMRA-labeled linear probe and a stem-loop probe in real
time PCR measured at various probe concentrations. This figure
illustrates that the linear probe generates a significantly
stronger signal than the probe that forms a stem-loop structure.
(Example 8)
[0041] FIG. 6C depicts a comparison of the performance of doubly a
6-ROX-labeled linear and a stem-loop probe in real time PCR with
various probe concentrations. This figure illustrates that the
linear probe generates a significantly stronger signal than the
probe that forms a stem-loop structure. (Example 8)
[0042] FIG. 7 illustrates the chemical equilibria involved in
nucleic acid detection using a beacon probe. (Example 8)
[0043] FIG. 8A depicts the spectra of three probes labeled with a
combination of FAM/FAM, FAM/CR110 and CR110/CR110 dye pairs,
respectively (Example 9).
[0044] FIG. 8B illustrates amplification plots of the GAPDH gene
detected with the probes depicted in FIG. 8k (Example 9)
[0045] FIG. 9A illustrates data collected from amplifying the GAPDH
gene by PCR and detected the product with doubly 5-CR110-labeled
forward primer of GAPDH (SEQ ID No. 15). (Example 10)
[0046] FIG. 9B illustrates data collected from amplifying the GAPDH
gene by PCR and detected the product with doubly 5-CR110-labeled
forward primer of GAPDH (SEQ ID No. 16). (Example 10)
[0047] FIG. 9C illustrates data collected from amplifying the GAPDH
gene by PCR and detected the product with 5-CR110-labeled reverse
primer (SEQ ID No. 17). (Example 10)
[0048] FIG. 9D illustrates data collected from amplifying the GAPDH
gene by PCR and detected the product with doubly 5-CR110-labeled
forward primer (SEQ ID No. 15) and the same forward primer singly
labeled with 5-CR110 at 5' end (SEQ ID No. 18). This graph
demonstrates that only the doubly labeled oligonucleotide functions
as a fluorogenic primer. (Example 10)
[0049] FIG. 9E is a comparison of data collected by amplifying the
GAPDH gene by PCR and detecting the product with a doubly
5-CR110-labeled forward primer (SEQ ID No 16) and the same forward
primer singly labeled at 3' end (SEQ No 19). This graph
demonstrates that only a doubly labeled primer functions as a
fluorogenic primer for PCR detection. (Example 10)
[0050] FIG. 9F illustrates a possible secondary structure that may
allow a 5' G to act as a quencher as in a LUX primer. This figure
also illustrates that fluorogenic primers of some embodiments of
the present invention may work in accordance with a mechanism
different from that which is responsible for the functioning of the
LUX primers. (Example 10)
[0051] FIG. 10 illustrates SNP typing of model estrogen receptors.
(Example 11)
[0052] FIG. 11 illustrates data that was collected by amplifying
the HCV gene by PCR and detecting the products using a doubly
ROX-labeled probe (SEQ ID No. 33). As illustrated in the figure the
signal was enhanced by treating the product with an enzyme, which
exhibits an exo-minus activity, such as Taq DNA polymerase.
(Example 12)
[0053] TABLE 1. A concise listing of some of the sequences referred
to throughout the text as well as the structures of some of linking
molecules suitable for practicing some embodiments.
[0054] TABLE 2. A partial listing of reactive groups including some
electrophilic and nucleophilic groups that can be used in some
embodiments to attach labeling molecules and quenching molecules to
oligonucleotides.
[0055] SEQUENCE LISTING. An attached set of pages listing some of
the sequences used in greater detail.
DETAILED DESCRIPTION
[0056] For the purposes of promoting an understanding of the
principles of the invention, reference will now be made to the
embodiments described herein and specific language will be used to
describe the same. It will, nevertheless, be understood that no
limitation of the scope of the invention is thereby intended. Any
alterations and further modifications in the described devices,
systems, and treatment methods, and any further applications of the
principles of the invention as described herein, are contemplated
as would normally occur to one skilled in the art to which the
invention relates. While aspects of the invention may be discussed
in terms of specific or general theories or principles, the
invention is in no way bound by these theories or principles. Such
discussion is purely illustrative and in no way limiting.
[0057] Because nucleic acid polymers play an essential role in
modern medicine and the life sciences, a wide variety of reagents,
including fluorescent dyes have been developed for use in processes
for detecting, sequencing and measuring nucleic acid polymers.
Similarly, a wide variety of methods have been developed for using
these dyes to create ever more sensitive nucleic acid assays. One
area that has generated intense study is the development of
extremely sensitive assays for measuring the accumulation of
nucleic acid polymers produced by PCR.
[0058] DNA amplification via PCR may be quite complex as the
process is affected by many factors. One factor that effects PCR is
the gradual depletion of starting components such as dNTP, primers,
the effective concentration of Mg.sup.2+ that occurs as the
reaction progresses. Another factor is the inhibitory effect of the
accumulating end product. During the PCR process these various
components interact with each other in an interrelated dynamic
fashion. Typically DNA amplification comprises three sequential
phases: the exponential phase, the linear phase and the plateau
phase. Each phase is different and may exhibit a different
amplification efficiency than the other two. As a practical matter
only data collected during the exponential phase can be used to
reliably estimate the initial concentration of target
oligonucleotide.
[0059] One method for monitoring PCR in real time that often uses
fluorescent molecules is known commercially as the TaqMan assay.
TaqMan assays exploit the 5'-exonuclease activity of the Taq
polymerase to monitoring DNA amplification in real time. Further
discussion of this well known assay is provided by Holland et al.,
Proc. Natl. Acad. Sci. USA (1991); Lee et al., Nucleic Acids Res.
(1993); and U.S. Pat. Nos. 5,210,015; 5,538,848; 6,258,569 and
5,691,146). The TaqMan assay detects the accumulated PCR product
via hybridization and subsequent cleavage of a fluorogenic probe
(the "TagMan" probe) during the amplification reaction. The probe
is an oligonucleotide whose sequence is complementary to the target
DNA to be detected. The probe is labeled with a single fluorescent
reporter dye and a single fluorescence quencher. The reporter and
quencher dyes are attached to the oligonucleotide with a separation
of typically about 15 to about 60 nucleotides for optimal
fluorescence quenching and 5'-exonuclease activity. The dye can be
attached to either the nucleotide bases or the backbone of the
oligonucleotide or a combination thereof. Typically, one of the
reporter/donor labels is attached to the 3' terminal backbone
phosphate and the other attached to the 5' terminal backbone
phosphate. The quencher can be either a non-fluorescent dye or a
fluorescent dye of appropriate wavelengths. In both cases,
quenching of the fluorophore bound to the probe occurs via
fluorescence resonance energy transfer (FRET).
[0060] In order to briefly discuss how to select a
fluorophore/quencher pair and design a TaqMan probe that will
exhibit optimal performance, it is useful to review some basic
photophysics. Firstly, when a dye receives a photon of sufficient
energy from an external source, it is electronically excited to the
singlet excited state. The excited state exists for a finite time,
during which period the electronic energy is partially dissipated
into vibrational and rotational modes of the molecule.
Consequently, when the dye emits the photon, the energy of the
photon is lower and thus the wavelength of the photon emitted by
the molecule is longer than the wavelength of the photon absorbed
by the molecule. This energy or wavelength difference between
excitation, or absorption, and emission is called the Stokes shift
and is common among fluorescent dyes. Secondly, FRET-based
quenching is governed by Forster's resonance energy transfer
theory, which states that the efficiency of photo energy transfer
is positively related to the overlap of emission spectrum of the
donor and absorption spectrum of the acceptor. Further discussions
of this is provided in Forster, Ann. Phys. (1948); and Stryer et
al., Proc. Natl. Acad. Sci. (1967). The combination of the Stokes
shift and the spectral overlap requirement necessitates that the
fluorescent donor and acceptor be different dyes. This is true even
if the donor and quencher molecules are both fluorescent dyes.
[0061] In fact, a quencher molecule that is also a fluorescent dye
should ideally be selected to have a wavelength of emission that is
significantly longer than the emission wavelength of the donor
(reporter) dye. Using this combination ensures a dark background.
An additional gain in sensitivity can be achieved by using a filter
that selectively blocks all or most of the signal emitted by the
quencher molecule. For optimal performance then, quenchers are
preferably chosen from molecules that are themselves completely
non-fluorescent. Indeed, achieving a low or zero background for
TaqMan assays has been the focus of considerable research effort.
To this end, many non-fluorescent quencher molecules have been
developed. Well known commercially available non-fluorescent
quenchers include, for example, Black Hole Quencher (BHQ) dyes from
Biosearch, which are non-fluorescent azo dyes and Eclipse Dark
Quencher (DQ) from Epoch (Eurogentec catalog number OL-0273-DQ02),
and IOWA Black (IWB) from Integrated DNA Technologies. Further
discussion of these molecules is provided by Johansson, M. K, et
al, J. Am. Chem., Soc., (2002). These and similar non-fluorescent
quenchers improve the sensitivity of TaqMan probes by suppressing
background fluorescence, thereby increasing the signal gain
following enzymatic cleavage of the probe. Examples of popular
donor/acceptor (fluorophore/quencher pairs used in TaqMan probe
design include FAM/TAMRA, VIC/BHQ1, HEX/BHQ2, and TET/DHQ and the
like.
[0062] In addition to the spectral overlap requirement stated
above, a second parameter critical to FRET-based fluorescence
quenching is the distance between the donor and acceptor. FRET is
dependent on the inverse sixth power of the intermolecular
separation.
[0063] Prior to hybridization with a target nucleic acid, a typical
TaqMan probe assumes a random conformation, wherein the donor and
quencher are able to move close enough to one another for efficient
FRET to occur. Because of their proximity to one another and their
spectral characteristic the reporter molecule, is quenched and
therefor the probe, is non-fluorescent. Upon hybridization with the
segment of a target DNA being amplified, the probe is stretched and
consequently becomes fluorescent because the increased distance
between the donor and quencher makes FRET energy transfer
impossible. Alternatively, and often preferably, the probe is
digested during amplification by, for example, using a polymerase
that has 51-exonucleas activity. Digestion completely frees the
donor dye from being in proximity to the quencher, thereby further
reducing quenching and further enhancing the fluorescence signal
from the donor dye. As more DNA copies are made during the PCR
amplification, more probes are hybridized and then cleaved, which
in turn increases the fluorescence signal A variation of this
scheme is the use of a two-enzyme system. In this system one enzyme
is responsible for polymerizing the oligonucleotide and the second
enzyme is responsible for cleaving the reporter dye from the PCR
product. One such commercially available product that exploits this
strategy is the Full Velocity kit available from Stratagene.
[0064] Another variation of probe design that exploits FRET to
create probes with good signal to noise ratios is described in U.S.
Pat. No. 6,492,346. This variation employs a minor groove binding
molecule (MGB) covalently linked to the 3'-end, 5'-end, or any
other position within the oligonucleotide probe. Probes containing
a MGB may have a shorter nucleotide sequence than probes that do
not use MGBs and still exhibit very efficient fluorescence
quenching. In addition, probes labeled with a MGB tend to have
improved specificity, efficiency and enhanced discrimination
against mismatches relative to probes labeled by certain other
labeling molecules that do not selectively bind to the minor
groove.
[0065] Still another method of detecting amplification products
uses the so-called "molecular beacon probe" which is the subject of
U.S. Pat. Nos. 5,925,517; and 5,118,801 and 5,312,728. Molecular
beacon probes have stem-loop structures including a central
target-recognition sequence (loop region) that is flanked by a pair
of complimentary 3' and 5' terminal sequences that hybridize to
each other to form a stem structure. A fluorescence donor dye and
quencher molecule are attached to the 3' and 5' terminals,
respectively. In the absence of a complementary target sequence the
beacon probe stays in its closed conformation, and the fluorescence
of the donor dye is quenched by the quencher dye via FRET. Upon
hybridization with a target sequence, the beacon probe stretches to
an open conformation, thereby separating the donor dye and
quencher, and increasing the fluorescence signal of the donor dye.
As additional copies of a target DNA are generated during the
course of a PCR reaction, more beacon probes adopt the open
conformation by hybridizing to the target DNA and the fluorescence
signal rises accordingly. Further discussion of this technique is
provided by Tyagi et al., Nature Biotechnol. (1996). Unlike TaqMan
probes, which are "consumed" via enzymatic cleavage of the probes
during PCR, molecular beacon probes remain chemically unchanged
throughout the DNA amplification process; only the conformation of
the beacon probes changes from the closed form (stem-loop) to the
open form.
[0066] The two conformations of the beacon probes--open and
closed--exist in a dynamic equilibrium with one another. During PCR
the equilibrium depends on three competing hybridization reactions
between each pair of complementary strands. The three equilibria
are between: 1) the two complementary strands of the target DNA; 2)
the complementary 3' and 5' ends (stem) of the probe; and 3) the
target-recognition sequence of the probe (loop) and the target
sequence. Only the third hybridization reaction favors the open
beacon conformation and thus only one produces a detectable
fluorescence signal. Both the first and second hybridization
reactions favor the closed beacon conformation and in turn reduce
the fluorescence signal. Because of the competing hybridization
interactions (1 and 2), a molecular beacon probe is inherently less
sensitive in nucleic acid detection than a TaqMan probe labeled
even when both are labeled with the same or similar donor/quencher
dye pair. However, a beacon probe hybridized to a target sequence
can also be cleaved from the oligo as is a TaqMan, probe if an
enzyme with 5'-exonuclease activity is used in the reaction. In
this instance, a beacon probe has become a de facto TaqMan
probe.
[0067] Still another method is disclosed in U.S. Pat. No.
6,174,670, this method uses an energy transfer system in which
energy transfer occurs between two hybridization probes. For
example, the first probe is labeled at the 3' end with a
fluorescence donor dye, while the second probe is labeled at the 5'
end with a acceptor dye. The acceptor molecule on the second primer
emits energy at a longer wavelength than the donor dye on the first
primer. When employed in PCR, the two probes hybridize to one of
the two complementary strands of a target DNA in a head to toe
arrangement. Because of how the two donor and acceptor dyes are
positioned FRET occurs between these two molecules. Accordingly, by
measuring the fluorescence emission of the acceptor dye, one can
relate the fluorescence intensity to the amount of a target DNA
being generated as the PCR progresses.
[0068] Yet another method of detecting amplification products is
disclosed in U.S. Pat. No. 6,635,427. This method uses an internal
guanosine (G) nucleotide as a quencher (acceptor) for a single
reporter dye attached to an oligonucleotide probe. In the absence
of a target sequence, the guanosine nucleotide, which is usually
strategically positioned near to the position of the reporter dye,
quenches the fluorescence of the reporter dye. Upon hybridization
with the target sequence, quenching by guanosine is reduced,
leading to an increase in the fluorescence signal.
[0069] PCR can also be monitored by using of fluorogenic primers
that become fluorescent upon incorporation into the amplification
products. One such method that uses a beacon-like hairpin primer
labeled with a donor/quencher dye pair is known commercially as
Ampliphore, and is disclosed in U.S. Pat. No. 5,866,336. Prior to
hybridizing to its target DNA sequence, the primer exists in the
closed hairpin conformation, which quenches the fluorescence of the
donor dye via FRET. Once hybridized to the target DNA sequence and
incorporated into the amplification product, the primer assumes an
extended open conformation and the primer becomes fluorescent
because FRET is diminished. In order to achieve the required
specificity of a primer as well as to maintain the necessary
hairpin structure, a beacon-like primer is usually quite long. The
length of the probe imposes restrictions on primer design and adds
to the cost of synthesizing the primer.
[0070] Another method of monitoring PCR uses a primer known
commercially as the LUX primer. Further discussion of this
technique is provided by Nazarenko et al., published in Nucleic
Acid Research (2002); and -Marras et al., Nucleic Acid Research
(2002). LUX primers are labeled with a single fluorescent dye
(donor molecules) positioned strategically near an internal
guanosine (G) nucleotide that acts to quench the fluorescence of
the dye. The labeled primer in its free form is non-fluorescent or
weakly fluorescent due to the interaction between the dye and the G
nucleotide that is near to it. When the primer is extended and the
G nucleotide is internalized in double stranded DNA, fluorescence
quenching is reduced or eliminated and the fluorescent signal from
the donor molecule increases. LUX primers have the advantage of
being relatively simple in design. One problem with the LUX system
is that it requires the presence of a G nucleotide near the dye
attachment site (usually thymidine) this restricts choices in
primer design. Furthermore, the fluorescence quenching by G only
works well with a very few dyes of selected wavelength. The
restriction on primer sequence and, especially, the incompatibility
with longer wavelength dyes make it difficult to develop a set of
LUX primers for use in multiplex PCR.
[0071] More recently, BD Biosciences developed a fluorescence-based
primer kit known commercially as DNAzyme for use in RT-PCR. This
method employs a combination of a fluorogenic oligonucleotide and
primers and is disclosed in US Patent Application Publication No.
2001/0001063. In addition to the normal initiation sequence for the
extension reaction, one of the primer sequences encodes an enzyme
that cleaves the fluorogenic oligonucleotide during primer
extension. The result, as with the TaqMan assay, is in an increase
in the fluorescence signal. The fluorogenic oligonucleotide used in
the primer kit is similar in design to a typical TaqMan.RTM. probe
except that the former does not have a sequence complimentary to
the target sequence and as a result this technique lacks the
specificity of TaqMan based assays. Furthermore, because of the
need to code for the requisite enzyme, the DNAzyme primer can be
easily over 50 nucleotides long. The need for a longer nucleotide
increases the cost of manufacturing the primer.
[0072] One major advantage of fluorogenic probes over fluorogenic
primers is that fluorescence signal detected from probes derives
only from hybridization between probe and target. Non-specific
amplification of signal due to mis-priming or primer-dimer
artifacts, as sometimes occurs with primers and does not generate
useful signals, does not generally occur with probes. Fluorogenic
probes can be labeled with different, distinguishable reporter
dyes. By using several probes each labeled with a unique reporter
dye, amplifications of multiple targets with distinct sequences can
be detected in a single PCR reaction. This method is commonly
referred to as multiplex PCR. The development of fluorogenic probes
has also made it possible to eliminate post-PCR processing, thereby
eliminating the possibility of cross-contamination, which is a
critical factor for clinical diagnostics and forensic
applications.
[0073] One disadvantage of fluorogenic probes is their cost. A
fluorogenic oligonucleotide is at least 10 times more expensive
than the corresponding unlabeled oligonucleotide. Furthermore,
probe design is relatively complex and one often needs to consider
many factors such as the length of probe, annealing temperature and
proper spectral matching of quencher to fluorophore when
constructing a suitable probe.
[0074] Compared with fluorogenic oligonucleotide probes,
particularly TaqMan probes, fluorogenic primers tend to generate a
weaker signal because they are not cleaved during PCR to allow
permanent separation of the reporter dye from the quencher.
Additionally, non-specific PCR products resulting from mis-priming
and primer dimer formation also contribute to increasing noise in
the assay. For these reasons, fluorogenic probes are generally
preferred over fluorogenic primers for use in real time
quantitative PCR.
[0075] Numerous methods have been developed for labeling
oligonucleotides. Typically a fluorescent donor dye and a quencher
are attached to the oligonucleotides in a stepwise fashion. These
processes often involve expensive reagents and complex
protection/de-protection steps and the yields are often quite low.
Moreover, the choice of dyes and quenchers as well as the order in
which they are attached to the oligonucleotides are limited and
inflexible, in part, because not all dyes can tolerate the harsh
chemical conditions of oligonucleotide syntheses.
[0076] Typically, a dye is incorporated into an oligonucleotide via
one of two methods: 1) by using a dye-modified nucleoside or
deoxynucleoside phosphoramidite during automated synthesis; or 2)
in a post-synthesis labeling by reacting an amine- or
thiol-modified nucleotide or deoxynucleotide with an amine- or
thiol-reactive dye. For example, an oligonucleotide labeled with a
FAM/TAMRA dye pair at the 3' and 5' termini is typically
synthesized by starting with a TAMRA-labeled modifier attached to
CPG solid support, followed by successive buildup of the remaining
oligonucleotide. The donor dye FAM is attached to the oligo at the
last coupling step via standard phosphoramidite chemistry. This
approach has two drawbacks: first, dye-labeled phosphoramidite is
costly; second, it diminishes the quenching ability of TAMRA
because rhodamine dyes in general, and TARMA, in particular are
unstable under the standard oligonucleotide synthesis condition. To
address this problem, it is a common practice to avoid starting
with TAMRA linked to CPG support. One approach is to start the
synthesis with a protected 3'-amino-modifer linked CPG and the
donor dye FAM is attached to the 5'-end as usual at the last step
of the automated synthesis via standard phosphoramidite chemistry.
Following subsequent oligonucleotide cleavage from the solid
support and a 3'-amine de-protection step, the amine-modified
oligonucleotide is reacted with a TAMRA succinimidyl ester. To
ensure the performance of the probe or primer, purification steps
using either HPLC or polyacrylamide gel electrophoresis are
necessary both before and after to the second dye attachment
reaction. The limitations and inflexibility imposed by the designs
of the probes and primers as well as the nature of the labeling
chemistry make these fluorogenic oligonucleotides very expensive to
manufacture.
[0077] An increasing important type of PCR assay that makes use of
labeled oligonucleotides is real-time PCR. One important parameter
for a real-time PCR monitoring is the so-called threshold cycle
point, or Ct value. The Ct value is the theoretical number of
reaction cycles needed for the fluorescent signal of the PCR
product to reach a pre-set value above the baseline. The higher the
concentration of a target DNA in the sample, the smaller the Ct
value will be. FIG. 1 shows a representative amplification plot and
defines the terms used in the quantification analysis. An
amplification plot is the plot of fluorescence signal versus cycle
number. In the initial cycles of PCR, there is little change in
fluorescence signal. This relatively "flat region" defines the
baseline for the amplification plot. An increase in fluorescence
above the baseline indicates the detection of accumulated PCR
product. The logarithm of initial target copy number is reversibly
correlated to Ct value in a linear fashion, and this relationship
forms the mathematical basis for real-time quantitative PCR.
DEFINITIONS OF SOME OF THE TERMS USED HEREIN
[0078] The terms "oligonucleotide" and "oligo" are used
interchangeably and refer to a sequence of nucleic acids,
2'-deoxynucleic acids, peptide nucleic acids (PNA), locked nucleic
acid (LNA) and other unnatural nucleic acids which include pyrazolo
pyrimidine. In general oligonucleotides are of a length suitable
for use as primers or probes. Most oligonucleotides are
polynucleotides generally less than 100 nucleotides long, many are
less than 50 nucleotides long and a number of oligonucleotides are
comprised of 25 or fewer nucleotides. For a more thorough
discussion of the term the reader is directed to the following
references Kutyavin, I., et al., N. A. R. 30, 2002:4952-4959; and
He J. & Seela F., N. A. R. 30, 2002:5485-5496 and references
therein.
[0079] "Primer" refers to an oligonucleotide that is capable of
acting as a starting point to extend along a complementary strand.
Primers usually are used as a set in PCR, one forward and one
reverse. The forward primer contains a sequence complementary to a
region of one strand of target nucleic acid and guides the
synthesis along this strand. Similarly the reverse primer contains
a sequence complementary to the opposite stand of the target
nucleic and guides the synthesis along the opposite strand of
target nucleic acid.
[0080] "Probe" refers to a labeled oligonucleotide containing a
sequence complementary to a region of the target nucleic acid,
wherein the labeled oligonucleotide anneals to the target sequence
and generates a signal indicating the presence of the region of the
target. The probe is generally blocked at the 3' terminus and is
not extended into products.
[0081] The term "photometric labeling molecule" refers to molecules
that generate a detectable change signal due to change in the
molecule's physical or chemical environment and used to label
another molecule. The change may be in the amount of light absorbed
or in the wavelength of light absorbed. Photometric molecules
include, for example, fluorescent dye molecules that absorb light
at one length and emit light at another wavelength. In the case of
photometric labeling molecules that are fluorescent dyes the
molecules may also exhibit a change in the amount of light emitted
or in the wavelength of the light emitted.
[0082] The term `reactive groups` refers to chemical moieties that
may be useful in attaching various labeling groups including
fluoropores and quenching molecules to oligonucleotides. The choice
of reactive group used to attach the dye to an oligo typically
depends on the functional group on the oligo to be labeled.
[0083] The bond formation reaction between a reactive group of, for
example, a dye molecule and a functional group of a oligo is
typically a reaction between a nucleophile and an electrophile.
Accordingly, a reactive group can be either a nucleophile or a
electrophile, and correspondingly a functional group can be either
an electrophile or a nucleophile. A non-exhaustive list of pairs of
electrophile/nucleophile can be found in Table 2. For a further
discussion of reactive pairs the reader is directed to U.S. Pat.
No. 6,130,101.
[0084] Typical functional groups present on an oligo include, but
are not limited to, amines, thiols, alcohols, phenols, aldehydes,
ketone, hydrazines, hydroxylamines, disubstituted amines, halides,
or carboxylic acids. More typical functional groups on an oligo are
amines, thiols, alcohols, aldehydes or a ketones.
[0085] Common reactive groups attached to a dye molecules include,
but are not limited to: acrylamide, an activated ester of a
carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an
alkyl halide, an amine, an anhydride, an aniline, an arylhalyde, an
azide, an aziridine, a carboxylic acid, a haloacetamide, a
halotriazine, a hydrazine, a hydrazide, an imido ester, an
isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a
sulfonyl halide or a thiol. Other reactive groups include
succinimidyl ester, an amine, a haloacetamide, a hydrazine, an
isothiocyanate, maleimide, or a phosphoramidite. When the
functional groups present on the oligonucleotide are amines one
commonly used reactive group on the dye used to attach the dye to
the oligonucleotide is a succinimidyl ester.
[0086] Some of the abbreviations used for various reagents
including dyes are as follows: 5-CR110 refers to
5-carboxyrhodamine-110; 6-CR110 refers to 6-carboxyrhodamine-110;
5-FAM refers to 5-carboxyfluorescein; 6-FAM refers to
6-carboxyfiuorescein; 5-R6G refers to 5-rhodamine 6G; 5-ROX refers
to 5-carboxy-X-rhodamine; 6-ROX refers to 6-carboxy-X-rhodamine;
5-TAMRA refers to 5-carboxytetramethylrhodamine; 6-TAMRA refers to
6-carboxytetramethyl-rhodamine; JOE refers to
2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein
[0087] The term "spectrally similar dyes", for the purpose of the
present invention, refers to fluorescent dyes that may or may not
have similar chemical structures but possess similar excitation
and/or excitation spectral properties. The emission spectra of the
dyes, however, may or may not be similar. An example of one such
pair is 5-FAM and 5-CR110. 5-FAM has absorption/emission
wavelengths at 495/519 nm while 5-CR110 has absorption/emission at
502/524 nm. For the purpose of the present invention, absorption or
excitation wavelengths having a difference within 15 nm are
considered to be similar.
[0088] The term "spectrally identical dyes" refers to fluorescent
dyes that may or may not have the same chemical structures but have
either emission profiles or excitation or both emission and
excitation profiles that are spectrally indistinguishable.
[0089] The term "same dyes" refers to fluorescent dyes that are
both chemically and spectrally identical.
[0090] The term "reporter dye" or "fluorescent reporter dye" refers
to a fluorescent dye whose fluorescence is monitored during an
assay. When a quencher dye is also used to label the same
biomolecule, a reporter dye may be referred to as a donor dye and
the quencher dye may sometimes be referred to as an acceptor,
acceptor dye or acceptor molecule.
[0091] As used herein, the terms "quench" or "quenches" or
"quenching" or "quenched" refer to reducing the signal produced by
a molecule, it includes, but is not limited to, reducing the signal
produced to zero or to below a detectable limit. Hence, a given
molecule can be "quenched" by, for example, another molecule and
still produce a detectable signal albeit the size of the signal
produced by the quenched molecule will be smaller when the molecule
is quenched than when the molecule is not quenched.
[0092] The term "quencher" or "quencher dye" or "quencher molecule"
refers to a dye or an equivalent molecule, such as nucleoside
guanosine (G) or 2'-deoxyguanosine (dG), which is capable of
reducing the fluorescence of a fluorescent reporter dye or donor
dye. A quencher dye may be a fluorescent dye or non-fluorescent
dye. When the quencher is a fluorescent dye, its fluorescence
wavelength is typically substantially different from that of the
reporter dye and the quencher fluorescence is usually not monitored
during an assay.
[0093] Some embodiments of the present invention disclose methods
for constructing fluorogenic oligonucleotides and their uses as
primers and probes for the purpose of nucleic acid detection,
particularly nucleic acid detection in real-time qPCR. Compared
with existing technologies, fluorogenic oligonucleotides of some
embodiments are significantly easier to manufacture and
substantially more sensitive than many commercially available
technologies. Furthermore, unlike conventional fluorogenic
oligonucleotides, such as the TaqMan probes, whose choice of a
reporter dye is limited by the availability of the accompanying
quencher dye, oligonucleotides of various embodiments of the
present invention can accommodate fluorescent dyes of virtually any
wavelength. This flexibility in dye selection facilitates syntheses
of fluorogenic primers or probes of different fluorescent
wavelengths and allows multiplex detection in a single-tube
format.
[0094] Some embodiments are probes that are single-stranded
oligonucleotides labeled with a plurality of spectrally identical
or similar fluorescent dyes. The probes may be further labeled with
one or more quenchers. In the absence of a target sequence, the
probes assume a random coiled conformation, and are either
non-fluorescent or only weakly fluorescent. Generally, an
oligonucleotide with a random coiled conformation is substantially
free of secondary structure that serves to bring the two termini of
the oligonucleotide in proximity with one another. In contrast, an
example of oligonucleotide probes having a secondary structure is
the so-called molecular beacon probe.
[0095] In the presence of a target sequence, probes of various
embodiments readily hybridize to the target sequence. In the
presence of specific reagents or enzymes with 5' exonuclease
activity the dye molecules are subsequently cleaved from the
oligonucleotide. Cleavage results in permanent separation of the
dye molecules resulting in a large increase in fluorescence.
[0096] Maximal fluorescent increase is dependent on the
cleavability of the phosphate backbone linking each neighboring dye
pair by the 5'-exonuclease or an equivalent nuclease. When the dye
attachment sites are too close to each other, hybridization of the
oligonucleotide to the target and 5'-exonuclease activity may be
hindered. On the other hand, when the dye attachment sites are too
far away from each other, background fluorescence may be high.
Thus, to achieve minimal fluorescence background in the absence of
a target sequence and optimal 5'-exonuclease activity following
probe/target hybridization, each neighboring pair of dye attachment
sites should be separated by 3 to 60 nucleotides, preferably by 12
to 35 nucleotides, and most preferably by 15 to 25 nucleotides.
[0097] Useful enzymes for use with these embodiments include Taq
polymerase or a stand-alone exonuclease, or any other enzyme that
can cut between the two labeling molecules. In some embodiments,
permanently separating the labeling molecules substantially
increased fluorescent emission.
[0098] In contrast to the multiple labeling molecules incorporated
into oligos labeled according to the invention, fluorogenic probes
such as the TaqMan probes disclosed in U.S. Pat. No. 5,538,848,
Molecular Beacons disclosed in U.S. Pat. No. 5,925,517 and the Hyb
probes disclosed in U.S. Pat. No. 6,174,670 include only single
fluorescent reporter. As a result, the maximum fluorescent signal
that currently available probes can generate is the signal produced
by a single fluorophore. On the other hand, probes of various
embodiments of the present invention include at least two dyes.
Accordingly, hybridization with a target sequence and cleavage of
the probes of the present invention results in the generation of
signal from multiple fluorescent dyes. Accordingly, probes of
various embodiments of the present invention are capable of
generating fluorescent signal many times stronger than the signals
generated by probes made in accordance with the currently available
methods.
[0099] FIG. 3A illustrates the result of comparing the kinetic
fluorescence measurements of a typical MCG gene amplification
reaction detected with a TagMan probe (JOE as the fluorophore and
TAMRA as the quencher) and the same reaction detected with a probe
made in accordance with one embodiment of the present invention.
The probe made in accordance with one embodiment of the instant
invention was labeled with two R6G dyes at the two termini (See
Example 4). The same sequence was used in both assays.
[0100] As the data in FIG. 3A illustrate the fluorescence signal
from the doubly labeled probe is twice that of the signal measured
using the TagMan probe. It is a surprising observation that an
oligonucleotide labeled with two or more identical or spectrally
similar fluorophores is sufficiently quenched (without forming dye
aggregates) that separation of the fluorophores from one another by
cleavage of the probe generates a signal high enough above
background to be useful in tracking the amplification of DNA.
Because this observation with oligonucleotides was so unexpected,
we looked for an explanation to techniques that use fluorescent
dyes to measure the level of another biopolymer polypeptides.
[0101] Proteins are sometimes labeled with antibodies tagged with
fluorescent dyes. In these assays it is common to attach two or
more identical fluorophores to a single polypeptide. The additional
of each dye molecule increases the overall fluorescence of the
labeled proteins, although the fluorescence increase over the
number of dyes may not be linear due to fluorescence quenching
related to physical touching of the dyes. In protein labeling
experiments significant fluorescence quenching occurs, only when an
excess number of dye molecules are attached to the protein. In such
cases, the quenching is often a result of the physical interaction
among the dye molecules namely dye aggregation. Further discussion
of this technique is provided in Haugland, R P Handbook of
Fluorescent Probes and Research Products 9.sup.th edition, pp.
20-74 and references therein.
[0102] In contrast to relatively highly structured polypeptides,
oligonucleotides lacking complimentary internal sequences are
expected to assume unstructured, unpacked, extended structures.
This conformation is generally referred to as random coil and is
characterized by a lack of readily definable internal secondary
structure. Oligonucleotides are believed to adopt the random coil
conformation in order to minimize inter molecular electrostatic
repulsion due to the highly negatively charged phosphate backbone
of the molecule. Consequently, the dyes used to label, for the
example, the termini of an oligonucleotide with a substantially
random coil shape are not expected to interact with one another and
should not be expected to demonstrate fluorescence signal
quenching. Therefore, based on what is widely known about protein
labeling and oligonucleotide structure, one would have expected
that oligonucleotides of various embodiments of the present
invention would exhibit too little fluorescence quenching to be
useful in detecting the presence of or level of oligonucleotide in
a given sample. Based on what is taught in the art it was expected
that the background fluorescence of these molecules would be so
high that they could not be used to monitor real-time PCR. Indeed,
researchers have gone to great length to design elaborate oligo
structures that position labeling fluorophores so as to facilitate
fluorophore aggregation to quench fluorescence. For example, both
U.S. Pat. Nos. 6,150,097 and 6,037,137 mentioned the possibility of
designing a real-time PCR probe based on Molecular Beacon structure
that brings two reporter fluorophores into physical contact with
one another.
[0103] Another unexpected observation gleaned from using one
embodiment of the present invention is that oligonucleotide
secondary structure that promotes dye aggregation is both
unnecessary and undesirable. We observed that aggregation of
identical reporter dyes is not only unnecessary, but in many
instances detrimental to the performance of real-time PCR probes.
Probes of one embodiment of the present invention, that have the
simplest structure, are oftentimes much more sensitive than probes
made in accordance with methods disclosed in much of the prior art.
Additionally, probes that are designed to have complex internal
secondary structure are generally more difficult to design and
manufacture than are probes that are substantially devoid of
internal secondary structure.
[0104] It has been widely reported that certain fluorescent or
non-fluorescent dyes tend to form ground state complexes. These
complexes are likely to form, in aqueous solvents at high
concentrations or when the molecules are within close proximity to
one another. Further discussion of this is provided by West et al.,
J Phys Chem (1965); Rohatgi et al., J Phys Chem (1966); Rohatgi et
al., Chem Phys Lett (1971); and Khairutdinov et al., J Phys Chem.
(1997).
[0105] The formation of either a homodimer between two identical
dyes, or heterodimer between two different dyes leads to a distinct
change in the absorption spectrum of the dyes. This is believed to
be the result of coupling of the excited state energies of the
dyes. A particular type of dimer called an H-dimer that forms
between two fluorescent dyes or between a fluorescent dye and a
non-fluorescent dye is characterized by a blue shift in the
absorption maximum of the dyes and fluorescence quenching.
Fluorescence quenching due to H-dimer formation has been exploited
to construct fluorogenic peptidase substrates. Further discussion
of this subject is provided by Packard et al., Proc. Natl. Acad.
Sci. (1996); Geoghegan et al., Bioconjugate Chem (2000); Tyagi et
al., Nat. Biotechno. (1998); Bernacchi et al, Nucleic Acids Res.
(2001); Marras et al., Nucleic Acids Res. (2002); Johansson et al.,
J. Am. Chem. Soc. (2002); U.S. Pat. No. 6,037,1376 and 150,097.
[0106] In all of the aforementioned references, the labeled
peptides or oligonucleotides are constructed in a manner designed
to ensure that the molecules are physically close enough to one
another so that signal from the donor dye is quenched prior to
enzymatic cleavage. This is apparently the case with peptidase
substrates, and with molecular beacon probes before they hybridize
to their target sequences. For example, the fluorogenic peptides
disclosed in U.S. Pat. No. 6,037,137, require so-called,
"conformation determining regions" that introduce bends into the
peptides so that the dye pair are held in close proximity for
efficient "contact fluorescence quenching". Similarly, U.S. Pat.
Nos. 6,150,097 and 6,037,137 disclose molecular beacons having a
fluorescent reporter dye attached to one terminal and a quencher
dye attached to the other terminal. Alternatively these probes have
one reporter dye attached to one terminal and an identical reporter
dye attached to the other terminal, wherein the dye pair is in
physical contact to effect fluorescence quenching. In a variation
of this method one group reportedly achieved contact quenching by
labeling a linear oligonucleotide with a fluorophore and a highly
hydrophobic quencher that favors formation of a heterodimer with
the fluorophore. See Johansson, et al, J. Am. Chem. Soc.
(2002).
[0107] In contrast to these approaches, oligonucleotide probes of
some embodiments of the present invention are substantially devoid
of clearly definable secondary structure or other conformation
determining structures that result in the probes assuming a
particular rigid conformation. Furthermore, it is not necessary to
form a dimer or other structures that facilitate physical
"touching" between the dyes in the probes of many embodiments of
the present invention comprising two or more fluorescent reporter
dyes.
[0108] The absence of proximal quenching between the dye pair of
various embodiments is demonstrated by a lack of significant
alteration in the absorption profile of the doubly labeled probe
before and after enzymatic digestion. See, for example, Traces 29
vs. 28 in FIG. 4A and further discussion in Example 5. The small
overall wavelength shift from Trace 28 to Trace 29, is different
from a change in the shape or profile of the spectrum, and is
caused by a difference in the micro environment that the dye
experiences. This is referred to as the "solvent effect". The
solvent effect is confirmed by the nearly superimposable spectra of
the probe doubly labeled with CR110 (Trace 29) and another
oligonucleotide of the same sequence labeled with a single CR110 at
either 5' (Trace 31 in FIG. 4B) or at 3' (Trace 33 in FIG. 4C). In
this instance the dyes experience a similar solvent effect. Once
again, the similarity in the absorption spectra between the doubly
labeled probe and the two singly labeled oligonucleotides indicates
that there is substantially no dye aggregation in the doubly
labeled probe.
[0109] To illustrate the spectral change upon dimer formation, we
synthesized three molecular beacon probes with loop sequences that
are identical to that of the linear probe. One beacon probe was
labeled with two 5-CR110 dyes at the 3' and 5' terminals (SEQ ID No
12), another beacon probes was labeled with two 5-TAMRA dyes at the
3' and 5' termini (SEQ ID No 13. And still another beacon probe was
labeled with two 5-ROX dyes at the 3' and 5' termini (SEC) ID No
14). As shown in FIGS. 5A, 5B and 5C and further detailed in
Example 7. The absorption spectra of all beacon probes (Trace 38,
40, and 42) have been altered significantly from those of linear
probes (Traces 37, 39, and 41), with each forming a new shorter
wavelength peak characteristic of H-dimer formation. For a more
thorough discussion of this please see Blackman et al.,
Biochemistry (2002); Packard et al., Proc. Natl. Acad. Sci. (1996).
Upon S1 nuclease digestion, the spectra changed back to that of
free dye. Although the fluorescence of beacon probe labeled with
two reporter dyes is well quenched as a result of dye dimer
formation, the probe has relatively low sensitivity for nucleic
acid detection. FIGS. 6A, 6B, and 6C compares the kinetic
fluorescence measurements between the molecular beacons and the
linear probe each labeled with two identical dyes of 5-CR110,
5-TAMRA, 5-ROX, respectively according to various embodiments of
the present invention.
[0110] As the data indicate, the probes made according to various
embodiments of the present invention are often times 2-10 times
more sensitive than the corresponding beacon probes. The
significantly weaker sensitivity of the beacon probes may be
explained in terms of competing equilibria that exist during the
PCR detection process. As illustrated in FIG. 7, (details in
Example 8), there are three competing equilibria these are between:
1) single stranded target DNA and double stranded target DNA
(K.sub.A); 2) beacon in the open conformation and beacon in the
closed conformation (K.sub.B); and 3) the probe-target
hybridization product and the two reactants, single stranded target
DNA and beacon in random conformation (KO.
[0111] Formation of the probe-target hybrid separates the two dyes,
and as a result, fluorescent signal is generated. Clearly, the
higher the concentrations of the single stranded target DNA and
greater the amount of probe in the random coil conformation, the
more the equilibrium K.sub.c will shift toward the formation of the
probe-target hybrid product, thereby increasing the fluorescent
signal. However, at a given PCR cycle number and therefore at a
given concentration of the single stranded target DNA, the amount
of hybrid formation is proportional to the concentration of the
probe in random conformation, which is in equilibrium with the
beacon in the closed conformation. Therefore, the very existence of
the closed beacon conformation reduces the concentration of the
probe in random conformation that can form the hybrid product and
this reduces the strength of the fluorescent signal. Additionally,
juxtaposing a pair of dyes at the probe terminals to form a dimer
is likely to increase the melting temperature of the stem-loop
structure, further stabilizing the closed conformation and making
formation of the fluorescent probe-target hybrid even more
unfavorable. The fluorescent signal of a beacon probe can be
improved if an enzyme with 5'-exonuclease activity is used in the
reaction; following probe-target hybrid formation, 5'-exonuclease
cleaves the probe and generates irreversibly stable fluorescent
signal. Still, the equilibrium between the closed and random
conformations of the beacon slows down the rate at which the
fluorescent product is formed. Within the time frame of each PCR
cycle, typically 10-30 seconds, only a fraction of
thermodynamically allowable amount of cleaved product is formed,
resulting in a relatively weak signal as shown in FIGS. 6A, 6B and
6C (details in Example 8). On the other hand, homo-doubly labeled
probes according to various embodiments of the present invention
stay in an open random conformation, and therefore can readily form
fluorescent probe-target hybrids. Cleavage of the hybridized probe
by 5'-exonuclease further enhances the signal because it produces
an irreversibly de-quenched stable fluorescent product.
[0112] A major distinction between probes of some of the
embodiments of the present invention and probes such as the TaqMan
probe is that probes of many embodiments of the present invention
have two or more reporter dyes while TaqMan probes have only a
single reporter dye and a single quencher. Although the quencher
itself can also be a fluorescent dye, such as TAMRA in the
FAM/TAMRA donor/quencher pair, only the emission of the donor dye
FAM is detected and only its fluorescence signal is correlated to
the amount of DNA produced. In TaqMan assays the fluorescence of
the quencher is either ignored or not even detected. Real-time PCR
detection using TaqMan probes relies upon FRET-based fluorescence
quenching of the donor fluorophore to lower the background signal
in the assay. Prior to hybridization to a target DNA and/or
hydrolytic cleavage of the probe by 5-exonucelase activities, the
fluorescence of the donor is quenched and thus no signal or very
weak signal is detected. Following probe hybridization and/or
enzymatic cleavage of the probe, the fluorescence of the donor is
released and thus a positive signal corresponding to an increase to
the amount of DNA produced is detected.
[0113] Because the maximum signal that can be generated using a
TaqMan is produced by a single donor molecule the net signal gain,
(the ultimate performance) of a TaqMan probe is largely determined
by the efficiency of fluorescence quenching before and the
fluorescence yield of the fluorophore after probe cleavage.
Therefore, ideally in the TaqMan assay, the quencher should
completely quench the fluorescence of the donor until the reporter
is cleaved from the oligonucleotide. Complete quenching is usually
required to ensure that the real-time PCR assay starts with a dark
background. As discussed earlier, in accordance with to the
well-known principles of FRET, the efficiency of FRET-based
quenching is positively related to the overlap of the emission
spectrum of the donor molecule and absorption spectrum of the
acceptor (quencher) molecule. For a more thorough discussion of
FRET the reader is directed to references such as Forster, Ann.
Phys. (1948); Stryer et al., and Proc. Natl. Acad. Sci. (1967).
[0114] Furthermore considering that the fluorescence emission
wavelength of a dye is always longer than its absorption wavelength
(as defined by its Stokes shift) the best quencher molecule for a
given dye molecule will necessarily be a different dye. This is so
because the absorption spectra of the quencher must match with the
emission spectra of the donor. In fact, the larger the Stokes shift
of the donor dye is, the better the donor dye is because the donor
can then be excited at its absorption maximum without having it
interfere with its emission. This is the rationale for a second
method used to increase signal output. Given the principles of
FRET-based quenching, the advantage of having a donor dye with a
large Stokes shift, and the need to have maximal FRET-based
quenching, one would have necessarily choose a quencher with an
emission wavelength substantially different the emission wavelength
of the donor dye.
[0115] In sum based on the basic principles of FRET and its wide
spread use in the construction of oligonucleotide including primers
and probes the current art appears to teach away from various
embodiments of the present invention.
[0116] Accordingly, the embodiments of the invention are nonobvious
in view of the cited art as illustrated by the results obtained
with probes that were either homo-doubly labeled with FAM or
sulfonated Cy5. FAM and Cy5 are two of the most widely used
reporter dyes. However, neither of these dyes produced homo-doubly
labeled probes, which exhibited significant signal changes, and low
background fluorescence (Data not shown). In practice, in order to
increase the sensitivity of TaqMan probes, most commercial effort
has been focused on the development of more efficient quenchers
particularly quenchers based on non-fluorescent dyes. Examples of
highly efficient commercially available non-fluorescent quenchers
include azo dye-based BHQ quenchers from BioSearch, Inc., polynitro
cyanine dyes from Amersham, Inc. and the rhodamine-based YSQ dyes
from Molecular Probes.
[0117] Probes labeled with two reporter dyes according to various
embodiments do not have to be designed so as to position the dye
molecules in close physical proximity to one another. However,
aggregation is more likely to occur among probes that are labeled
with multiple dye molecules and optional quencher molecules than
among the same oligonucleotide sequences labeled with a single
reporter and quencher molecule. Accordingly, at least some of the
oligonucleotides, labeled with at least two signaling dyes (and
optionally with one or more quencher molecules) according to
various embodiments of the invention may exhibit low background
fluorescence and high signal output upon permanent separation of
the dyes as result of the probe cleavage, because of the large
number of signaling molecules per oligonucleotide.
[0118] Preferably, in some embodiments, oligonucleotides are
labeled with a plurality of spectrally identical or similar
fluorescent dyes. A mixture of fluorescent reporter dyes may be
used for labeling a particular probe as long as the dyes have
similar absorption or excitation spectra so that they can all be
efficiently excited with a single excitation light. For example, a
probe of the present invention may comprise both Cy3 (Glen
Research, Sterling, Va.) and TAMRA (Biotium, Inc. Hayward, Calif.),
both of which have similar spectra and can be efficiently excited
at 540 nm. As another example, a particular probe may comprise both
CR110, and FAM, both of which also have similar spectra and can be
well excited by the 488 nm argon laser line. Although probes
labeled with mixed dyes are relatively more difficult to
synthesize, in certain cases it can be advantageous if such mixed
dyes promote fluorescence quenching prior to hybridization with a
target sequence. For example, a probe of the present invention can
be labeled with a mixture of one dye with a net negative charge and
another dye of similar spectrum but with a net positive charge. A
mixture of dyes with opposite charges may promote fluorescence
quenching. Methods of adding charges to dyes are well known to
anyone skilled in the art. For example, negative charges can be
added to dyes by sulfonation, while positive charges can be created
on dyes by adding secondary, tertiary, or quaternized amines to
dyes. For a more thorough discussion of these molecules the reader
is directed to see Mujumdar et al., 1993, Bioconjugate Chem.
[0119] More preferably, probes of some embodiments of the present
invention are oligonucleotides labeled with a plurality of
identical fluorescent dye molecules. Oligonucleotide probes thereof
have the advantage of being easily and economically manufactured
because dyes can be conjugated to the oligonucleotide in a single
step. Extensive research efforts were made in the 80's to develop
efficient techniques for labeling nucleic acids. These techniques
have been well documented. For a more thorough discussion of this
subject the reader is directed to the following references:
Connolly et al., Nucleic Acids Res. (1985); Dreyer et al., Proc.
Natl. Acad. Sci. (1985); Nelson et al., Nucleic Acids Res. (1989);
Sproat et al, Nucleic Acids Res. (1987) and Zuckerman et al.,
Nucleic Acids Res. (1987).
[0120] Probes of existing technologies having a reporter/quencher
dye pair require separate labeling steps and expensive reagents.
For example, the first label, dye or quencher, is typically
attached to an oligonucleotide, either by starting the
oligonucleotide synthesis with a protected dye linked to a CPG
solid support, or by incorporating the dye during the
oligonucleotide synthesis by using a dye-labeled nucleoside (or
2'-deoxynucleoside) phosphoramidite. The second quencher or dye
molecule is attached to the oligonucleotide by using a dye- or
quencher-labeled nucleoside phosphoramidite during the standard
oligo synthesis. Alternatively and more typically, the second dye
or quencher molecules is attached to the oligo by first
incorporating an amino group into the oligo during the oligo
synthesis and then reacting a succinimidyl ester dye or quencher
with the amine-modified oligo. In contrast to this multi-step
labeling procedure, probes of one embodiment of the present
invention comprising multiple identical dyes. Labeling with a
single dye requires only a single dye-labeling step, typically by
mixing in a buffer for 1.about.2 hours a reactive form of the dye
with an oligonucleotide containing a desired number of a reactive
groups capable of reacting with the dye. Typically, reactive groups
are first incorporated into an oligonucleotide via standard
phosphoramidite chemistry using commercially available
reagents.
[0121] Most preferably, probes of some embodiments of the present
invention are oligonucleotides labeled with two identical
fluorescent dyes whose fluorescence is quenched without formation
of dye aggregates. And preferably, the dyes are attached to the 3'-
and 5'-terminals of the oligonucleotides, respectively. In one
embodiment of the invention, one dye is attached to the 3' terminal
backbone phosphate via a flexible aliphatic linker, and another
identical dye attached to the 5' terminal backbone phosphate via
another flexible aliphatic linker. The flexible linkers are C2 to
C30 linear or branched, saturated or unsaturated hydrocarbon
chains, optionally substituted by heteroatoms, aryls, lower alkyls,
lower hydroxylalkyls and lower alkoxys. Preferably, the linkers are
C4 to C12 linear or branched, saturated hydrocarbon chains
optionally substituted by heteroatoms, lower alkyls and lower
hydroxylalkyls.
[0122] In another embodiment of invention, probes labeled with a
plurality of reporter dyes and quenchers further comprise a nucleic
acid binding group. Examples of nucleic acid binding groups include
minor grove binders (MGB), nucleic acid interculators, and
polyamines. In cases in which an nucleic acid binding group is
incorporating into probes of various embodiments of the present
invention, the oligonucleotide sequence of the probes can be me
made shorter and still produce a useful signal. Shorter probes
translate into lowering manufacturing costs. Methods of
incorporating a nucleic acid binding group into an oligonucleotide
probe or primer have been well documented see, for example, U.S.
Pat. Nos. 5,801,155; 6,472,153; 6,486,308; 6,492,346 and numerous
publications such as Afonina et al, N. A. R. (1997); Kumar, et al.,
N. A. R. (1998) and Kutyavin, et al, N. A. R. (2000). Preferably,
the nucleic acid binding group is a minor grove binder (MGB). And
preferably, probes comprising a nucleic acid binding group are
labeled with two or more identical reporter dyes. More preferably,
probes comprising a nucleic acid binding group are labeled with two
identical dyes that may or may not physically touch each other.
[0123] In still another embodiment of the invention,
oligonucleotide probes comprise a plurality of fluorescent reporter
dyes, in which at least some of the reporter dyes are attached to a
G nucleotide or near to a G nucleotide located within the labeled
oligonucleotide. In this arrangement the fluorescence of the dye or
dye molecules nearest to the G nucleotides are quenched before the
oligo hybridizes to its complementary sequence.
[0124] In one embodiment of the invention, the primer is a single
stranded linear oligonucleotide comprising a plurality of
spectrally identical or similar fluorescent reporter dyes. In the
absence of a target sequence, said primer assumes a random coiled
conformation and is non-fluorescent or weakly fluorescent. An
oligonucleotide primer in the random coiled conformation is
substantially devoid of internal secondary structure.
Oligonucleotides substantially devoid of internal secondary
structure do not readily from secondary structures such as of
stem-loops, hairpins and the like.
[0125] Once primers labeled with photometric molecules such as
fluorescent molecules are incorporated into the amplification
product the fluorophores are further separated from each other due
to the more extended conformation of the amplification product and
therefore the fluorescence signal from the photometric molecule
increases. When a primer is made in accordance with the embodiments
of the present invention the primer includes at least two
fluorophores per primer. Accordingly, primers made in accordance
with the present invention can be used to assay for complimentary
oligonucleotides with greater sensitivity than assays that use
primers labeled with only a single signaling molecule.
[0126] Upon hybridization with a target sequence and subsequent
incorporation into the amplification product, the fluorescence of
some of the primers made in accordance with embodiments of the
present invention increases. Accordingly, one advantage of primers
of some embodiments of the present invention is that multiple dye
molecules present on the oligos produce signal when they hybridize
to their targets. As there are at least two signaling molecules per
oligonculetide the signal they produce is greater than the signal
produced by a primer that has only a single dye molecule attached
to it. This helps to make some of the primer made in accordance
with the methods of the present invention more sensitive nucleic
acid detectors than primers of existing technologies that include
only a single signaling molecule.
[0127] The dye molecules are typically attached to the bases of
nucleotides or to a combination of the 5' terminal backbone
phosphate and the bases of an oligonucleotide via an aliphatic
linker. The dye attachment sites are spaced in a manner to achieve
maximal fluorescence quenching before hybridization and maximal
fluorescence following hybridization and incorporation into the
amplification product. Typically, the optimal spacing between any
two adjacent pair of fluorescently labeled nucleotides or between
the 5' terminal and an adjacent fluorescently labeled nucleotide is
10 to 50 nucleotides; Preferably, the spacing is about 15 to 30
nucleotides; Most preferably, the spacing is about 15 to 25
nucleotides.
[0128] In one embodiment is a primer labeled with two spectrally
identical or similar fluorescent dyes. Typically, one dye is
attached at or near the 5' ends via a linker and another dye
attached at or near the 3' ends via another linker. Preferably, one
dye is attached to the 5' terminal backbone phosphate via a linker
and, with proper spacing, another dye to the base of a nucleotide,
such as a T via another linker. Typically, linker molecules are C2
to C12 linear or branched, saturated hydrocarbon chains optionally
substituted by heteroatoms, aryls, lower alkyls and lower
hydroxylalkyls. The two dye molecules may be separated by about 15
to about 25 bases.
[0129] In one embodiment, the primer is a linear oligonucleotide
labeled with two identical dyes with one dye attached to the 5'
terminal backbone phosphate via a linker and another dye attached
to the base of a nucleotide T at or near the 3' end via another
linker. Said linkers are C4 to C12 linear or branched saturated
hydrocarbon chains optionally substituted by heteroatoms, lower
alkyls and lower hydroxylalkyls. Fluorogenic primers labeled with
two identical dyes according to the present invention are
significantly easier to manufacture than primers of existing
technologies. This is especially true when a donor dye and a
quencher molecule need to be attached in separate steps using
expensive reagents, or wherein a long, low-yielding nucleotide
needs to be synthesized to form a required hairpin structure.
[0130] Similarly, primers made in accordance with various
embodiments of the present invention are less expensive to
manufacture than primers made by many of the currently used
methods. In contrast to the multi-step labeling procedure required
to synthesize primers that include labeling molecules with
different chemistries, primers comprising two identical dyes
require only a single dye-labeling step. Typically labeled
oligonucleotides of various embodiments of the present invention
can be made by mixing a reactive form of the dye with a primer
containing two reactive groups capable of reacting with the dye in
a suitable buffer for 1.about.2 hours. Reactive groups are first
incorporated into the oligo via standard phosphoramidite chemistry
using commercially available reagents.
[0131] A major advantage of the fluorogenic oligonucleotides
according to various embodiments of the present invention is the
freedom to use fluorescent dyes of virtually any class and any
wavelengths without having to match up a report dye with a
particular quencher. This may be so because some embodiments appear
not to rely on classic FRET-based fluorescence quenching to produce
an assay with a useable signal. Examples of suitable classes of
fluorescent dyes useful in the present invention include, but are
not limited to, coumarins, xanthene dyes, cyanines, pyrenes, styryl
dyes, BODIPY dyes, stilbenes and derivatives thereof; the widely
used rhodamines, fluoresceins and rhodols belong to the class of
xanthene dyes. Preferable dyes include neutral dye molecules and
dye molecules that have a delocalized positive or negative charge.
Neutral dyes are dyes that do not bear any charge or dyes that bear
an equal number of positive charges and negative charges, neutral
dyes of latter type are also referred to as zwiterionic dyes.
Examples of neutral dyes include, but are not limited to, BODIPY
dyes, rhodamines, zwiterionic cyanine dyes, and derivatives thereof
as shown in the following representative structures:
##STR00001##
wherein R is a reactive group.
[0132] Examples of dyes having a delocalized positive charge
include, but are not limited to, rosamines, cyanines, and
derivatives thereof as shown in the following representative
structures:
##STR00002##
wherein R is a reactive group.
[0133] Examples of dyes having a delocalized negative charge
include, but are not limited to, fluorescein and resorufin
derivatives as in the following representative structures:
##STR00003##
wherein R is a reactive group.
[0134] Alternatively, oligonucleotides of some embodiments are
labeled with a combination of negatively charges dyes and
positively charged dyes wherein the numbers of negatively charged
dyes and positively charged dyes are in proximately 1:1 ratio.
Examples of negatively charged dyes include fluorescein derivatives
and sulfonated dyes such as those described in U.S. Pat. Nos.
5,696,157; 6,130,101; 5,268,486; and 6,133,445, and in US patent
applications UA2002006479A1 and UA20020077487A1. Examples of
positively charged dyes include the abovementioned rosamine dyes
and cyanine dyes as well as dyes modified with a tertiary or
quaternary amines using standard chemistry.
[0135] In another embodiment of the present invention, suitable
dyes for synthesizing said oligonucleotides include energy transfer
dyes such as those described in U.S. Pat. Nos. 5,800,996;
6,479,303B1; and 6,545,164B1 as well as International Publication
WO 00/13026. Combinations of probes or primers labeled with
different energy transfer dyes and non-energy transfer dyes can be
used for multiplex detection in a single closed tube.
[0136] Suitable dyes include for use in various embodiments
include, but are not limited to, rhodamine xanthene dyes having the
following structure:
##STR00004## [0137] wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4
are independently H, F, Cl, C1-C18 alkyl or C2-C18 alkenyl groups;
R.sub.6 is H; R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are
independently H, C1-C18 alkyl groups, or C2 to C18 alkenyl groups
optionally substituted with a reactive group; said structure
further includes between 0 and 4 additional saturated or
unsaturated 5 or 6 membered rings selected from the group of rings
consisting of rings that include; R.sub.1 in combination with
R.sub.11, R.sub.2 in combination with R.sub.13, R.sub.3 in
combination with R.sub.10, and R.sub.4; each said additional ring
may be substituted with one or more lower alkyl groups; Q is
CO.sub.2.sup.-, or SO.sub.3.sup.-, or a reactive group; R.sub.7 and
R.sub.8 are independently H, F Cl, or a reactive group; and R.sub.6
and R.sub.9 are independently H, F, or Cl. Suitable reactive groups
for attaching these and other dyes and quenchers to
oligonucleotides include, but are not limited to electrophiles and
nucleophiles as listed in Table 2 and other groups listed in the
definitions section. Other means for attaching photometric
molecules and quenchers include aliphatic linker groups.
[0138] Still other dyes for use in various embodiments include but
are not limited to cyanine dyes with the following structure:
##STR00005##
wherein, R.sub.1 and R.sub.2 are independently selected from the
groups consisting of H, F, Cl, Br, CN, carboxylic acid group,
carboxamide, sulfonate, sulfonamide, lower alkyl groups, or at
least one additional fused aromatic rings, said additional fused
rings include atoms independently selected from the group
consisting of: C, N, O and S, a reactive group, and lower alkoxy
groups substituted with H or a reactive group; R.sub.3 and R.sub.4
are independently lower alkyl groups substituted with either H or a
reactive group; X and Y are independently selected from the group
consisting of: O, S, NR.sub.5 and CR.sub.6R.sub.7; R.sub.5,
R.sub.6, and R.sub.7 wherein R.sub.5, R.sub.6, and R.sub.7, R.sub.5
are independently H, C1 to C18 alkyl groups; and "bridge" is either
a methane or polymethine group. Reactive groups suitable for
attaching the molecule to an oligonucleotide include but are not
limited to the nucleophilic and electrophilic groups listed in
Table 2 and other groups listed in the definitions section. In
addition to various reactive groups, suitable dyes and quenching
molecules may also be attached to oligonucleotides by for example
aliphatic linkers.
[0139] Alternatively, suitable dyes are energy transfer dyes
wherein one of the dye pair is a rhodamine dye or cyanine dye.
EXAMPLES
Example 1
Oligonucleotide Synthesis and Labeling
Materials and Equipment
[0140] All anhydrous solvents and phosphoramidite reagents
including phosphoramidites of nucleosides and protected linkers
were purchased from Proligo, Boulder, Colo. or Glen Research,
Sterling, Va. All unlabeled and amine-modified oligonucleotides
were synthesized on an Expedite 8909 oligo synthesizer by Applied
Biosciences (Foster City, Calif.).
Synthesis of Unlabeled Oligonucleotides
[0141] All unlabeled oligonucleotides (primers) were synthesized by
starting with a protected nucleoside on CPG support with a glass
bead pore size of 500 .ANG.. Deprotection, coupling and oxidation
steps were all carried out by following standard protocols provided
by the manufacturers. Cleavage of oligonucleotides from CPG support
and deprotections were carried out by incubating the CPG beads in
ammonium hydroxide at 55.degree. C. for 16-18 hours. Once removed
from the solid support, the oligonucleotides were concentrated down
via a SpeedVac to remove the excess ammonia, and then purified by
passing the crude products through a Sephadex G-25 column or a C18
reverse phase cartridge. Final purifications, if necessary, were
carried out with HPLC (See Purification below).
Synthesis of Amine-modified Oligonucleotides
[0142] Amine-modified oligonucleotides were synthesized by using a
CPG-supported amino modifier and appropriate phosphoramidite
reagents containing a protected amine during the normal automated
oligo synthesis.
[0143] A variety of commercially available CPG-supported
amino-modifiers with different spacer arms can be used. These
products allow one to introduce an amino group at the 3' end. The
CPG-supported amino modifier, 3'-amino-modifier C7 CPG, was used in
some of the examples listed in Table 1 and it has the following
structure:
##STR00006##
(with L.sub.1 spacer) wherein, Fmoc and DMT are protection groups
for the amine and hydroxy groups, respectively, and "succinyl-lcaa"
is a spacer between the solid support and the modifier. The
base-labile Fmoc group was removed during ammonium treatment to
remove oligos from CPG support. The reagent introduces a 7-carbon
branched spacer between the amine group and the 3'-end phosphate.
For reference purpose, we refer to this spacer as L.sub.1. One
skilled in the art can appreciate that there are many other forms
of modifier reagents on a solid support that can be used to
introduce an amine with a different spacer, or to introduce a
different reactive group other than an amine.
[0144] Amine-containing phosphoramidite reagents include
phosphoramidites of protected amino-deoxynucleosides and protected
amino-modifiers. The most widely used and also least expensive
phosphoramidites of protected amino-deoxynucleosides is
phosphoramidite of trifluoroacetylamino-2'-deoxythymidine, or
amino-modifier C6 dT, which was used for making T-modified
oligonucleotides in some of the examples given Table 1. Shown below
is the structure of amino-modifier C6 dT:
##STR00007##
(with L.sub.2 spacer)
[0145] This reagent introduces a 10-atom linear aliphatic spacer
between dT and the amine group, or between dT and a dye. For
reference purpose, we refer to the 10-atom spacer as L.sub.2.
[0146] Alternatively, an amine can be introduced to the 5'-end by
using a phosphoramidite of a protected amine at the last step of
the automated synthesis. Two amino modifier reagents,
5'-amino-modifier C6-TFA and 5'-amino-modifer C12, were used for
synthesizing 5'-end labeled oligonucleotides shown in this
disclosure. The structures are shown below:
##STR00008##
[0147] For reference purpose, the linear 6-carbon spacer of
5'-amino-modifier C6-TFA is referred to as L.sub.3, and similarly
the linear 12-carbon spacer of 5'-amino-modifer C12 is referred to
as L.sub.4.
[0148] There are many other forms of modifier reagents that can be
used to introduce an amine with a different spacer, or to introduce
a different reactive group other than an amine.
Synthesis of Dye-Labeled Oligonucleotides
[0149] Labeling reactions were conducted by adding a solution of a
succinimidyl ester dye (Biotium, Inc., Hayward, Calif.) in DMF at
-40 mg/mL to an amino oligo dissolved in 0.1 M NaHCO.sub.3 (pH 8.5)
at -1 mg/ml and vortexing the solution at room temperature for
.about.2 h. The molar ratio of dye NHS ester to each amino group in
the oligo was about 20-40 to 1. Unreacted dye was effectively
removed by a Sephadex G-25 spin column. The crude products thus
obtained were subject to further purification by HPLC (See
below).
Purification of Labeled Oligonucleotides
[0150] Labeled oligonucleotides were purified by reverse phase HPLC
on a Hitachi D7000 HPLC System.
Typical HPLC condition:
Column: C18 YMC ODS-A 5 um 12 nm 150.times.4.6 mm, or C18 Microsorb
5 um 30 nm 200.times.4.6.
[0151] Column temperature: 45.degree. C. Gradient: 10%B to 50% B in
20 min-30 min @ 1 ml/min. A: 100 mM TEAA PH7.0; B: 100%
CH.sub.3CN.
Determination of Degree of Labeling
[0152] The absorbance from 230 nm to 700 nm of purified dye labeled
oligonucleotides was measured on a spectrophotometer, whereby
A.sub.max for the dye (A.sub.max) and A.sub.260 were determined.
The concentration of the dye was determined by measuring A.sub.max
values, while the oligonucleotide concentration was calculated
based on A.sub.260 after factoring in the absorbance of the dye at
260 nm. The ratio of dye to oligo concentrations defines the degree
of labeling (DOL). In our experiments, DOL for single label is
close to one (e.g. FIGS. 4B & 4C) and that for doubly labeling
is close to two (e.g. FIG. 4A). Based on this calculation we
conclude that the doubly or singly labeled probes or primers
detailed in current invention are generally over 90 to 95%
pure.
Example 2
Monitoring of MCG Gene Amplification Using Doubly Labeled
Probes
[0153] The first set of experiment in this example demonstrates the
use of a doubly 6-ROX-labeled probe in RT-PCR detection of a MCG
gene. The amplifications were performed in 20 .mu.l reaction
solution containing 10 mM Tris (pH 8.0), 50 mM KCl, 3.5 mM
MgCl.sub.2, 2 mM each of dNTP, and 1 unit of AmpliTaq Gold (ABI,
Foster City, Calif.). A MCG gene fragment (SEQ ID 25) in pTOPO
plasmid was amplified with 0.5 .mu.M forward primer
5'-TCAAGAGGTGCCACGTCTCC-3' (SEQ ID No. 4), 0.5 .mu.M reverse primer
6-CTGATCTGTCTCAGGACTCTGACACTGT-3' (SEQ ID No. 5). A doubly
6-ROX-labeled MCG probe, 5'-(6-ROX-L.sub.3-CAGCACAACT ACGCAGCGCC
TCC(-L.sub.1-6-ROX)-3' (SEQ ID No. 6, see Table 1) was used for
following the reaction. The thermal regimen was set at 95.degree.
C. for 7 minutes followed by 50 cycles of 15-second duration at
95.degree. C. and 20 second duration at 60.degree. C. Fluorescence
was measured at the 60.degree. C. step. A series of 10-fold
dilutions of the template was made to create titration curves of
the amplification plot. FIG. 2A shows amplification plots of
aforementioned reactions starting with 10.sup.7 copies of template
(Trace 1) down to 10.sup.1 copies of template (Trace 7). Two NTC
(no template control, Traces 8 and 9) are also shown in the figure.
The insert shows that the Ct value is reversibly correlated with
the logarithm of starting copy number (Trace 10).
[0154] In the second set of experiments the probe (SEQ ID No 36)
was doubly labeled with non-sulfonated cyanine dyes and the
experiment was carried out at two template concentrations, 100,000
copies (Trace 151) and 0 copy (Trace 152). All other reagents and
conditions were the same as in the first set of experiments. This
set demonstrates the use of a doubly cyanine-labeled probe in
RT-PCR detection of the MCG gene.
Example 3
Monitoring of MCG and GAPDH Gene Amplification Using a Probe Doubly
Labeled with 6-CR110 from Complex Templates
[0155] Amplifications of MCG gene fragment from human genomic DNA
were performed as in Example 2 except (1) a 6-CR110 probe,
5'-(6CR110-L.sub.3-) CAGCACAACT ACGCAGCGCC TCC(-L.sub.1-6-CR110)-3'
(SEQ ID No. 7, see Table 1) and (2) a series of 10-fold dilutions
of human DNA were used. FIG. 2B shows amplification plots of the
reactions starting with 10.sup.5 copies of human DNA (Trace 11)
down to 10.sup.1 copies of human DNA (Trace 15). A NTC (Traces 16)
is also shown in the figure. The insert shows that the Ct value is
reversibly correlated with the logarithm of starting copy number
(Trace 17).
[0156] Another titration (FIG. 2C) using cDNA as template was
carried out as above except that GAPDH primers,
5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID No. 1) and
5'-GAAGATGGTGATGGGATTTTC-3'(SEQ ID No. 2), and a GAPDH probe,
5'-(6CR110-L.sub.3-)CAAGCTTCCCGTTCTCAGC(-L.sub.1-6-CR110)-3' (SEQ
ID No. 21) were used. Starting with 0.2 .mu.l of human brain cDNA
(Invitrogen, Carlsbad, Calif.), a series of 2-fold dilutions were
made. All PCR reactions were carried out in 10 .mu.l volume. The
thermal regimen was 95.degree. C. (7-minutes), 45 cycles of
95.degree. C. (15-second) and 56.degree. C. (20-seconds).
Example 4
Comparison of a TaqMan Probe with a Doubly Labeled Probe of the
Present Invention
[0157] A GAPDH gene fragment was amplified from a pTOPO plasmid
containing GAPDH gene fragment (SEQ ID No. 24, Table 1) with a
forward primer, 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID No. 1, Table 1)
and a reverse primer, 5'-GAAGATGGTGATGGGATTTTC-3'(SEQ ID No. 2,
Table 1). A TagMan probe with JOE as the reporter dye and TAMRA as
the quencher (5'-(6-JOE-L.sub.3-)
CAAGCTTCCCGTTCTCAGC(-L.sub.1-6-TAMRA)-3'; SEQ ID No. 8, See Table
1) or a probe according to the present invention doubly labeled
with 5-R6G
(5'-(5-R6G-L.sub.3-)CAAGCTTCCCGTTCTCAGC(-L.sub.1-5-R6G)-3'; SEQ ID
No. 9, See Table 1) was used for monitoring the reaction under
reaction condition identical to that used in Example 2 except that
the annealing/extension temperature was lowered to 56.degree.
C.
[0158] All amplification reactions were carried out with 1 million
copies of the template, and concentrations of 125 nM, 250 nM and
500 nM were used for each probe, respectively. As shown in FIG. 3A,
the signal strength of the probe made according to the present
invention is twice as strong as that of the corresponding TaqMan
probe (Trace 18 vs 21, Trace 19 vs 22 and Trace 20 vs. 23). R6G and
JOE have comparable spectra as well as similar fluorescence quantum
yield and extinction coefficient. Therefore, the observed
performance difference between the two probes is not due to the
dyes themselves but a reflection of the superior design of the
probe according to the present invention.
[0159] A FAM labeled MCG TaqMan probe with FAM at the 5' end and
TAMRA at 3' end was made and compared with doubly labeled CR110
probe (5'-(6CR110-L.sub.3-) CAGCACAACT ACGCAGCGCC TCC
(-L.sub.1-6CR110)-3' (SEQ ID No. 7) with identical sequence in PCR.
FIG. 3B shows the amplification plots of the said homo-doubly
labeled CR110 probe with TaqMan probe of 1000 nM (Trace 115 vs
Trace 119), 500 nM (Trace 116 vs Trace 120), 250 nM (Trace 117 vs
Trace 121) and 125 nM (Trace 118 vs Trace 122) respectively. All
amplifications start with one million copies of a plasmid
containing MCG fragment. At saturated concentration (1000 nM),
homo-doubly labeled probe outperformed said TaqMan probe in signal
strength by 60%. Doubly labeled CR110 probe uses one half of TaqMan
probe in concentration to get the same signal strength.
[0160] A FAM labeled cMyc TaqMan probe was currently provided by
ABI in a 20.times. mixture of primer and probe. The probe has
undisclosed sequence and comprised of a MGB at its 3' end. A
comparison was made between said TaqMan probe with doubly labeled
CR110 probe (5'-(6CR110-L.sub.3-) CAGCACAACT ACGCAGCGCC
TCC(-L.sub.1-6CR110)-3' (SEQ ID No. 7). FIG. 3C shows the
amplification plots of the said TaqMan probe (Trace 110) and
homo-doubly labeled CR110 probe of 1000 nM (Trace 111), 500 nM
(Trace 112), 250 nM (Trace 113) and 125 nM (Trace 114). All
amplifications used identical concentrations of human brain cDNA
from Invitrogen. At saturated concentration (1000 nM), homo-doubly
labeled probe outperformed said TaqMan probe in signal strength.
Incorporation of MGB may further improve the performance of the
probes in current invention.
Example 5
UV/Vis Absorption Spectra of Doubly Dye-labeled and Singly
Dye-labeled Oligonucleotides and S1 Nuclease Digested
Oligonucleotides Thereof
[0161] The purpose of this experiment is to demonstrate that there
is no physical touching between dyes in an oligonucleotide labeled
with two reporter dyes according to the present invention. In order
to demonstrate that the dyes need not touch one another we
synthesized three CR110-labeled oligonucleotides of the same
sequence: 1) a GAPDH probe doubly labeled at 3' and 5' (SEQ ID No.
3, Table 1); 2) a GAPDH probe sequence singly labeled at the 5'
(SEQ ID No. 7, Table 1); and 3) a GAPDH probe sequence singly
labeled at 3' (SEQ ID No. 8, Table 1). The two singly labeled
oligonucleotides were made to serve as controls.
[0162] The spectra of the three labeled oligonucleotides in S1
buffer are shown in FIGS. 4A (trace 29), 4B (trace 31) and 4C
(trace 33), respectively. To assess how the spectrum of the dye is
also affected by the microenvironment surrounding the dye, the
labeled oligonucleotides were digested by S1 and then spectra were
taken (FIGS. 4A (Trace 28), 4B (Trace 30) and 4C (Trace 32)).
Digestions were carried out by adding 20 units of S1 nuclease
(Promega, Madison, Wis.) to a 100 .mu.l reaction solution
containing 250 to 500 nM probes in S1 buffer (50 mM sodium acetate
(pH 4.5), 280 mM NaCl, and 4.5 mM ZnSO.sub.4). S1 digestion was
completed almost instantaneously after the S1 addition, as
confirmed by following the UVN is absorption spectrum change.
However, spectra were taken after 1-hour incubation at 37.degree.
C. to ensure complete digestion.
[0163] As the figures show, all three labeled oligonucleotides have
nearly identical absorption spectra in the visible wavelength
range, indicating that the doubly labeled probe does not have dye
aggregation, which is typically characterized by a significant
alteration in the shape or profile of the absorption spectrum (See
Example 7). The fact that the shape of the doubly labeled probe
(FIG. 4A, Trace 29) and that of the digested probe (FIG. 4A, Trace
28) are similar is further evidence for the lack of dye dimer
formation. The slight overall wavelength shift from Trace 29 to
Trace 28, as opposed to a change in the shape of the absorption
peak, is due to a difference in the microenvironment that surrounds
the dye, similar to solvent effect. As one would expect, this
"solvent effect" is similar for all three labeled oligonucleotides
(Trace 28 vs. Trace 29 in FIG. 4A; Trace 30 vs. Trace 31 in FIG.
4B; and Trace 32 vs. Trace 33 in FIG. 4C).
Example 6
Amplification of GAPDH Monitored with a Homo-Doubly Labeled Probe
and Two Singly Labeled Control Oligonucleotides
[0164] The purpose of this experiment is to demonstrate that the
superior performance of the probes according to the present
invention is not due to any uniqueness of the dyes employed but the
novel design of the probes. We synthesized three labeled
oligonucleotides all having the same GAPDH probe sequence: 1) GAPDH
oligo doubly labeled with 5-CR110 at 3' and 5' ends (SEQ ID No. 3,
Table 1); 2) GAPDH oligo singly labeled with 5-CR110 at 5' end
(SEQ. ID No. 10, Table 1); and 3) GAPDH oligo singly labeled with
5-CR110 at 3' end (SEQ ID. No. 11, Table 1). The labeled
oligonucleotides were then tested for their utilities as potential
probes for the amplification of a GAPDH gene fragment using
condition identical to that in Example 3.
[0165] FIG. 4D shows the kinetic profiles of GAPDH gene
amplification using the 3 labeled oligonucleotides as potential
probes. The doubly labeled probe gave a typical kinetic profile
(Trace 34 in FIG. 4D), whereas under the same amplification
condition the two singly labeled oligonucleotides failed to respond
to the amplification kinetics (Traces 35 and 36 in FIG. 4D).
Successful gene amplifications for all three reactions were
confirmed by agarose gel electrophoresis using ethidium bromide as
the stain. Therefore, the lack of response from the singly labeled
oligonucleotides is not caused by the absence of amplification
products. These results suggest that the superior performance of
the probes made according to various embodiments of the present
invention are a result of the novel design of the probes. The
improvements are not caused by the unique nature of the dyes or the
manner at which an individual dye is attached to the
oligonucleotides, or by the interaction between the dye and
oligonucleotide.
Example 7
Spectral Comparison of Homo-Doubly Labeled Probes According to the
Present Invention with Similar Molecular Beacon Probes
[0166] Here we compare the absorption spectra of probes doubly
labeled with a reporter dye according to the present invention with
probes having a physically touching dye pair according to prior
art. The purpose is to further demonstrate that unlike probes of
prior art, doubly labeled probes of the present invention do not
form dye aggregates.
[0167] A GAPDH stem-loop sequence having an amine group at the 3'
and 6' ends respectively,
5'-(Am-L.sub.3-)CCAAGCGGCTGAGAACGGGAAGCTTGGCTTGG(-L.sub.1-Am)-3'
was synthesized, where the underlined nucleotides indicate the
stem-forming sequences. This amine-modified sequence was then used
to synthesize three homo-doubly labeled molecular beacon probes by
reacting with the succinimidyl esters of 6-CR110 (SEQ ID No 12,
Table 1)), 6-TAMRA (SEQ ID No 13, Table 1) and 6-ROX (SEQ ID No 14,
Table 1), respectively. Similarly, three corresponding homo-doubly
labeled probes according to the present invention were made by
reacting a double amine-modified sequence of (Am-L.sub.3-)
CAAGCTTCCC GTTCTCAGC(-L.sub.1-Am) with the succinimidyl esters of
6-CR110 ((SEQ ID No 21, Table 1), 6-TAMRA (SEQ ID No 22, Table 1)
and 6-ROX (SEQ ID No 23, Table 1), respectively. The spectra of
aforementioned stem-loop probes and their counterparts according to
the present invention were measured at -0.5 .mu.M in 10 mM Tris
buffer (pH 8.0) at 25.degree. C. on a Shimadzu 1201 UVN is
spectrophotometer. For easy comparison, spectra for each pair of a
beacon probe and the corresponding probe of this invention were
shown in FIGS. 5A, 5B and 5C, respectively. All homo-doubly labeled
beacon probes showed a shorter wavelength shoulder peak, which
indicates dye dimer formation (Blackman et al., 2002, Biochemistry;
Packard et al., 1996, Proc. Natl. Acad. Sci.). On the other hand,
probes of the present invention had spectra similar to those of
singly labeled oligonucleotides or digested labeled
oligonucleotides (See example 5)
Example 8
Signal Strength Comparison of Doubly Labeled Probes According to
the Present Invention with Corresponding Homo-Doubly Labeled Beacon
Probes
[0168] This experiment demonstrates that homo-doubly labeled probes
according to the present invention are several times more sensitive
than the corresponding doubly labeled beacon probes.
[0169] A GAPDH gene fragment was amplified from a pTOPO plasmid
containing GAPDH gene fragment (SEQ ID 24) using primers and
conditions identical to that in Example 4. Each of the six probes
from Example 7 (three homo-doubly labeled probes of this invention
and the three corresponding beacon probes) was used to follow the
amplification reaction at four different probe concentrations, 125
nM, 250 nM, 500 nM and 1 mM, respectively. Amplifications were
performed in extra cycles to ensure all reactions are complete. In
addition, gel-electrophoresis revealed that equal amount of
amplified PCR products were formed for all three reactions. The
kinetic profiles for each pair of a homo-doubly labeled probe of
this invention and the related beacon probe are shown in FIGS. 6A,
6B and 6C respectively.
[0170] The data in FIGS. 6A, 6B and 6C clearly show that probes
according to this invention are several fold more sensitive than
the corresponding beacon probes when used at the same
concentration. Also shown in the figures is that the beacon probes
did not become fully saturated even at 1 mM concentration while the
related probes of this invention displayed saturation at or near
250 nM. This delayed saturation is due to the equilibrium between
the open and dosed beacon conformations that makes only a fraction
of the total amount of the probe available for hybridization with
the target sequence at a given time (FIG. 7).
Example 9
Probes Labeled with a Mixture of FAM and CR110
[0171] This experiment demonstrates that oligonucleotides of the
present invention can be labeled with a mixture of reporter
dyes.
[0172] To synthesize a probe labeled with a single 6-FAM and a
single 6-CR110, a double amine-modified GAPDH probe sequence of
5'-(Am-L.sub.3-)CAAGCTTCCC GTTCTCAGC(-L.sub.1-Am)-3' was reacted
with a 1:1 mixture of 6-FAM SE and 6-CR110SE. The labeling reaction
produced four doubly labeled products: 1)
5'-(6-FAM-L.sub.3-)CAAGCTTCCC GTTCTCAGC(-L.sub.1-6-FAM)-3'; 2)
5'-(6-FAM-L.sub.3-) CAAGCTTCCC GTTCTCAGC(-L.sub.1-6-CR110)-3'; 3)
5'-(6-CR110-L.sub.3-) CAAGCTTCC GTTCTCAGC(-L.sub.1-6-FAM)-3'; 4)
5'-(6-CR110-L.sub.3-) CAAGCTTCCC GTTCTCAGC(-L.sub.1-6-CR110)-3'.
Products were purified by C18 RP HPLC and peaks corresponding to
products 1) and 4) were identified by comparing the HPLC retention
times of individually prepared products. Peaks that had retention
times between those of product 1) and product 4) were assigned to
those of the two hetero-doubly labeled probes, and fractions were
collected and analyzed by UVN is spectroscopy. FIG. 8A shows the
UV/Vis spectra of product 1) (Trace 80), products 2) and 3) (Trace
81) and product 4) (Trace 82). The spectrum of the hetero-doubly
labeled probes (products 2) and 3) falls in between those of the
two homo-doubly labeled probes as one would have expected. The
isolated probes were then used as probes for the amplification of
the GAPDH gene under condition identical to that used in Example 4.
FIG. 8B shows the kinetic profiles of GAPDH gene amplification
using the isolated probes.
[0173] Alternatively, a hetero-doubly labeled oligonucleotide can
be made via the traditional method for synthesizing FRET-based
probes or primers by attaching the dyes in separate steps. However,
the synthesis procedure described in this example may serve as a
rapid way of screening for optimal dye pairs that may yield the
best performance of the labeled oligonucleotides.
Example 10
Homo-doubly Labeled Primers
[0174] These experiments demonstrate the use of oligonucleotides
according to the present invention as fluorogenic primers for
RT-PCR monitoring.
[0175] In a first experiment, a fluorogenic forward primer
(5'-(5-CR110-L.sub.3-) GAAGGTGAAGGTCGGAGT (-L.sub.2-5-CR110)C-3',
SEQ No. 15, Table 1) for a GAPDH gene amplification was synthesized
by reacting 5-CR110SE with a diamine-modified primer
5'-(Am-L.sub.3-)GAAGGTGAAGGTCGGAGT(-L.sub.2-Am)C-3', wherein one
amine is attached to the 5' phosphate via a C6 aliphatic linker and
another amine attached to the base of No. 18 deoxynucleotide dT via
a 10-atom aliphatic linker (See Example 1 for synthesis details).
Amplification of the GAPDH gene was carried out using conditions
identical to those used in Example 4 except that: 1) no probe was
used; 2) the forward primer was replaced with the above homo-doubly
labeled fluorogenic primer; and 3) three template copy numbers were
used: one million, one thousand and zero (control). FIG. 9A shows
the amplification profiles, where Traces 87, 88, and 89 represent
one million, one thousand copies of templates and NTC,
respectively.
[0176] In a second experiment, we synthesized another doubly
labeled fluorogenic primer identical to the above forward
fluorogenic primer except that the G at the very 5' end is omitted,
(5'-(5-CR110-L.sub.3-) AAGGTGAAGGTCGGAGT(-L.sub.2-5-CR110)C-3', SEQ
ID No 16, table 1). Similarly, PCR reactions were carried out using
the same three template copy numbers as in the first experiment.
FIG. 9B shows the amplification profiles, where Traces 90, 91, and
92 represent one million, one thousand and zero copies of
templates, respectively. The result indicates that the successful
application of the homo-doubly labeled primers according to the
present invention is not due to G-nucleotide-associated
fluorescence quenching/de-quenching, the working mechanism of the
LUX primers (Nazarenko et al., 2002, Nucleic Acid Research)
[0177] In a third experiment, we synthesized a homo-doubly labeled
reverse primer 5'-(5-CR110-L.sub.3-)GAAGATGGTGATGGGATT(-L.sub.3
5-CR110)TC-3' (SEQ No 17, Table 1) by reacting 5-CR110SE with a
diamine-modified reverse primer
5'-(Am-L.sub.2-)GAAGATGGTGATGGGATT(-L.sub.2Am)TC-3', wherein one
amine is attached to the 5' phosphate via a C6 aliphatic linker and
another amine attached to the base of No. 18 nucleotide dT via a
10-atom aliphatic linker. Similarly, the GAPDH gene was amplified
using condition identical to that used in the first experiment
except that 1) a regular forward primer was used and 2) the above
homo-doubly labeled reverse primer was used. FIG. 9C shows the
amplification profiles, where Traces 93, 94, and 95 represent one
million, one thousand and zero copies of templates, respectively.
The data from the first and second experiments indicates that
either a forward primer or a reverse primer can be fluorogenically
labeled according to the present invention for nucleic acid
detection.
[0178] To exclude the possibility that the fluorescence
quenching/dequenching of the homo-doubly labeled primers was caused
by a difference in the interaction between the dye and the
oligonucleotide before and after hybridization with the target, we
synthesized two control primers, one with a single 5' end label
(5'-(5-CR110-L.sub.3-)GAAGGTGAAG GTCGGAGTC-3', SEQ No 18, Table 1),
and another with a single 5-CR110 attached to the base of No. 17 dT
via a 10-atom linker (5'-(AAGGTGAAGG
TCGGAGT(-L.sub.2-5-CR110)C)-3', SEQ No 19, Table 1). As FIGS. 9D
and 9E show, neither the 5'-end labeled primer (Trace 97 in FIG.
9D) nor the dT-labeled primer (Trace 99 in FIG. 9E) responded to
the PCR reaction, although gel electrophoresis of the end products
revealed that both PCR reaction proceeded normally.
[0179] In still another control experiment, we synthesized a singly
labeled forward primer with the dye 5-CR110 attached to the No. 18
dT nucleotide via a 10-atom flexible linker
(5'-GAAGGTGAAGGTCGGAGT(-L.sub.2-5-CR110)C-3', SEQ No 20, Table 1).
This primer did respond positively to the amplification reactions
as it was incorporated into PCR product, similar to the homo-doubly
labeled primers in experiments 1) and 3). A possible explanation
for this observation is that the dG nucleotide at the very 5' end
may loop over to the 3' end to quench the fluorophore as in the
case of LUX primers (FIG. 9F). To test this hypothesis, we
synthesized a singly labeled forward primer with the 5'-end dG
removed (5'-AAGGTGAAGGTCGGAGT(-L.sub.2-5-CR110)C-3', SEQ No 19,
Table 1). As shown in FIG. 9E, this primer failed to respond to the
amplification reaction. This result, along with the result from the
second experiment in this example, indicates that in the absence of
nucleotide G-associated fluorescence quenching/de-quenching
fluorogenic oligonucleotides of the present invention require at
least two reporter dyes.
Example 11
SNP Typing with a Pair of Homo-Doubly Labeled Probes
[0180] Tapp et al have shown SNP typing by using a pair of TaqMan
probes, each labeled with FAM and TET respectively for C to T
transition of the estrogen receptor gene in codon 10. This
experiment demonstrates a pair of AllGlo probes, labeled with CR110
or R6G, in replacement of FAM and TET respectively work equally
well for this purpose.
SNP typing reactions were carried out in 20 .mu.l reactions
containing 10 mM Tris (pH 8.0), 50 mM KCl, 3.5 mM MgCl.sub.2, 2 mM
each of dNTP, 1 unit of AmpliTaq Gold (ABI, Foster City, Calif.),
0.5 .mu.M forward primer 5'-CCACGGACCATGACCATGA-3' (SEQ ID No. 26),
0.5 .mu.M reverse primer 5'-TCTTGAGCTGCGGACGGT-3' (SEQ ID No. 27),
0.2 .mu.M ERcodon10C probe,
5'-(6-CR110-L.sub.4-CCAAAGCATCCGGGATGGCC(-L.sub.1-6-CR110)-3' (SEQ
ID No. 28), 2 .mu.M ERcodon10T probe,
5'-(5-R6G-L.sub.3-CCAAAGCATCTGGGATGGCC (-L.sub.1-5-R6G)-3' (SEQ ID
No. 29), and model plasmid DNA to be typed. The reaction profile
was set at 95.degree. C. for 7-minutes followed by 50 cycles of 15
second at 95.degree. C. and 20 second at 60.degree. C. Fluorescence
was measured at the 60.degree. C. step simultaneously from both FAM
and TET channels. The homozygote CC model genotype consists of
10.sup.6 copies of a plasmid pER(C), a pTOPO plasmid containing an
106 by insert flanking the codon10 of estrogen receptor gene, where
the SNP is C (SEQ ID No. 30); the homozygote TT model genotype
consists of 10.sup.8 copies of a plasmid pER(T), a pTOPO plasmid
containing an 106 by insert flanking the codon10 of estrogen
receptor gene, where the SNP is T (SEQ ID No. 31); the hoterozygote
CT genotype consist of 0.5.times.10.sup.5 copies of pER(C) and
0.5.times.10.sup.5 copies of pER(C). The three genotypes exhibited
three distinct amplification profile patterns, these patterns are a
follows: Homozygote CC had a high CR110 signal (Trace 131, FIG.
10A) and very low R6G signal (Trace 132, FIG. 10A); Homozygote TT
has low CR110 signal (Trace 135, FIG. 10C) and high R6G signal
(Trace 136, FIG. 10C); Heterozygote CT has mid-level CR110 signal
(Trace 133, FIG. 10B) and mid-level R6G signal (Trace 134, FIG.
10B).
Example 12
Amplification Using an Exo.sup.- DNA Polymerase
[0181] This experiment demonstrates the use of an exo.sup.- DNA
polymerase in real time PCR where the fluorescent signal was
monitored by hybridization instead of cleavage of the probes. The
amplifications were performed in 20 .mu.l reaction solution
containing premixed buffer and Titanium Taq (BD Biosciences,
Mountain View, Calif.). A HCV gene fragment (SEQ ID 32) in pTOPO
plasmid was amplified with 2 .mu.M forward primer
5'-GCACGAATCCTAAACCTCAAAA-3' (SEQ ID No. 33), 0.2 .mu.M reverse
primer 5'-GGCAACAAGTAAACTCCACCAA-3' (SEQ ID No. 34). A doubly
6-ROX-labeled HCV probe,
5'-(6-ROX-L.sub.3-ATCTGACCACCGCCCGGGAAC-(-L.sub.1-6-ROX)-3' (SEQ ID
No. 35) at final 0.5 .mu.M was used for each of the reactions. The
thermal regimen was set at 95.degree. C. for 2 minutes followed by
50 cycles of 15-second duration at 95.degree. C., 20-second
duration at 60.degree. C. and 5 second duration at 72.degree. C.
Fluorescence was measured at the 60.degree. C. step. A series of
10-fold dilutions of the template was made to create titration
curves of the amplification plot. FIG. 12 shows amplification plots
of aforementioned reactions starting with 10.sup.6 copies of
template (Trace 140) down to 1 copy of the template (Trace 146). An
NTC (no template control, Traces 148) is also shown in the figure.
The inset shows that the Ct value is reversibly correlated with the
logarithm of starting copy number (Trace 148).
[0182] All publications, patents, and patent applications cited in
this specification are incorporated herein by reference as if each
individual publication, patent, or patent application was
specifically and individually indicated to be incorporated by
reference and set forth in its entirety herein.
[0183] Unless specifically identified to the contrary, all terms
used herein are used to include their normal and customary
terminology. Further, while various embodiments of diagnostic tests
and medical treatment devices having specific components and steps
are described and illustrated herein, it is to be understood that
any selected embodiment can include one or more of the specific
components and/or steps described for another embodiment where
possible.
[0184] Further, any theory of operation, proof, or finding stated
herein is meant to further enhance understanding of the present
invention and is not intended to make the scope of the present
invention dependent upon such theory, proof, or finding.
[0185] While the invention has been illustrated and described in
detail in the drawings and foregoing description, the same is to be
considered as illustrative and not restrictive in character, it
being understood that only the preferred embodiments have been
shown and described and that all changes and modifications that
come within the spirit of the invention are desired to be
protected. And while the invention was illustrated using specific
examples, and theoretical arguments, protein and DNA sequences,
accounts and illustrations these examples, arguments, illustrations
sequences, accounts and the accompanying discussion should by no
means be interpreted as limiting the invention. The Abstract of the
Disclosure is included for the convenience of the persons searching
for the document; the Abstract is not a summary of the invention it
should not be used to interpret or to limit the claims or
specification.
Sequence CWU 1
1
36119DNAArtificial Sequenceprimer 1gaaggtgaag gtcggagtc
19220DNAArtificial Sequenceprimer 2gaagatggtg atgggatttc
20319DNAArtificial Sequenceprobe 5CR 110 labeled at 5'- and 3'-
terminal amines 3caagcttccc gttctcagc 19420DNAArtificial
Sequenceprimer 4tcaagaggtg ccacgtctcc 20528DNAArtificial
Sequenceprimer 5ctgatctgtc tcaggactct gacactgt 28623DNAArtificial
Sequenceprobe 6ROX labeled at 5'- and 3'- terminal amines
6cagcacaact acgcagcgcc tcc 23723DNAArtificial Sequenceprobe 6CR110
labeled at 5'- and 3'- terminal amine 7cagcacaact acgcagcgcc tcc
23819DNAArtificial Sequenceprobe JOE labeled at 5'- terminal amine
and TAMRA at at 3'- terminal amine 8caagcttccc gttctcagc
19919DNAArtificial Sequenceprobe 5R6G labeled at 5'- and 3'-
terminal amine 9caagcttccc gttctcagc 191019DNAArtificial
Sequenceprobe 5CR110 labeled at 5'- terminal amines 10caagcttccc
gttctcagc 191119DNAArtificial Sequenceprobe 5CR110 labeled at 3'-
terminal amines 11caagcttccc gttctcagc 191232DNAArtificial
sequenceprobe 6CR110 labeled at 5'- and 3'- terminal amines
12ccaagcggct gagaacggga agcttggctt gg 321332DNAArtificial
sequenceprobe 6TAMRA labeled at 5'- and 3'- terminal amines
13ccaagcggct gagaacggga agcttggctt gg 321432DNAArtificial
Sequenceprobe 6ROX labeled at 5'- and 3'- terminal amines
14ccaagcggct gagaacggga agcttggctt gg 321519DNAArtificial
Sequenceprimer 5CR110 Labeled at 5'-end amine and #18 dT
15gaaggtgaag gtcggagtc 191618DNAArtificial Sequenceprimer 5CR110
Labeled at 5'- terminal amine and #17 base of dT 16aaggtgaagg
tcggagtc 181720DNAArtificial Sequenceprimer 5CR110 labeled at 5'-
terminal amine and #17 base of dT 17gaagatggtg atgggatttc
201819DNAArtificial Sequenceprimer 5CR110 labeled at 5'- terminal
amine 18gaaggtgaag gtcggagtc 191918DNAArtificial Sequenceprimer
5CR110 labeled at #17 dT 19aaggtgaagg tcggagtc 182019DNAArtificial
Sequenceprimer 5CR110 Labeled at #18 dT 20gaaggtgaag gtcggagtc
192119DNAArtificial Sequenceprobe 6CR110 labeled at 5'- and 3'-
terminal amines 21caagcttccc gttctcagc 192219DNAArtificial
Sequenceprobe 6TAMRA labeled at 5'- and 3'- terminal amines
22caagcttccc gttctcagc 192319DNAArtificial Sequenceprobe 6ROX
labeled at 5'- and 3'- terminal amines 23caagcttccc gttctcagc
1924226DNAHomo sapiens 24gaaggtgaag gtcggagtca acggatttgg
tcgtattggg cgcctggtca ccagggctgc 60ttttaactct ggtaaagtgg atattgttgc
catcaatgac cccttcattg acctcaacta 120catggtttac atgttccaat
atgattccac ccatggcaaa ttccatggca ccgtcaaggc 180tgagaacggg
aagcttgtca tcaatggaaa tcccatcacc atcttc 22625121DNAHomo sapiens
25tcaagaggtg ccacgtctcc acacatcagc acaactacgc agcgcctccc tccactcgga
60aggactatcc tgctgccaag agggtcaagt tggacagtgt cagagtcctg agacagatca
120g 1212619DNAArtificial Sequenceprimer 26ccacggacca tgaccatga
192718DNAArtificial Sequenceprimer 27tcttgagctg cggacggt
182820DNAArtificial Sequenceprobe 6CR110 labeled at 5'- and 3'-
terminal amines 28ccaaagcatc cgggatggcc 202920DNAArtificial
Sequenceprobe 5-R6G labeled at 5'- and 3'- terminal amines
29ccaaagcatc tgggatggcc 2030106DNAHomo sapiens 30ccacggacca
tgaccatgac cctccacacc aaagcatccg ggatggccct actgcatcag 60atccaaggga
acgagctgga gcccctgaac cgtccgcagc tcaaga 10631106DNAHomo sapiens
31ccacggacca tgaccatgac cctccacacc aaagcatctg ggatggccct actgcatcag
60atccaaggga acgagctgga gcccctgaac cgtccgcagc tcaaga
10632109DNAHepatitus C virus 32gcacgaatcc taaacctcaa agaaaaacca
aacgtaacac caaccgccgc ccacaggacg 60tcaagttccc gggcggtggt cagatcgttg
gtggagttta cctgttgcc 1093322DNAArtificial Sequenceprimer
33gcacgaatcc taaacctcaa aa 223422DNAArtificial Sequenceprimer
34ggcaacaagt aaactccacc aa 223521DNAArtificial Sequenceprobe 6ROX
labeled at 5'- and 3'- terminal amines 35atctgaccac cgcccgggaa c
213623DNAArtificial Sequenceprobe non-sulfonated Cy-5 labeled at
5'- and 3'- terminal amines 36cagcacaact acgcagcgcc tcc 23
* * * * *