U.S. patent application number 11/970445 was filed with the patent office on 2010-09-09 for oligonucleotides containing pyrazolo[3,4-d] pyrimidines for hybridization and mismatch discrimination.
Invention is credited to Irina A. Afonina, Igor V. Kutyavin, Rich B. Meyer, JR..
Application Number | 20100228017 11/970445 |
Document ID | / |
Family ID | 21993802 |
Filed Date | 2010-09-09 |
United States Patent
Application |
20100228017 |
Kind Code |
A1 |
Meyer, JR.; Rich B. ; et
al. |
September 9, 2010 |
OLIGONUCLEOTIDES CONTAINING PYRAZOLO[3,4-D] PYRIMIDINES FOR
HYBRIDIZATION AND MISMATCH DISCRIMINATION
Abstract
Oligonucleotides in which one or more purine residues are
substituted by pyrazolo[3,4-d]pyrimidines exhibit improved
hybridization properties. Oligonucleotides containing
pyrazolo[3,4-d]pyrimidine base analogues have higher melting
temperatures than unsubstituted oligonucleotides of identical
sequence. Thus, in assays involving hybridization of an
oligonucleotide probe to a target polynucleotide sequence, higher
signals are obtained. In addition, mismatch discrimination is
enhanced when pyrazolo[3,4-d]pyrimidine-containing oligonucleotides
are used as hybridization probes, making them useful as probes and
primers for hybridization, amplification and sequencing procedures,
particularly those in which single- or multiple-nucleotide mismatch
discrimination is required.
Inventors: |
Meyer, JR.; Rich B.;
(Bothell, WA) ; Afonina; Irina A.; (Mill Creek,
WA) ; Kutyavin; Igor V.; (Bothell, WA) |
Correspondence
Address: |
JACKSON WALKER LLP
901 MAIN STREET, SUITE 6000
DALLAS
TX
75202-3797
US
|
Family ID: |
21993802 |
Appl. No.: |
11/970445 |
Filed: |
January 7, 2008 |
Related U.S. Patent Documents
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Patent Number |
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10302608 |
Nov 22, 2002 |
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11970445 |
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09431385 |
Nov 1, 1999 |
6485906 |
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10302608 |
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09054830 |
Apr 3, 1998 |
6127121 |
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09431385 |
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Current U.S.
Class: |
536/24.3 |
Current CPC
Class: |
C12Q 1/6832 20130101;
C12Q 1/6827 20130101; C12Q 1/6818 20130101; C12Q 1/6827 20130101;
C12Q 2525/117 20130101; C12Q 2527/107 20130101 |
Class at
Publication: |
536/24.3 |
International
Class: |
C07H 21/00 20060101
C07H021/00 |
Claims
1-47. (canceled)
48. An oligonucleotide probe capable of hybridizing with a target
sequence comprising a modified target binding sequence wherein one
or more purine residues of said target binding sequence are
substituted by a pyrazolo[3,4-d]pyrimidine residue, and further
comprising an attached minor groove binder, said modified target
binding sequence corresponding to and capable of binding with a
target sequence containing no pyrazolo[3,4-d]pyrimidine
residues.
49. An oligonucleotide probe of claim 48, wherein the minor groove
binder is selected from the group consisting of a trimer of
1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) and a
pentamer of N-methylpyrrole-4-carbox-2-amide (MPC5).
50. An oligonucleotide probe of claim 48, further comprising a
detectable label.
51. An oligonucleotide probe of claim 50 wherein the detectable
label is selected from radioactive isotopes, chromophores,
fluorophores, chemiluminescent agents, electrochemiluminescent
agents, magnetic labels, immunologic labels, ligands and enzymatic
labels.
52. An oligonucleotide probe of claim 50, wherein the label is
located is located at the oligonucleotide 5' end.
53. An oligonucleotide probe of claim 50, wherein the label is
located is located at the oligonucleotide 3' end.
54. An oligonucleotide probe of claim 50, wherein the detectable
label is a fluorescent label.
55. An oligonucleotide probe of claim 54 further comprising a
quenching agent which quenches the fluorescence emission of the
fluorescent label.
56. An oligonucleotide probe of claim 50, comprising multiple
fluorescent labels.
57. An oligonucleotide probe of claim 56, wherein the emission
wavelengths of one of the fluorescent labels overlaps the
absorption wavelengths of another of the fluorescent labels.
58. An oligonucleotide probe according to claim 48, wherein the
purine residues are selected from guanine residues and adenine
residues.
59. An oligonucleotide probe according to claim 48, wherein the
purine residues are guanine residues, and all of the guanine
residues are substituted by a pyrazolo[3,4-d]pyrimidine
residue.
60. An oligonucleotide probe according to claim 48, comprising four
guanine residues in sequence that are each substituted by a
pyrazolo[3,4-d]pyrimidine residue.
61. An oligonucleotide probe according to claim 48, comprising six
guanine residues in sequence that are each substituted by a
pyrazolo[3,4-d]pyrimidine residue.
62. An oligonucleotide probe according to claim 48 comprising at
least 12 but less than 21 bases.
Description
TECHNICAL FIELD
[0001] This application is in the field of molecular biology
relating to the use of oligonucleotides as probes and primers. It
relates further to the use of modified nucleic acid bases to
improve the hybridization properties and discriminatory abilities
of oligonucleotides that are used as probes and primers.
BACKGROUND
[0002] Many techniques currently in use in molecular biology
utilize oligonucleotides as probes and/or primers. It is often
advantageous, in the practice of these techniques, to be able to
distinguish between two or more sequences which are related but
which differ by one or more nucleotides. For example, many
mutations of clinical significance differ by only a single
nucleotide from the wild-type sequence. Polymorphisms in mammalian
genomes are also often characterized by sequence differences of one
or a few nucleotides. The ability to make such a distinction is
known as mismatch discrimination. In practical terms, mismatch
discrimination describes the property by which a defined sequence
oligonucleotide, at a given stringency, hybridizes strongly (one
manifestation of which is that the hybrids have a high melting
temperature) to a target sequence with which it is complementary
along its entire length (a perfect hybrid or perfect match), but
hybridizes detectably more weakly to a target sequence that is
non-complementary to the sequence of the oligonucleotide at one or
a few nucleotides (a mismatch). The differences in hybridization
strength are such that a particular stringency can be selected at
which a perfect match is detectable as a hybrid and a mismatch
fails to form a hybrid.
[0003] In a nucleic acid duplex, each base pair contributes to
stability. Hence, the shorter the duplex, the greater the relative
contribution of each individual base pair to the stability of the
duplex. As a result, the difference in stability between a perfect
match and a mismatch will be greater for shorter oligonucleotides.
However, short oligonucleotides hybridize weakly, even to a
perfectly complementary sequence, and thus must be hybridized under
conditions of reduced stringency. Thus, the potential
discriminatory power of short oligonucleotides cannot be easily
realized except under conditions of low stringency, which
counteract their discriminatory ability. It would constitute a
substantial advance in the art if it were possible to achieve
mismatch discrimination, particularly for single-nucleotide
mismatches, under conditions of high stringency; for example, at
the elevated temperatures characteristic of most amplification
reactions.
[0004] Stabilization of duplexes by pyrazolopyrimidine base
analogues has been reported. Seela et al. (1988) Helv. Chim. Acta.
71:1191-1198; Seela et al. (1988) Helv. Chim. Acta. 71:1813-1823;
and Seela et al. (1989) Nucleic Acids Res. 17:901-910.
Pyrazolo[3,4-d]pyrimidine residues in oligonucleotides are also
useful as sites for attachment of various pendant groups to
oligonucleotides. See co-owned PCT Publication WO 90/14353, Nov.
29, 1990. In addition, oligonucleotides in which one or more purine
residues have been substituted by pyrazolo[3,4-d]pyrimidines
display enhanced triplex-forming ability, as disclosed, for
example, in Belousov et al. (1998) Nucleic Acids Res. 26:1324-1328.
Pyrazolopyrimidines, when incorporated into an oligonucleotide, may
provide improved duplex and triplex formation. U.S. Pat. No.
5,594,121.
DISCLOSURE OF THE INVENTION
[0005] It is an object of the present invention to provide new
oligonucleotide compositions with improved properties related to
hybridization and mismatch discrimination. It is a further object
of the present invention to provide improved methods for
hybridization, primer extension, hydrolyzable probe assays, PCR,
single-nucleotide mismatch discrimination, nucleotide sequence
analysis, array analysis and related techniques involving the use
of oligonucleotides as probes and/or primers.
[0006] Accordingly, in one aspect, the present invention provides
modified oligonucleotide compositions comprising one or more
pyrazolo[3,4-d]pyrimidine base analogues substituted for at least
one purine. In preferred embodiments, the guanine analogue
6-amino-1H-pyrazolo[3,4-a]pyrimidin-4(5H)-one (ppG) is substituted
for guanine, and/or the adenine analogue
4-amino-1H-pyrazolo[3,4-d]pyrimidine (ppA) is substituted for
adenine. In other embodiments, the guanine analogue
1H-pyrazolo[3,4-d]pyrimidin-4(5H) one (ppI) is substituted for
guanine. The pyrazolo[3,4-d]pyrimidine-substituted oligonucleotides
can comprise, in addition, other moieties such as detectable labels
and/or minor groove binders and/or other types of modified bases or
base analogues.
[0007] Another aspect of the invention is a method for
hybridization of nucleic acids, wherein at least one of the nucleic
acids is a modified nucleic acid wherein one or more purine
residues are substituted with a pyrazolo[3,4-d]pyrimidine base
analogue. This method provides higher melting temperatures and
enhanced mismatch detection. The improved hybridization methods
provided by the present invention can be used in techniques which
include, but are not limited to, hybridization, primer extension,
single-nucleotide polymorphism detection, hydrolyzable probe
assays, cDNA synthesis, nucleotide sequence determination,
amplification reactions, and other techniques such as are known to
those of skill in the art.
[0008] When the guanine bases in an oligonucleotide are replaced by
the guanine analogue ppG, the T.sub.m values of probes containing
the analogues are slightly higher than those of oligonucleotide
probes containing guanine. Hence, G-containing and ppG-containing
oligonucleotides perform similarly in hybridization assays.
However, when ppG-substituted oligonucleotides are used as
hydrolyzable probes (described infra and see U.S. Pat. No.
5,210,015), two properties are significantly enhanced. First,
ppG-substituted probes are more effective at mismatch
discrimination, as measured by higher signal-to-noise values
comparing the fluorescent signal obtained from a perfectly-matched
hybrid with that from a hybrid containing a single-nucleotide
mismatch. In addition, ppG-substituted probes provide higher
absolute signal from a perfectly-matched target.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows the nucleotide sequence of the E. coli supF
gene contained in the plasmid pSP189 (SEQ ID NO.: 1). Locations of
the target sequences for amplification primers are shown as "Primer
1" and "Primer 2." Also shown are the target sequences for the
probes (designated "12-mer," "15-mer" and "18-mer"), and the
single-nucleotide substitutions that were introduced into the probe
target sequences (shown underneath the probe target sequences).
[0010] FIG. 2 shows results of a hydrolyzable probe assay, using
minor groove binder (MGB)-conjugated 15-mers as probes. The target
was the E. coli supF gene. Annealing/elongation was conducted at
72.degree. C. for 20 sec per cycle.
[0011] FIG. 3 shows results of a hydrolyzable probe assay, using
MGB-conjugated 15-mers as probes. In this experiment, all guanine
bases in the probes were substituted with the guanine analogue ppG.
All probes also contained a conjugated MGB. The target was the E.
coli supF gene. Annealing/elongation was conducted at 72.degree. C.
for 20 sec per cycle.
[0012] FIG. 4 shows results of a hydrolyzable probe assay, using
MGB-conjugated 15-mer probes in which all guanine bases in the
probe were substituted with the guanine analogue ppG. The target
was the E. coli supF gene. Annealing/elongation was conducted at
75.degree. C. for 20 sec per cycle.
MODES FOR CARRYING OUT THE INVENTION
[0013] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques in organic chemistry,
biochemistry, oligonucleotide synthesis and modification,
bioconjugate chemistry, nucleic acid hybridization, molecular
biology, microbiology, genetics, recombinant DNA, and related
fields as are within the skill of the art. These techniques are
fully explained in the literature. See, for example, Maniatis,
Fritsch & Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL,
Cold Spring Harbor Laboratory Press (1982); Sambrook, Fritsch &
Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition,
Cold Spring Harbor Laboratory Press (1989); Ausubel, et al.,
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons
(1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996); Gait
(ed.), OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press
(1984); Eckstein (ed.), OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL
APPROACH, IRL Press (1991).
[0014] Modified oligonucleotides wherein one or more purine bases
(i.e., adenine and/or guanine) are substituted by their
pyrazolo[3,4-d]pyrimidine analogues form stronger hybrids (i.e.,
duplexes) than those formed by unmodified oligonucleotides.
Hybridization strength is generally assessed by determination of
the melting temperature (T.sub.m) of a hybrid duplex. This is
accomplished by exposing a duplex in solution to gradually
increasing temperature and monitoring the denaturation of the
duplex, for example, by absorbance of ultraviolet light, which
increases with the unstacking of base pairs that accompanies
denaturation. T.sub.m is generally defined as the temperature
midpoint of the transition from a fully duplex structure to
complete denaturation (i.e., formation of two isolated single
strands). Hybrids formed by oligonucleotides in which one or more
purine residues are substituted by pyrazolo[3,4-d]pyrimidines have
a higher (T.sub.m) than those formed by unsubstituted
oligonucleotides.
[0015] At the same time, modified oligonucleotides wherein one or
more purine bases are substituted by pyrazolo[3,4-d]pyrimidines
possess enhanced abilities for mismatch discrimination, compared to
unsubstituted oligonucleotides. Without wishing to be bound by any
particular theory, it is likely that one contribution to the
enhanced discriminatory ability of
pyrazolo[3,4-d]pyrimidine-modified oligonucleotides stems from the
decreased tendency for a pyrazolo[3,4-d]pyrimidine base to
participate in self-pairing or to pair with a non-standard
base-pairing partner (i.e., whereas G is capable of base-pairing
with G and T, ppG-G and ppG-T base pairs are much less likely).
[0016] Structure and Synthesis of Pyrazolo[3,4-D]Pyrimidine
Nucleotides
[0017] In preferred embodiments of the modified oligonucleotides of
the invention all, or substantially all, guanine-containing
nucleotide units are replaced by a
6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one containing nucleotide
(ppG). A ppG-containing portion of an oligonucleotide is
illustrated in Formula 1. In less preferred embodiments not
necessarily all, but nevertheless several guanine-containing
nucleotide units are replaced by ppG.
##STR00001##
[0018] Optionally adenine containing nucleotide units of the
oligonucleotide can also be replaced by the corresponding
pyrazolo[3,4-d]pyrimidine analog, to wit: by
4-amino-1H-pyrazolo[3,4-d]pyrimidine. The nucleotide unit
containing this adenine analog is termed ppA, and a ppA-containing
portion of the oligonucleotide is illustrated in Formula 2. Thus,
oligonucleotides where at least one guanine base has been replaced
with ppG and which include no ppA analogue at all, as well as
oligonucleotides which in addition to ppG also have some, or
possibly all adenines replaced by ppA, as well as oligonucleotides
which comprise at least one ppA analogue but no ppG, are within the
scope of the invention.
[0019] The 2-deoxy-.beta.-D-ribofuranosides of ppG and ppA, namely
6-amino-1-(2'-deoxy-.beta.-D-erythro-pentofuranosyl-(1H)-pyrazolo[3,4-d]p-
yrimidin-4-5(H)-one and
4-amino-1-(2'-deoxy-.beta.-D-erythropentofuranosyl-1H-pyrazolo[3,4-d]pyri-
midine are synthesized and the corresponding activated
phosphorous-containing analogs (phosphoramidites) suitable for
oligonucleotide synthesis in a state-of-the-art automatic
oligonucleotide synthesizer, are obtained in accordance with the
literature procedures of Seela et al. (1986a) Helvetica Chimica
Acta 69:1602-1613; Seela et al. (1988a) Helvetica Chimica Acta
71:1191-1198; Seela et al. (1988b) Helvetica Chimica Acta
71:1813-1823; and Seela et al. (1989) Nucleic Acids Research
17:901-910. Each of these publications is specifically incorporated
herein by reference.
[0020] As a still further optional modification of the bases
present in the modified oligonucleotides of the invention, the
pyrazolo[3,4-d]pyrimidine 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(ppI) can replace one or more purine bases. ppI and the
corresponding nucleosides and nucleotides can be obtained by
methods related to those described. Seela et al. (1986b) Liebigs.
Ann. Chem.: 1213-1221; Seela et al. (1986a), supra; Seela et al.
(1988a), supra; Seela et al. (1988b), supra; and Seela et al.
(1989), supra.
[0021] In the presently preferred embodiments of the modified
oligonucleotides of the invention the sugar or glycosidic moieties
are 2-deoxyribofuranosides, and all internucleotide linkages are
the naturally occurring phosphodiester linkages. In alternative
embodiments however, instead of 2-deoxy-.beta.-D-ribofuranose,
other sugars, for example, .beta.-D-ribofuranose may be present. In
addition, .beta.-D-ribofuranose may be present wherein the 2-OH of
the ribose moiety is alkylated with a C.sub.1-6 alkyl group
(2-(O--C.sub.1-6 alkyl) ribose) or with a C.sub.2-6 alkenyl group
(2-(O--C.sub.2-6 alkenyl) ribose), or is replaced by a fluoro group
(2-fluororibose). Any sugar moiety compatible with hybridization of
the oligonucleotide can be used, such as are known to those of
skill in the art.
[0022] In a preferred embodiment, the sugar-phosphate backbone of
the modified oligonucleotides of the present invention comprises
phosphodiester bonds, as are found in naturally-occurring nucleic
acids. However, the sugar-phosphate backbone can also comprise any
structure that is compatible with hybridization of the
oligonucleotide including, but not limited to,
.alpha.-D-arabinofuranosides, .alpha.-2'-deoxyribofuranosides or
2',3'-dideoxy-3'-aminoribofuranosides. Oligonucleotides containing
.alpha.-D-arabinofuranosides can be obtained in accordance with the
teachings of U.S. Pat. No. 5,177,196, the disclosure of which is
expressly incorporated herein by reference. Oligonucleotides
containing 2',3'-dideoxy-3'aminoribofuranosides can be obtained in
accordance with the method of Chen et al. (1995) Nucleic Acids Res.
23:2661-2668, expressly incorporated herein by reference. The
phosphate backbone of the modified oligonucleotides of the
invention may also be modified so that the oligonucleotides contain
phosphorothioate linkages and/or methylphosphonates. Additional
backbone modifications are known to those of skill in the art.
[0023] The modified oligonucleotides of the present invention can
also comprise additional pendant groups such as, for example,
intercalators, lipophilic groups, minor groove binders, reporter
groups, chelating agents and cross-linking agents attached to one
or more of the internally located nucleotide bases, to the 3', to
the 5' end, to both ends, or can have such pendant groups attached
both internally and at one or both ends. The nature and attachment
of intercalator, lipophilic groups, minor grove binders, reporter
groups, chelating agents and cross-linking agents to
oligonucleotides are presently well known in the state-of-the-art,
and are described, for example, in U.S. Pat. Nos. 5,512,667,
5,419,966 and in the publication WO 96/32496, which are
incorporated herein by reference. The oligonucleotides of the
invention can also have a relatively low molecular weight "tail
moiety" attached either at the 3' or 5' end, or at both ends. By
way of example a tail molecule can be a phosphate, a phosphate
ester, an alkyl group, an aminoalkyl group, or a lipophilic group.
The tail moiety can also link the intercalators, lipophilic groups,
minor groove binders, reporter groups, chelating agents and
cross-linking functionalities to the oligonucleotides of the
invention.
[0024] The nature of tail moieties and methods for obtaining
oligonucleotides with various tail moieties are also described in
the above-referenced U.S. Pat. Nos. 5,512,667 and 5,419,966.
[0025] In a preferred embodiment, modified oligonucleotides of the
invention containing ppG substituted for guanine and/or ppA
substituted for adenine also comprise a conjugated minor groove
binder (MGB). Optimal single-nucleotide mismatch discrimination is
obtained using MGB-conjugated oligonucleotides containing ppG in
place of guanine, as shown in Examples 4 and 5, infra. Preferred
MGB moieties include the trimer of
3-carbamoyl-1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate
(CDPI.sub.3) and the pentamer of N-methylpyrrole-4-carbox-2-amide
(MPC.sub.5). Additional MGB moieties that will find use in the
practice of the present invention are disclosed in co-owned U.S.
Pat. No. ______ [allowed U.S. patent application Ser. No.
08/415,370], the disclosure of which is hereby incorporated herein
by reference.
[0026] Reactive precursors of pyrazolo[3,4-d]pyrimidines can be
obtained following procedures described supra, and these precursors
can be used in techniques of automated oligonucleotide synthesis.
Such techniques are routine and well-known to those of skill in the
art.
[0027] Methods of Use of the Invention
[0028] The present invention provides modified oligonucleotides
having new and surprising properties of superior mismatch
discrimination, compared to unmodified oligonucleotides. Modified
oligonucleotides of the invention are used as probes, wherein their
hybridization to a target sequence is detected, or as primers,
wherein their hybridization to a target sequence is followed by
polynucleotide synthesis initiated from the 3' terminus of the
modified oligonucleotide, and the synthesized product (i.e., the
extension product) is detected.
[0029] A target sequence refers to a nucleotide sequence which
comprises a site of hybridization for a probe or a primer. Target
sequences can be found in any nucleic acid including, but not
limited to, genomic DNA, cDNA and RNA, and can comprise a wild-type
gene sequence, a mutant gene sequence, a non-coding sequence, a
regulatory sequence, etc. A target sequence will generally be less
than 100 nucleotides, preferably less than 50 nucleotides, and most
preferably, less than 21 nucleotides in length.
[0030] Oligonucleotides are short polymers of nucleotides,
generally less than 200 nucleotides, preferably less than 150
nucleotides, more preferably less than 100 nucleotides, more
preferably less than 50 nucleotides and most preferably less than
21 nucleotides in length. Polynucleotides are generally considered,
in the art, to comprise longer polymers of nucleotides than do
oligonucleotides, although there is an art-recognized overlap
between the upper limit of oligonucleotide length and the lower
limit of polynucleotide length. With respect to the present
invention, "oligonucleotide" generally refers to a nucleic acid,
usually comprising a detectable label, that is used as a probe or
as a primer; while polynucleotide refers to a nucleic acid
containing a target sequence. Consequently, for the purposes of the
present invention, the terms "oligonucleotide" and "polynucleotide"
shall not be considered limiting with respect to polymer
length.
[0031] Hybridization of probes and/or primers to target sequences
proceeds according to well-known and art-recognized base-pairing
properties, such that adenine base-pairs with thymine or uracil,
and guanine base-pairs with cytosine. The property of a nucleotide
that allows it to base-pair with a second nucleotide is called
complementarity. Thus, adenine is complementary to both thymine and
uracil, and vice versa; similarly, guanine is complementary to
cytosine and vice versa. An oligonucleotide which is complementary
along its entire length with a target sequence is said to be
perfectly complementary, perfectly matched, or fully complementary
to the target sequence, and vice versa. An oligonucleotide and its
target sequence can have related sequences, wherein the majority of
bases in the two sequences are complementary, but one or more bases
are noncomplementary, or mismatched. In such a case, the sequences
can be said to be substantially complementary to one another. If
the sequences of an oligonucleotide and a target sequence are such
that they are complementary at all nucleotide positions except one,
the oligonucleotide and the target sequence have a single
nucleotide mismatch with respect to each other.
[0032] The modified nucleotides of the invention retain the
base-pairing specificity of their naturally-occurring analogues;
i.e., ppG is complementary to cytosine, while ppA is complementary
to thymine and uracil. The ppG and ppA analogues have a reduced
tendency for so-called "wobble" pairing with non-complementary
bases, compared to guanine and adenine.
[0033] Conditions for hybridization are well-known to those of
skill in the art and can be varied within relatively wide limits.
Hybridization stringency refers to the degree to which
hybridization conditions disfavor the formation of hybrids
containing mismatched nucleotides, thereby promoting the formation
of perfectly matched hybrids or hybrids containing fewer
mismatches; with higher stringency correlated with a lower
tolerance for mismatched hybrids. Factors that affect the
stringency of hybridization include, but are not limited to,
temperature, pH, ionic strength, and concentration of organic
solvents such as formamide and dimethylsulfoxide. As is well known
to those of skill in the art, hybridization stringency is increased
by higher temperatures, lower ionic strengths, and lower solvent
concentrations. See, for example, Ausubel et al., supra; Sambrook
et al., supra; M. A. Innis et al. (eds.) PCR Protocols, Academic
Press, San Diego, 1990; B. D. Hames et al. (eds.) Nucleic Acid
Hybridisation: A Practical Approach, IRL Press, Oxford, 1985; and
van Ness et al., (1991) Nucleic Acids Res. 19:5143-5151.
[0034] Thus, in the formation of hybrids (duplexes) between an
oligonucleotide and its target sequence, the oligonucleotide is
incubated in solution, together with a polynucleotide containing
the target sequence, under conditions of temperature, ionic
strength, pH, etc, that are favorable to hybridization, i.e., under
hybridization conditions. Hybridization conditions are chosen, in
some circumstances, to favor hybridization between two nucleic
acids having perfectly-matched sequences, as compared to a pair of
nucleic acids having one or more mismatches in the hybridizing
sequence. In other circumstances, hybridization conditions are
chosen to allow hybridization between mismatched sequences,
favoring hybridization between nucleic acids having fewer
mismatches.
[0035] The degree of hybridization of an oligonucleotide to a
target sequence, also known as hybridization strength, is
determined by methods that are well-known in the art. A preferred
method is to determine the T.sub.m of the hybrid duplex. This is
accomplished, as described supra, by subjecting a duplex in
solution to gradually increasing temperature and monitoring the
denaturation of the duplex, for example, by absorbance of
ultraviolet light, which increases with the unstacking of base
pairs that accompanies denaturation. T.sub.m is generally defined
as the temperature midpoint of the transition in ultraviolet
absorbance that accompanies denaturation. Alternatively, if
T.sub.ms are known, a hybridization temperature (at fixed ionic
strength, pH and solvent concentration) can be chosen that is below
the T.sub.m of the desired duplex and above the T.sub.m of an
undesired duplex. In this case, determination of the degree of
hybridization is accomplished simply by testing for the presence of
hybridized probe.
[0036] If a probe comprises a detectable label, assays for
hybridized probe are usually designed to detect the presence of
label in duplex material. This can be accomplished, for example, by
specifically selecting duplex material, specifically destroying
single-stranded material, or utilizing some combination of these
methods. For example, hybridization reaction mixtures can be
subjected to high-stringency conditions and/or single
strand-specific nucleases; or duplexes can be purified by affinity
techniques specific for double-stranded, as opposed to
single-stranded, nucleic acids. In a preferred embodiment of the
invention, duplexes are detected by release of label from a probe
under conditions in which label is released only when the probe is
in a duplex.
[0037] Detectable labels or tags suitable for use with nucleic acid
probes are well-known to those of skill in the art and include, but
are not limited to, radioactive isotopes, chromophores,
fluorophores, chemiluminescent and electrochemiluminescent agents,
magnetic labels, immunologic labels, ligands and enzymatic labels.
Suitable labels also include mass labels and those used in
deconvolution of combinatorial chemistry libraries, for example,
tags that can be recognized by high performance liquid
chromatography (HPLC), gas chromatography, mass spectrometry,
etc.
[0038] Methods for labeling of oligonucleotides are well-known to
those of skill in the art and include, for example, chemical and
enzymatic methods. By way of example, methods for incorporation of
reactive chemical groups into oligonucleotides, at specific sites,
are well-known to those of skill in the art. Oligonucleotides
containing a reactive chemical group, located at a specific site,
can be combined with a label attached to a complementary reactive
group (e.g., an oligonucleotide containing a nucleophilic reactive
group can be reacted with a label attached to an electrophilic
reactive group) to couple a label to a probe by chemical
techniques. Exemplary labels and methods for attachment of a label
to an oligonucleotide are described, for example, in U.S. Pat. No.
5,210,015; Kessler (ed.), Nonradioactive Labeling and Detection of
Biomolecules, Springer-Verlag, Berlin, 1992; Kricka (ed.)
Nonisotopic DNA Probe Techniques, Academic Press, San Diego, 1992;
Howard (ed.) Methods in Nonradioactive Detection, Appleton &
Lange, Norwalk, 1993. Non-specific chemical labeling of an
oligonucleotide can be achieved by combining the oligonucleotide
with a chemical that reacts, for example, with a particular
functional group of a nucleotide base, and simultaneously or
subsequently reacting the oligonucleotide with a label. See, for
example, Draper et al. (1980) Biochemistry 19:1774-1781. Enzymatic
incorporation of label into an oligonucleotide can be achieved by
conducting enzymatic modification or polymerization of an
oligonucleotide using labeled precursors, or by enzymatically
adding label to an already-existing oligonucleotide. See, for
example, U.S. Pat. No. 5,449,767. Examples of modifying enzymes
include, but are not limited to, DNA polymerases, reverse
transcriptases, RNA polymerases, etc. Examples of enzymes which are
able to add label to an already-existing oligonucleotide include,
but are not limited to, kinases, terminal transferases, ligases,
glycosylases, etc.
[0039] If an oligonucleotide is capable of acting as a primer, the
degree of hybridization of the oligonucleotide can also be
determined by measuring the levels of the extension product of the
primer. In the case, either the primer can be labeled, or one or
more of the precursors for polymerization (normally nucleoside
triphosphates) can be labeled. Extension product can be detected,
for example, by size (e.g., gel electrophoresis), affinity methods,
or any other technique known to those of skill in the art.
[0040] Nucleotide monomers containing one or more reactive groups
can be introduced into an oligonucleotide during automated
synthesis; and these nucleotides can be used as points of label
attachment. See Example 1, infra. Also, pyrazolo[3,4-d]pyrimidines
containing linker arms can be incorporated into oligonucleotides by
automated synthesis and serve as sites for attachment of various
labels. See Example 1, infra and WO90/14353.
[0041] In certain embodiments of the present invention,
oligonucleotides comprising fluorescent labels (fluorophores)
and/or fluorescence quenching agents are used. In a preferred
embodiment, an oligonucleotide contains both a fluorophore and a
quenching agent. Fluorescent labels include, but are not limited
to, fluoresceins, rhodamines, cyanines, phycoerythrins, and other
fluorophores as are known to those of skill in the art. Quenching
agents are those substances capable of absorbing energy emitted by
a fluorophore so as to reduce the amount of fluorescence emitted
(i.e., quench the emission of the fluorescent label). Different
fluorophores are quenched by different quenching agents. In
general, the spectral properties of a particular
fluorophore/quenching agent pair are such that one or more
absorption wavelengths of the quencher overlaps one or more of the
emission wavelengths of the fluorophore. A preferred
fluorophore/quencher pair is fluorescein/tetramethylrhodamine;
additional fluorophore/quencher pair can be selected by those of
skill in the art by comparison of emission and excitation
wavelengths according to the properties set forth above.
[0042] For use in amplification assays conducted at elevated
temperatures, such as a polymerase chain reaction, or other
procedures utilizing thermostable enzymes, the label is stable at
elevated temperatures. For assays involving polymerization, the
label is such that it does not interfere with the activity of the
polymerizing enzyme. Label can be present at the 5' and/or 3' end
of the oligonucleotide, and/or can also be present internally. The
label can be attached to any of the base, sugar or phosphate
moieties of the oligonucleotide, or to any linking group that is
itself attached to one of these moieties.
[0043] Exemplary Applications
[0044] The methods and compositions of the present invention can be
used with a variety of techniques, both currently in use and to be
developed, in which hybridization of an oligonucleotide to a target
sequence in another nucleic acid is involved. These include, but
are not limited to, 1) techniques in which hybridization of an
oligonucleotide to a target sequence is the endpoint; 2) techniques
in which hybridization of one or more oligonucleotides to a target
sequence precedes one or more polymerase-mediated elongation steps
which use the oligonucleotide as a primer and the target nucleic
acid as a template; 3) techniques in which hybridization of an
oligonucleotide to a target sequence is used to block extension of
another primer; 4) techniques in which hybridization of an
oligonucleotide to a target sequence is followed by hydrolysis of
the oligonucleotide to release an attached label; and 5) techniques
in which two or more oligonucleotides are hybridized to a target
sequence and interactions between the multiple oligonucleotides are
measured.
[0045] Hybridization Probes
[0046] In one aspect of the present invention, one or more modified
oligonucleotides are used as probe(s) to identify a target sequence
in a nucleic acid by assaying hybridization between the probe(s)
and the nucleic acid. A probe can be labeled with any detectable
label, or it can have the capacity to become labeled either before
or after hybridization, such as by containing a reactive group
capable of association with a label or by being capable of
hybridizing to a secondary labeled probe, either before or after
hybridization to the target. Conditions for hybridization of
nucleic acid probes are well-known to those of skill in the art.
See, for example, Sambrook et al., supra; Ausubel et al., supra;
Innis et al., supra; Hames et al., supra; and van Ness et al.,
supra.
[0047] Hybridization can be assayed (i.e., hybridized nucleic acids
can be identified) by distinguishing hybridized probe from free
probe by one of several methods that are well-known to those of
skill in the art. These include, but are not limited to, attachment
of target nucleic acid to a solid support, either directly or
indirectly (by hybridization to a second, support-bound probe)
followed by direct or indirect hybridization with probe, and
washing to remove unhybridized probe; determination of nuclease
resistance; buoyant density determination; affinity methods
specific for nucleic acid duplexes (e.g., hydroxyapatite
chromatography); interactions between multiple probes hybridized to
the same target nucleic acid; etc. See, for example, Falkow et al.,
U.S. Pat. No. 4,358,535; Urdea et al., U.S. Pat. Nos. 4,868,105 and
5,124,246; Freifelder, Physical Biochemistry, Second Edition, W. H.
Freeman & Co., San Francisco, 1982; Sambrook, et al., supra;
Ausubel et al., supra; Hames et al., supra; and other related
references.
[0048] The modified oligonucleotides disclosed herein are
particularly useful for distinguishing one among a group of related
target sequences. Related target sequences are those whose
sequences differ at one or more nucleotide positions, but which are
complementary over a majority of their length. In a preferred
embodiment of the invention, modified oligonucleotides are able to
distinguish related target sequences which differ by only a single
nucleotide. For example, it is possible to select hybridization
conditions in which perfectly-matched sequences form detectable
hybrids, but two sequences having a single-nucleotide mismatch do
not form detectable hybrids. See Example 5, infra.
[0049] Amplification Primers
[0050] Amplification procedures are those in which many copies of a
target nucleic acid sequence are generated, usually in an
exponential fashion, by sequential polymerization and/or ligation
reactions. Many amplification reactions, such as polymerase chain
reactions (PCR), utilize reiterative primer-dependent
polymerization reactions. A primer is a nucleic acid that is
capable of hybridizing to a second, template nucleic acid and that,
once hybridized, is capable of being extended by a polymerizing
enzyme (in the presence of nucleotide substrates), using the second
nucleic acid as a template. Polymerizing enzymes include, but are
not limited to, DNA and RNA polymerases and reverse transcriptases,
etc. Thermostable polymerases are preferred in most amplification
reactions. Conditions favorable for polymerization by different
polymerizing enzymes are well-known to those of skill in the art.
See, for example, SambroOk et al., supra; Ausubel, et al., supra;
Innis et al., supra. Generally, in order to be extendible by a
polymerizing enzyme, a primer must have an unblocked 3'-end,
preferably a free 3' hydroxyl group. The product of an
amplification reaction is an extended primer, wherein the primer
has been extended by a polymerizing enzyme.
[0051] Thus, in one embodiment of the invention, the methods and
compositions disclosed and claimed herein are useful in improved
amplification reactions such as PCR. See, e.g., U.S. Pat. Nos.
4,683,202; 4,683,195 and 4,800,159. The practice of the invention
will be especially useful in situations in which it is desired to
selectively amplify a particular sequence which differs from
undesired sequences by one or a small number of nucleotides.
[0052] The improvements provided by the present invention are
applicable to any type of assay or procedure in which PCR or a
related amplification technique is used, including, but not limited
to, priming with allele-specific oligonucleotides (ASOs), fragment
length polymorphism analysis, single nucleotide polymorphism (SNP)
analysis and microsatellite analysis, for example. These and other
techniques are useful in gene mapping, in the identification and
screening of disease-related genes, and in pharmacogenetics, to
name just a few applications.
[0053] Assays Utilizing Labeled Probes; Including Hydrolyzable
Probe Assays
[0054] Additional uses for modified oligonucleotides are found in
assays in which a labeled probe is hybridized to a target sequence
and/or an extension product comprising a target sequence, and a
change in the physical state of the label is effected as a
consequence of hybridization. By way of example, one assay of this
type, the hydrolyzable probe assay, takes advantage of the fact
that many polymerizing enzymes, such as DNA polymerases, possess
intrinsic 5'-3'exonucleolytic activities. Accordingly, if a probe
is hybridized to a sequence that can serve as a template for
polymerization (for instance, if a probe is hybridized to a region
of DNA located between two amplification primers, during the course
of an amplification reaction), a polymerizing enzyme that has
initiated polymerization at an upstream amplification primer is
capable of exonucleolytic digestion of the probe. Any label
attached to such a probe will be released as a consequence of the
exonucleolytic digestion of the probe. Released label is separated
from labeled probe and detected by methods well-known to those of
skill in the art, depending on the nature of the label. For
example, radioactively labeled fragments can be separated by
thin-layer chromatography and detected by autoradiography; while
fluorescently-labeled fragments can be detected by irradiation at
the appropriate excitation wavelengths with observation at the
appropriate emission wavelengths. See, e.g., U.S. Pat. No.
5,210,015.
[0055] In a preferred embodiment, a probe comprising a modified
oligonucleotide contains both a fluorescent label and a quenching
agent, which quenches the fluorescence emission of the fluorescent
label. In this case, the fluorescent label is not detectable until
its spatial relationship to the quenching agent has been altered,
for example by exonucleolytic release of the fluorescent label from
the probe. Thus, prior to hybridization to its target sequence, the
dual fluorophore/quencher labeled probe does not emit fluorescence.
Subsequent to hybridization of the fluorophore/quencher-labeled
probe to its target, it becomes a substrate for the exonucleolytic
activity of a polymerizing enzyme which has initiated
polymerization at an upstream primer. Exonucleolytic degradation of
the probe releases the fluorescent label from the probe, and hence
from the vicinity of the quenching agent, allowing detection of a
fluorescent signal upon irradiation at the appropriate excitation
wavelengths. This method has the advantage that released label does
not have to be separated from intact probe. Multiplex approaches
utilize multiple probes, each of which is complementary to a
different target sequence and carries a distinguishable label,
allowing the assay of several target sequences simultaneously.
[0056] This type of assay is becoming increasingly important,
especially in clinical applications, because it is a homogeneous
assay (i.e., no product separation steps are required for analysis)
in which the results can be monitored in real time. See, for
example, Wittwer et al. (1997) BioTechniques 22:130-138. Rapid,
fluorescence-based molecular assays find use in, for example,
real-time surgical and therapeutic applications, as well.
[0057] The enhanced ability of modified oligonucleotides to
discriminate between related target sequences will facilitate the
use of hydrolyzable probe assays in the identification of, for
example, single-nucleotide polymorphisms and the like. Examples 4
and 5, infra, disclose the use of modified oligonucleotides in a
hydrolyzable probe assay.
[0058] Additional assays involving the principles of fluorescence
quenching will be apparent to those skilled in the art, as will the
advantages of using modified oligonucleotides in such assays. It
will also be clear to those of skill in the art that
fluorescently-labeled modified oligonucleotides provide
improvements in discriminatory power in the practice of all types
of hybridization assays.
[0059] Fluorescence Energy Transfer
[0060] In further embodiments of the invention, modified
oligonucleotides are used in various techniques which involve
multiple fluorescently-labeled probes. In some of these assays,
fluorescence and/or changes in properties of a fluorescent label
are used to monitor hybridization. For example, fluorescence
resonance energy transfer (FRET) has been used as an indicator of
oligonucleotide hybridization. In one embodiment of this technique,
two probes are used, each containing a different fluorescent label.
One of the labels is a fluorescence donor, and the other is a
fluorescence acceptor, wherein the emission wavelengths of the
fluorescence donor overlap the absorption wavelengths of the
fluorescence acceptor. The sequences of the probes are selected so
that they hybridize to adjacent regions of a target sequence,
thereby bringing the fluorescence donor and the fluorescence
acceptor into close proximity, if target is present. In the
presence of target nucleic acid, irradiation at wavelengths
corresponding to the absorption wavelengths of the fluorescence
donor will result in emission from the fluorescence acceptor. These
types of assays have the advantage that they are homogeneous
assays, providing a positive signal without the necessity of
removing unreacted probe. For further details and additional
examples, see, for example, European Patent Publication 070685; and
Cardullo, et al. (1988) Proc. Natl. Acad. Sci. USA 85:
8790-8794.
[0061] Additional embodiments of the present invention will be
found in these and related techniques in which interactions between
two different oligonucleotides that are hybridized to the same
target nucleic acid are measured. The selection of appropriate
fluorescence donor/fluorescence acceptor pairs will be apparent to
one of skill in the art, based on the principle that, for a given
pair, the emission wavelengths of the fluorescence donor will
overlap the absorption wavelengths of the fluorescence acceptor.
The enhanced ability of modified oligonucleotides to distinguish
among related target sequences facilitates the use of FRET-based
techniques in the identification of single-nucleotide polymorphisms
and the like.
[0062] Assays Involving Oligonucleotide Ligation
[0063] Modified oligonucleotides are useful in assays in which two
or more oligonucleotides, complementary to adjacent sites on a
target nucleic acid, are hybridized to adjacent sites on the target
nucleic acid and ligated to one another. See, for example, European
Patent Publication 320,308; European Patent Publication 336,731;
and U.S. Pat. No. 4,883,750. Conditions for ligation are well-known
to those of skill in the art. See, for example, Sambrook et al.,
supra; Ausubel, et al., supra; Innis et al., supra. Ligated nucleic
acids can be identified, for example, by an increase in size of the
product compared to the starting oligonucleotides. As in the case
with hybridization assays, use of modified oligonucleotides in
assays involving ligation allows more efficient discrimination
among related target sequences; particularly between perfect
hybrids and single-base mismatches, which is especially important
in oligonucleotide ligation assays.
[0064] cDNA Synthesis
[0065] Synthesis of cDNA, as commonly practiced, utilizes a reverse
transcriptase enzyme to copy a mRNA template into cDNA. The primer
for reverse transcription is normally oligodeoxythymidylate, which
is complementary to the polyadenylate tail found at the 3' end of
most mRNA molecules. However, cDNA synthesis rarely proceeds all
the way to the 5' terminus of the template mRNA molecule. Thus,
most cDNA libraries are enriched for sequences near the 3' ends of
mRNAs and deficient in sequences near the 5' end. Consequently, to
obtain a complete cDNA representation of a mRNA sequence, one or
more additional synthesis reactions, primed at internal regions of
the mRNA template, must be conducted. Modified oligonucleotides can
be in these internal priming steps, allowing discrimination between
closely related mRNA sequences, such as might be found in different
members of a gene family.
[0066] In addition, synthesis of cDNA is often conducted under
conditions of low stringency, to promote the hybridization of the
oligodeoxythymidylate primer to the polyadenylate tail. Under such
conditions, mRNA molecules are known to readily adopt
intramolecular secondary structures, which can act as blocks to
elongation by reverse transcriptase, leading to production of
short, partial cDNA molecules. cDNA synthesis using modified
oligonucleotides as primers can, by contrast, proceed under more
stringent conditions, wherein secondary structure in the mRNA
template is minimized, leading to the synthesis of longer cDNA
products.
[0067] Nucleic Acid Sequencing
[0068] In one embodiment of the invention, a collection of all
possible n-mer oligonucleotides (where n is an integer less than
about 10) are used in a hydrolyzable probe assay to determine a
nucleotide sequence. Each oligonucleotide is uniquely labeled and
analysis of released label indicates which of the oligonucleotides
has hybridized to the target sequence. Alignment of the sequences
of the oligonucleotides which have hybridized provides the
nucleotide sequence. Modified oligonucleotides, with heightened
discriminatory properties, are particularly suitable for use in
this technique.
[0069] Modified oligonucleotides are also useful in
primer-dependent methods of DNA sequencing, such as the
chain-termination method and its derivatives, originally described
by Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467.
Use of modified oligonucleotides in chain-termination sequencing
enables a greater degree of mismatch discrimination during
sequencing, providing, for example, improved means for
distinguishing between two or more closely-related sequences.
[0070] Oligonucleotide Arrays
[0071] In another embodiment of the present invention, modified
oligonucleotides are used in procedures which utilize arrays of
oligonucleotides, such as sequencing by hybridization and
array-based analysis of gene expression. In sequencing by
hybridization, an ordered array of oligonucleotides of different
known sequences is used as a platform for hybridization to one or
more test polynucleotides, nucleic acids or nucleic acid
populations. Determination of the oligonucleotides which are
hybridized and alignment of their known sequences allows
reconstruction of the sequence of the test polynucleotide.
Alternatively, oligonucleotides comprising the wild-type sequence
and all possible mutant sequences for a given region of a gene of
interest can be placed on an array. Exposure of the array to DNA or
RNA from a subject or biological specimen, under hybridization
conditions, allows determination of wild-type or mutant status for
the gene of interest. See, for example, U.S. Pat. Nos. 5,492,806;
5,525,464; 5,556,752; and PCT Publications WO 92/10588 and WO
96/17957. Both of these techniques require discrimination between
related sequences, especially at the single-nucleotide level;
hence, the enhanced discriminatory properties of the modified
oligonucleotides of the invention will provide improvements in
these techniques. Materials for construction of arrays include, but
are not limited to, nitrocellulose, glass, silicon wafers, optical
fibers and other materials suitable for construction of arrays such
as are known to those of skill in the art.
[0072] An additional application of the present invention to array
technology is in the examination of patterns of gene expression in
a particular cell or tissue. In this case, oligonucleotides or
polynucleotides corresponding to different genes are arrayed on a
surface, and a nucleic acid sample from a particular cell or tissue
type, for example, is incubated with the array under hybridization
conditions. Detection of the sites on the array at which
hybridization occurs allows one to determine which oligonucleotides
have hybridized, and hence which genes are active in the particular
cell or tissue from which the sample was derived.
[0073] Array methods can also be used for identification of
mutations, where wild-type and mutant sequences are placed in an
ordered array on a surface. Hybridization of a polynucleotide
sample to the array under stringent conditions, and determination
of which oligonucleotides in the array hybridize to the
polynucleotide, allows determination of whether the polynucleotide
possesses the wild-type or the mutant sequence. Since many mutant
sequences of clinically-relevant genes differ from their wild-type
counterpart at only one or a few nucleotide positions, the enhanced
discriminatory powers of the modified oligonucleotides of the
invention will provide improvements in mutation detection.
[0074] In all of the above-mentioned applications of array
technology, the increased discriminatory abilities of modified
oligonucleotide provide significant improvements in sensitivity and
resolving power.
EXAMPLES
[0075] The following examples are intended to illustrate, not to
limit the invention.
[0076] In the hydrolyzable probe assay, a labeled probe is added to
a PCR reaction. The probe is complementary to a region between the
two PCR primers and is labeled with two fluorophores, one of which
quenches the fluorescence of the other. The probe is designed to
hybridize to its complementary target sequence on one of the PCR
product strands at or above the strand extension temperature
typically used in PCR (55-75.degree. C.). The polymerizing enzymes
normally used in PCR (Taq polymerase in particular) possess an
intrinsic 5'-exonuclease activity. During synthesis of new strands
in the extension stage of the PCR reaction, this 5'-exonuclease
activity will act on complementary strands bound to the template.
If a probe, labeled as described above, is bound to the template,
the 5'-exonuclease activity associated with the polymerizing enzyme
will liberate the bound fluorophore. Once liberated, its
fluorescence will no longer be quenched, and a fluorescent signal
will be obtained. See, for example, U.S. Pat. No. 5,210,015; Livak
et al. (1995) PCR Meth. App. 4:357-362; and Heid et al. (1996)
Genome Res. 6:986-994.
Example 1
Preparation of Dual-Labeled, MGB-Conjugated Hydrolyzable Probes
[0077] Synthesis of Oligonucleotide Probes Carrying a 5'-Reporting
Dye
[0078]
[(3',6'-dipivaloylfluoresceinyl)-6-carboxamidohexyl]-1-O-(2-cyanoet-
hyl)-(N,N-diisopropyl)-phosphoramidite (6-FAM) and
3'-CDPI.sub.3-tail (Scheme 1). Oligonucleotides with a conjugated
CDPI.sub.3 tail were prepared on a 1 .mu.mol scale using standard
3'-phosphoramidite chemistry on a CDPI.sub.3-CPG support
(.about.20-50 mg) Preparation of the CDPI.sub.3-CPG support is
disclosed in Lukhtanov et al. (1995) Bioconj. Chem. 6:418-426.
Oligonucleotides lacking a conjugated MGB were synthesized by
standard procedures. Synthesis was performed on an ABI 394
according to the protocol supplied by the manufacturer with one
exception: 0.01 M (instead of the standard 0.1 M) iodine solution
was utilized in the oxidation step to avoid iodination of the
CDPI.sub.3 moiety, when CDPI.sub.3-conjugated oligonucleotides were
being synthesized. An amino-linker for postsynthetic incorporation
of the TAMRA dye (see below) was introduced near the 3'-end of the
oligonucleotide by incorporating a protected aminopropyl ppG or
aminopropyl ppA phosphoramidite (see co-owned, allowed U.S. patent
application Ser. No. 08/334,490) in place of a G or A residue,
respectively, at the desired step of automated oligonucleotide
synthesis. To incorporate a FAM dye at the 5'-end of the probes,
5'-Fluorescein Phosphoramidite (6-FAM, Glen Research, Cat.
#10-5901) was used at the last step in the synthesis of an
oligonucleotide. After cleavage from the solid support and complete
deprotection by ammonia treatment (30% ammonia, 12-15 hrs,
50.degree. C.) reactions were filtered and dried by rotary
evaporation. Probes containing a CDPI.sub.3 tail were isolated by
RP-HPLC on a 4.6.times.250 mm, C-18, Dynamax-300A column (Rainin)
with a linear gradient of acetonitrile (0.fwdarw.60%, 20 min, 2
mL/min) in 0.1 M triethylammonium acetate buffer (pH 7.4).
Fractions containing CDPI.sub.3-tailed probe were concentrated with
butanol to a volume of 60-100 .mu.l in 1.5 ml plastic tubes,
precipitated in 2% NaClO.sub.4 (or LiClO.sub.4, 1.5 mL) in acetone,
washed with acetone, and dried in vacuo. The HPLC purified probes
were either (i) used directly for incorporation of a TAMRA dye or
(ii) additionally purified by 8% denaturing polyacrylamide gel
electrophoresis (see below).
[0079] Post-Synthetic Introduction of TAMRA Residue (Scheme 2).
[0080] An N-hydroxysuccinimide ester of Tetramethylrhodamine (TAMRA
NHS Ester, Glen Research, Cat. #50-5910) was incorporated at an
aminopropyl ppG or aminopropyl ppA residue of the 5'-FAM,
3'-CDPI.sub.3-tailed oligonucleotide probes (synthesized as
described above) using the protocol supplied by the manufacturer.
The reaction solution was then saturated with urea (.about.400
.mu.l) and loaded onto an 8% denaturing polyacrylamide gels
(1.5.times.270 mm packet, 38.times.50 cm plate, Bio-Rad
Laboratories; gel buffer contained 7 M urea, 2 mM EDTA, 90 mM
Tris-borate, pH 8.3). Gel purification was performed at a constant
power setting (100 Watts, 50-55.degree. C.). The desired products
of conjugation (i.e., probes carrying 6-FAM, TAMRA, and CDPI.sub.3
residues) were detected by the TAMRA-specific color and cut out of
the gel. Gel slices were incubated at 37.degree. C. overnight in
4-6 ml of 100 mM Tris-HCl, 10 mM triethylammonium acetate, 1 mM
EDTA, pH 7.8. Finally, the conjugates were isolated from the gel
extract either by (i) reverse phase HPLC as described above or (ii)
using MAXI-CLEAN C18 cartridges according to the protocol supplied
by manufacturer (Alltech Associates, Inc.). In either case the
probes were concentrated with butanol, precipitated in 2%
NaClO.sub.4 in acetone, washed with pure acetone, dried in vacuo,
dissolved in 100-400 .mu.l of water, and stored at -20.degree.
C.
[0081] Presence of the conjugated moieties in the oligonucleotide
probes was confirmed by absorbance at specific UV and visible
wavelengths. The following wavelengths were used: 255-265 nm for
detection of nucleic acid, 350 nm for detection of CDPI.sub.3,
460-480 nm for detection of 6-FAM, and 570 nm for detection of
TAMRA.
Example 2
Target, Primer and Probe Sequences
[0082] Strategy
[0083] The target sequence is located in the E. coli supF gene
contained in the plasmid pSP189 (FIG. 1, SEQ ID NO.: 1). See Parris
et al. (1992) Gene 117:1-5. Binding sites for the primers used for
amplification are indicated as Primer 1 and Primer 2, with Primer 1
having a sequence and polarity that is identical to that shown in
FIG. 1, and Primer 2 having a sequence and polarity that is the
reverse complement to that shown in FIG. 1. Three probes having
overlapping sequences, each labeled with FAM at the 5'-end and with
the quencher TAMRA at the 3'-end with were synthesized: a 12-mer, a
15-mer and an 18-mer. The 12-mer and 15-mer additionally contained
a conjugated minor groove binder (CDPI.sub.3) near the 3'-end of
the oligonucleotide. Finally, each probe contained either normal
guanine residues (indicated by G in the Tables) or all of its
guanine residues were substituted with ppG (indicated by ppG in the
Tables). These probes were used to determine the effect of
substitution of ppG for G on hybridization strength and mismatch
discrimination.
TABLE-US-00001 Primer sequences The forward amplification primer
has the sequence: 5'-CTGGGTGAGCAAAAACAGGAAGGC-3' SEQ ID No.: 2 The
reverse primer has the sequence: 5'-TGTGATGCTCGTCAGGGGGG-3' SEQ ID
No.: 3 Sequences of probes: The 12-mer probe has the following
sequence: 5'-TTCCCGAGCGGC SEQ ID NO.: 4 The 15-mer probe has the
following sequence: 5'-GGGTTCCCGAGCGGC SEQ ID NO.: 5 The 18-mer
probe has the following sequence: 5'-GTGGGGTTCCCGAGCGGC SEQ ID NO.:
6
[0084] Template Sequences:
[0085] The 18-nucleotide region of the template that is
complementary to the probes used in this study was modified to
generate a series of point mutations, as shown in FIG. 1. Each of
the mutant templates was used in a separate assay with each of the
three probes. The mutant sequences within this region of the
template were as follows, with the mismatched nucleotide indicated
by bold underlining:
TABLE-US-00002 SEQ ID NO.: 7 5'-GTGGGGTTCCCGAGCGGC (perfect match)
SEQ ID NO.: 8 5'-GTGGAGTTCCCGAGCGGC (32 G-A mismatch) SEQ ID NO.: 9
5'-GTGGGGTTTCCGAGCGGC (36 C-T mismatch) SEQ ID NO.: 10
5'-GTGGGGTTGCCGAGCGGC (36 C-G mismatch) SEQ ID NO.: 11
5'-GTGGGGTTACCGAGCGGC (36 C-A mismatch) SEQ ID NO.: 12
5'-GTGGGGTTCTCGAGCGGC (37 C-T mismatch) SEQ ID NO.: 13
5'-GTGGGGTTCACGAGCGGC (37 C-A mismatch) SEQ ID NO.: 14
5'-GTGGGGTTCCCCAGCGGC (39 G-C mismatch) SEQ ID NO.: 15
5'-GTGGGGTTCCCGTGCGGC (40 A-T mismatch) SEQ ID NO.: 16
5'-GTGGGGTTCCCGAACGGC (41 G-A mismatch) SEQ ID NO.: 17
5'-GTGGGGTTCCCGACCGGC (41 G-C mismatch) SEQ ID NO.: 18
5'-GTGGGGTTCCCGAGCAGC (43 G-A mismatch) SEQ ID NO.: 19
5'-GTGGGGTTCCCGAGCTGC (43 G-T mismatch) SEQ ID NO.: 20
5'-GTGGGGTTCCCGAGCGTC (44 G-T mismatch)
Example 3
Hydrolyzable Probe Assay
[0086] Hydrolyzable probe assays with fluorescent monitoring were
performed in an Idaho Technologies Light Cycler. Wittwer et al.
(1997a) BioTechniques 22:130-138, and Wittwer et al. (1997b)
BioTechniques 22:176-181. Each reaction mixture contained:
[0087] 40 mM NaCl [0088] 20 mM Tris-Cl, pH 8.9 [0089] 5 mM
MgSO.sub.4 [0090] 0.05% (w/v) Bovine Serum Albumin [0091] 125 .mu.M
each dATP, dGTP, dCTP, dTTP [0092] 0.5 .mu.M each primer [0093] 0.5
.mu.M probe [0094] 0.5 U/10 .mu.L Taq Polymerase
[0095] Cycling conditions were 40 cycles of 0 sec at 94.degree. C.
(i.e., temperature was raised to 94.degree. C. and immediately
lowered to the annealing/extension temperature), then 15 sec at the
annealing/extension temperature (which varied from 55-75.degree. C.
in individual experiments; see below and in figure legends for
details). Fluorescent output was expressed as the ratio of
fluorescence at 515-560 nm (fluorescein) to that at 560-630 nm
(rhodamine), as analyzed by the manufacturer's software that was
provided with the light cycler.
[0096] Melting temperatures (Table 3) were determined on a Perkin
Elmer .lamda.2S UV/VIS spectrophotometer, equipped with a PTP-6
temperature controller, using the PECSS software package.
Example 4
Effect of ppG Substitution on T.sub.m and on Single Nucleotide
Mismatch Discrimination
[0097] Oligonucleotides of 12, 15 or 18 nucleotides, spanning the
same target sequence region, were used as hydrolyzable probes and
tested for hybridization to a perfectly-matched target sequence,
and to different single nucleotide mismatched target sequences
(FIG. 1). Each probe was tested with and without substitution of
all G residues by ppG. The 12-mer and 15-mer oligonucleotides
additionally contained a conjugated MGB. A common pair of primers
was used to amplify The segment of the template containing the
target sequence. Fluorescence values are given, in Table 1, for
assays in which either the wild type sequence, to which the probe
is perfectly matched (labeled "match" in the table), or one of the
mismatched mutant sequences, is used as template. Table 1 shows the
amount of fluorescent signal generated after 40 cycles of PCR, in
arbitrary fluorescence units. This value provides an estimate of
the number of copies of target present in the original sample
(Wittwer et al., supra) and, for equivalent amounts of initial
target, provides an approximate measure of the efficiency of the
hydrolyzable probe in the assay. The results are presented in two
ways. Table 1 shows the absolute fluorescence measured in the assay
after 40 cycles of amplification. In Table 2, the fluorescence
value (after 40 cycles) of each mismatched probe/template hybrid is
given as a percentage of the value obtained for the perfectly
matched hybrid.
[0098] Two advantages resulting from the use of the ppG
substitution are evident from the data presented in Tables 1 and 2.
First, the substitution of ppG for G in a probe enhances the
intensity of the measured signal obtained with that probe. While
the signal from any particular probe will depend on conditions used
for the assay, the fact that addition of ppG to the probe always
enhances the signal could imply that hybrids formed with
ppG-containing oligonucleotides have higher T.sub.m values. Table 3
shows that this is indeed the case. In all cases tested, the
T.sub.m for a hybrid containing a ppG-substituted oligonucleotide
is 1-4.degree. C. higher than that of a hybrid formed with an
unsubstituted oligonucleotide.
[0099] The second advantage of using ppG-substituted
oligonucleotides is that the presence of ppG in the probe
significantly enhances single-nucleotide mismatch discrimination.
When fluorescence obtained with a given probe/template pair is
expressed as percentage of the fluorescence obtained in an assay
with the perfectly-matched probe/template pair (Table 2), it can be
seen that, in general, inclusion of ppG in place of G reduces the
ratio of signal obtained from mismatched targets to signal obtained
from a perfectly-matched target. Without wishing to be bound by any
particular theory, it is suggested that the enhanced mismatch
discrimination obtained with ppG-substituted oligonucleotides may
be related to the propensity of guanine to form unusual base pairs
(i.e., with bases other than cytosine), a property that ppG may not
have.
Example 5
Effect of ppG Substitution on Single Nucleotide Mismatch
Discrimination
[0100] FIG. 2 shows a time-course for fluorescence release in a
hydrolyzable probe assay (as described in Example 3) when
annealing/elongation was conducted at 72.degree. C. with
MGB-conjugated 15-mer probes. Although the perfect match (denoted
"match" in the Figure) provides the highest level of signal,
detectable signals are also obtained from many of the probes
harboring a single-nucleotide mismatch with the target. However, if
the assay is conducted under identical conditions except that all
guanine residues in the MGB-conjugated oligonucleotide probes are
replaced by ppG, generation of signal by probes containing a
single-base mismatch is significantly reduced, while the amount of
signal generated by the perfectly-matched probe is unaffected (FIG.
3). If, in addition, ppG-modified, MGB-conjugated, oligonucleotide
probes are used in an assay in which the annealing/elongation
temperature is raised to 75.degree. C., generation of signal by
probes with a single-base mismatch is completely suppressed, again
with no effect on the level of signal generated by the
perfectly-matched probe (FIG. 4).
[0101] Thus, the combination of MGB conjugation, substitution with
pyrazolo[3,4-d]pyrimidine base analogues, and appropriate reaction
conditions enable facile discrimination between a perfect-matched
hybrid and a hybrid containing a single-nucleotide mismatch, at
high stringency, allowing a heretofore unparalleled degree of
specificity to be obtained in hybridization reactions with short
oligonucleotides.
TABLE-US-00003 TABLE 1 Fluorescence release during amplification of
the supF gene 12-mer, 68.degree. C., 15-mer, 75.degree. C., 18-mer,
68.degree. C., +MGB +MGB -MGB Sequence: G ppG G ppG G ppG Match
4.85 5.62 1.01 4.67 0.58 2.39 32 G-A 0.12 0.55 0.04 0.17 36 C-T
0.12 0.10 0.00 0.15 0.00 0.00 36 C-G 0.02 0.00 0.00 0.09 0.00 0.00
36 C-A 0.00 0.00 0.00 0.00 0.00 0.00 37 C-T 0.05 0.05 0.02 0.05
0.01 0.03 37 C-A 0.01 0.00 0.04 0.11 0.00 0.00 39 G-C 0.03 0.00
0.02 0.05 0.07 0.02 40 A-T 0.56 0.51 0.16 0.37 0.03 0.03 41 G-A
0.18 0.01 0.06 0.33 0.05 0.03 41 G-C 0.55 0.34 0.10 0.23 0.06 0.05
43 G-A 0.12 0.05 0.29 0.24 0.06 0.04 43 G-T 0.13 0.01 0.15 0.14
0.04 0.10 44 G-T 0.57 0.63 0.65 0.24 0.13 0.23
TABLE-US-00004 TABLE 2 Fluorescence as a percentage of
perfectly-matched probe for oligonucleotides .+-. ppG 15-mer +
18-mer - 12-mer + MGB, MGB, MGB, 68.degree. C. 75.degree. C.
68.degree. C. Sequence: G ppG G ppG G ppG Match 100 100 100 100 100
100 32 G-A 11 12 6 7 36 C-T 2 2 0 3 0 0 36 C-G 0 0 0 2 0 0 36 C-A 0
0 0 0 0 0 37 C-T 1 1 2 1 1 1 37 C-A 0 0 4 2 0 0 39 G-C 1 0 2 1 12 1
40 A-T 11 9 16 8 5 1 41 G-A 4 0 6 7 9 1 41 G-C 11 6 9 5 10 2 43 G-A
3 1 29 5 11 1 43 G-T 3 0 14 3 7 4 44 G-T 12 11 64 5 23 10
TABLE-US-00005 TABLE 3 Melting temperatures of hybrids formed by
MGB- conjugated 15-mer oligonucleotides .+-. ppG T.sub.m Sequence:
G ppG .DELTA.T.sub.m Match 71 74 3 32 G-A 65 67 2 36 C-T 62 65 3 36
C-G 62 64 2 36 C-A 66 67 1 37 C-T 68 69 1 37 C-A 71 73 2 39 G-C 60
61 1 40 A-T 60 64 4 41 G-A 60 64 4 41 G-C 60 63 3 43 G-A 57 59 2 43
G-T 59 61 2 44 G-T 66 69 3
[0102] While the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent to those skilled in the art
that various changes and modifications can be practiced without
departing from the spirit of the invention. Therefore the foregoing
descriptions and examples should not be construed as limiting the
scope of the invention.
Sequence CWU 1
1
201510DNAEscherichia colimisc_featuren is a, c, g, or t 1aaaactctca
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 60caactgatct
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
120gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
tcatactctt 180cctttttcaa tattattgaa gcatttatca gggaattcga
gagccctgct cgagctgtgg 240tggggttccc gagcggccaa agggagcaga
ctctaaatct gccgtcatcg acttcgaagg 300ttcgaatcct tcccccacca
ccacggccga aattcggtac ccggatcctt agcgaaagct 360aagatttttt
ttacgcgtga gctcgactga ctccnnnnnn nngagctcaa ttcggtcgag
420gtcgggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
catcacaaaa 480atcgacgctc aagtcagagg tggcgaaacc 510224DNAArtificial
SequenceReverse amplification primer 2ctgggtgagc aaaaacagga aggc
24320DNAArtificial SequenceForward amplification primer 3tgtgatgctc
gtcagggggg 20412DNAArtificial SequenceProbe sequence 4ttcccgagcg gc
12510DNAArtificial SequenceProbe sequence 5gggttcccga
10618DNAArtificial SequenceProbe sequence 6gtggggttcc cgagcggc
18718DNAArtificial SequenceComplementary template to probes
7gtggggttcc cgagcggc 18818DNAArtificial SequenceComplementary
template to probe with one mismatch 8gtggagttcc cgagcggc
18918DNAArtificial SequenceComplementary template to probes with
one mismatch 9gtggggtttc cgagcggc 181018DNAArtificial
SequenceComplementary template to probes with one mismatch
10gtggggttgc cgagcggc 181118DNAArtificial SequenceComplementary
template to probes with one mismatch 11gtggggttac cgagcggc
181218DNAArtificial SequenceComplementary template to probes with
one mismatch 12gtggggttct cgagcggc 181318DNAArtificial
SequenceComplementary template to probes with one mismatch
13gtggggttca cgagcggc 181418DNAArtificial SequenceComplementary
template to probes with one mismatch 14gtggggttcc ccagcggc
181518DNAArtificial SequenceComplementary template to probes with
one mismatch 15gtggggttcc cgtgcggc 181618DNAArtificial
SequenceComplementary template to probes with one mismatch
16gtggggttcc cgaacggc 181718DNAArtificial SequenceComplementary
template to probes with one mismatch 17gtggggttcc cgaccggc
181818DNAArtificial SequenceComplementary template to probes with
one mismatch 18gtggggttcc cgagcagc 181918DNAArtificial
SequenceComplementary template to probes with one mismatch
19gtggggttcc cgagctgc 182018DNAArtificial SequenceComplementary
template to probes with one mismatch 20gtggggttcc cgagcgtc 18
* * * * *