U.S. patent application number 11/926108 was filed with the patent office on 2008-07-03 for methods and compositions for nucleic acid analysis.
This patent application is currently assigned to Promega Corporation. Invention is credited to Mary Ann Brow, David A. Casimir.
Application Number | 20080160529 11/926108 |
Document ID | / |
Family ID | 34526745 |
Filed Date | 2008-07-03 |
United States Patent
Application |
20080160529 |
Kind Code |
A1 |
Brow; Mary Ann ; et
al. |
July 3, 2008 |
Methods and Compositions for Nucleic Acid Analysis
Abstract
The present invention relates to improved methods and
composition for nucleic acid analysis. In particular, the present
invention provides improved methods and compositions for carrying
out nucleic acid analysis using modified nucleotides.
Inventors: |
Brow; Mary Ann; (Madison,
WI) ; Casimir; David A.; (Verona, WI) |
Correspondence
Address: |
Casimir Jones, S.C.
440 Science Drive, Suite 203
Madison
WI
53711
US
|
Assignee: |
Promega Corporation
Madison
WI
|
Family ID: |
34526745 |
Appl. No.: |
11/926108 |
Filed: |
October 28, 2007 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11699088 |
Jan 29, 2007 |
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11926108 |
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10969032 |
Oct 20, 2004 |
7282333 |
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11699088 |
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60512638 |
Oct 20, 2003 |
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Current U.S.
Class: |
435/6.12 ;
435/193; 536/23.1; 536/24.33; 536/25.3 |
Current CPC
Class: |
C12Q 1/6818 20130101;
C12Q 1/6858 20130101; C12Q 1/6858 20130101; C12Q 1/6858 20130101;
C12Q 2525/161 20130101; C12Q 2525/161 20130101; C12Q 2525/301
20130101; C12Q 2565/101 20130101; C12Q 2525/186 20130101; C12Q
2525/161 20130101; C12Q 2565/101 20130101; C12Q 1/6818 20130101;
C12Q 2565/101 20130101; C12Q 2525/186 20130101 |
Class at
Publication: |
435/6 ; 536/23.1;
536/24.33; 435/193; 536/25.3 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12N 9/10 20060101
C12N009/10 |
Claims
1. A kit comprising an extension disabled non-naturally occurring
nucleotide.
2. The kit of claim 1, further comprising a oligonucleotide
primer.
3. The kit of claim 1, further comprising a polymerase.
4. The kit of claim 3, wherein said polymerase is a DNA
polymerase.
5. The kit of claim 4, wherein said DNA polymerase is a
thermostable polymerase
6. The kit of claim 5, wherein said thermostable polymerase lacks
5'-nuclease activity.
7. The kit of claim 1, further comprising reagents for conducting a
polymerase chain reaction.
8. The kit of claim 1, wherein said extension disabled
non-naturally occurring nucleotide comprises a dideoxy
nucleotide.
9. The kit of claim 1, wherein said non-naturally occurring
nucleotide comprises dideoxy iso-G.
10. A method for analyzing a target nucleic acid, comprising:
exposing a target nucleic acid to polymerase, a first amplification
primer having a first non-natural nucleotide, a second
amplification primer, and an extension disabled second non-natural
nucleotide complementary to said first non-natural nucleotide under
conditions such that an extension product is generated from at
least said second amplification primer, wherein said extension
product incorporates said second non-natural nucleotides.
11. The method of claim 10, wherein said first and second
amplification primers are selected to hybridize to a portion of
said target nucleic acid to define an amplification region, wherein
said amplification region is selected to minimize the presence of T
nucleotides in said amplification region.
12. A method for analyzing a target nucleic acid using non-natural
nucleotides, comprising: a) providing: i) a first non-natural
nucleotide; ii) a sample suspected of comprising a target nucleic
acid; iii) a first amplification primer having a second non-natural
nucleotide, said primer having a sequence selected to be upstream
of a region of said target nucleic acid to be amplified, wherein
said region is selected to avoid or minimize the presence of
natural nucleotides that can base-pair with said first non-natural
nucleotide; iv) a second amplification primer; and v) a polymerase;
b) exposing said sample to said first non-natural nucleotide, said
first and second amplification primers, and said polymerase under
condition an extension product is generated from at least said
second amplification primer, wherein said extension product
incorporates at least one said first non-natural nucleotide.
13. The method of claim 12, wherein said first non-natural
nucleotide is iso-G.
14. The method of claim 13, wherein said natural nucleotides
comprise thymidine.
15. The method of claim 12, further comprising the step of
detecting the presence or absence of said target nucleic acid in
said sample.
16. The method of claim 12, wherein said region does not contain
said natural nucleotides in 20 bases upstream of said first
amplification primer.
17. The method of claim 12, wherein said region does not contain
said natural nucleotides in 10 bases upstream of said first
amplification primer.
18. The method of claim 12, wherein said region does not contain
said natural nucleotides in 5 bases upstream of said first
amplification primer.
19. The method of claim 12, wherein said first non-natural
nucleotide comprises an extension disabled non-naturally occurring
nucleotide.
20. The method of claim 12, wherein said region is selected by the
use of a processor configured to analyze a sequence characteristic
of said region selected from the group consisting of: location of
said natural bases in said region, number of said natural bases in
said region, and presence of said natural bases in said region.
21. A method for manufacturing oligonucleotide design data for use
in selecting said region of claim 12, comprising: a) providing a
sequence of said target nucleic acid to a processor, wherein said
processor is configured to analyze a sequence characteristic of
said region selected from the group consisting of: location of said
natural bases in said region, number of said natural bases in said
region, and presence of said natural bases in said region; and b)
generating oligonucleotide design data using said analyzed sequence
characteristic.
22. A method for manufacturing a primer for use in the method of
claim 12, comprising: selecting a primer sequence so as to amplify
said region, wherein said region is selected to avoid or minimize
the presence of natural nucleotides that can base-pair with said
first non-natural nucleotide; and manufacturing a primer having
said sequence.
23. The method of claim 22, wherein said region is selected by the
use of a processor configured to analyze a sequence characteristic
of said region selected from the group consisting of: location of
said natural bases in said region, number of said natural bases in
said region, and presence of said natural bases in said region.
24. A method for detecting a target nucleic acid, comprising: a)
contacting the sample with a polymerase; a first oligonucleotide
primer comprising a 3' region complementary to a first portion of
the target nucleic acid and a 5' region comprising a tag sequence;
a second oligonucleotide primer comprising a 3' region comprising a
sequence complementary to a second portion of the target nucleic
acid, a 5' region comprising the tag sequence, and a 5' terminal
region comprising a non-natural base; b) conducting a polymerase
chain reaction to produce an amplified sample under conditions
wherein the target nucleic acid, if present in the sample, is
amplified using the first and second oligonucleotide primers to
generate amplification products comprising a target amplification
product having (i) a double-stranded region and (ii) a
single-stranded region that comprises the non-natural base, c)
contacting said amplified sample with a reporter comprising a label
and a non-natural base that is complementary to the non-natural
base of the single-stranded region; d) incorporating the reporter
into the amplification product opposite the non-natural base of the
single-stranded region; and e) detecting the incorporation of the
reporter; wherein the detection of the incorporation of the
reporter correlates with presence of the target nucleic acid in the
sample.
25. The method of claim 24, further comprising step e) treating the
amplified sample under conditions wherein non-target amplification
product consisting essentially of primer-dimer product is treated
to separate the strands, wherein each separated strand forms a
duplex region comprising a tag sequence and a region that is
complementary to the tag sequence.
26. The method of claim 24, wherein each of said second
oligonucleotide primer and said reporter comprise interactive
labels.
27. The method of claim 26, wherein said interactive labels
comprise a fluorophore.
28. The method of claim 26 wherein said interactive labels comprise
a quencher.
29. The method of claim 26, wherein said second oligonucleotide
primer comprises a fluorophore and said reporter comprises a
quencher.
30. The method of claim 26, wherein said second oligonucleotide
primer comprises a quencher and said reporter comprises a
fluorophore.
31. The method of claim 26, wherein said second oligonucleotide
primer comprises a first fluorophore and said reporter comprises a
second fluorophore.
32. The method of claim 24, wherein the step of contacting the
sample with a reporter comprises contacting the sample with a
reporter comprising a label and a nucleoside triphosphate of a
non-natural base that is complementary to the non-natural base of
the single-stranded region.
33. The method of claim 24, wherein the step of contacting the
sample with a reporter comprises contacting the sample with a
reporter consisting essentially of a label and a nucleoside
triphosphate of a non-natural base that is complementary to the
non-natural base of the single-stranded region.
34. The method of claim 24, wherein the incorporating step
comprises incorporating the reporter into the amplification product
opposite the non-natural base of the single-stranded region using a
nucleic acid polymerase.
35. The method of claim 24, wherein the incorporating step
comprises incorporating the reporter into the amplification product
opposite the non-natural base of the single-stranded region using a
ligase.
36. A kit comprising: a first oligonucleotide primer comprising a
3' region complementary to a first portion of the target nucleic
acid and a 5' region comprising a tag sequence, a second
oligonucleotide primer comprising a 3' region comprising a sequence
complementary to a second portion of the target nucleic acid, a 5'
region comprising the tag sequence, and a 5' terminal region
comprising a non-natural base; and a reporter comprising a label
and a non-natural base that is complementary to the non-natural
base in said second oligonucleotide primer.
37. The kit of claim 26, further comprising a third oligonucleotide
primer comprising a 3' portion consisting essentially of said tag
sequence.
38. The kit of claim 36, wherein the reporter comprises an
oligonucleotide comprising the non-natural base.
39. The kit of claim 36, wherein the reporter does not include any
base other than the non-natural base.
40. The kit of claim 36, wherein the second region of the second
oligonucleotide primer further comprises a label and the labels of
the reporter and the second region of the second oligonucleotide
primer comprise a pair of fluorophores where the emission of one of
the fluorophores stimulates the emission of the other
fluorophore.
41. The kit of claim 36, wherein the second region of the second
oligonucleotide primer further comprises a label and the labels of
the reporter and the second region of the second oligonucleotide
primer comprise a signal generating element and a signal quenching
element.
42. The kit of claim 36, further comprising a polymerase.
Description
[0001] The present Application claims priority to Provisional
Application Ser. No. 60/512,638, filed Oct. 20, 2003, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to improved methods and
composition for nucleic acid analysis. In particular, the present
invention provides improved methods and compositions for carrying
out nucleic acid analysis using modified nucleotides.
BACKGROUND OF THE INVENTION
[0003] The development and use of modified nucleotides has expanded
the range of nucleic acid analysis techniques. For example, the
modified non-natural nucleotides of Eragen Corporation expand the
alphabet of bases that may be used in nucleic acid technologies
(See e.g., U.S. Pat. Nos. 5,216,141, 5,432,272, 5,958,702,
5,965,364, 6,001,983, 6,037,120, and 6,140,496 and patent
application Ser. Nos. 09/415,966, 09/538,338, 09/993,757,
60/205,712, 60/240,398, 60/282,831, 60/240,397, 60/252,783, and
60/253,382, each of which is incorporated herein in its entirety).
While these modified nucleotides have been found useful,
compositions and methods that provide improved sensitivity and
flexibility in using such products in nucleic acid analysis methods
are needed.
[0004] Modified nucleotides also find use in nucleic acid
amplification reactions such as the polymerase chain reaction. See,
e.g., U.S. patent Ser. No. 09/861,292, published as Publication No.
2002/0150900, and U.S. patent Ser. No. 10/977,615, published as
Publication No. 2004/0106108, each of which is incorporated herein
by reference in its entirety. While these applications have been
found useful, methods and compositions that reduce background
signal and improve assay sensitivity are needed.
SUMMARY OF THE INVENTION
[0005] The present invention relates to improved methods and
compositions for nucleic acid analysis. In particular, the present
invention provides improved methods and compositions for carrying
out nucleic acid analysis using modified nucleotides. Still more
particularly, the present invention provides improved methods and
compositions for carrying out nucleic acid analysis using
non-naturally occurring nucleotides that are complementary to other
non-naturally occurring nucleotides but that are capable of
base-pairing to one or more naturally occurring nucleotides.
[0006] In some embodiments, the present invention provides a kit
comprising an extension-disabled non-naturally occurring
nucleotide. In some preferred embodiments, the kit further
comprises one or more of an oligonucleotide primer, a polymerase
(e.g., a DNA polymerase, a thermostable polymerase, a polymerase
lacking 5' nuclease activity, etc.), and reagents for conducting a
polymerase chain reaction. In some preferred embodiments, the
extension-disabled non-naturally occurring nucleotide comprises a
dideoxy nucleotide (e.g., a dideoxy iso-G).
[0007] The present invention further provides methods for analyzing
a target nucleic acid, comprising: exposing a target nucleic acid
to polymerase, a first amplification primer having a first
non-natural nucleotide, a second amplification primer, and an
extension-disabled second non-natural nucleotide complementary to
said first non-natural nucleotide under conditions such that an
extension product is generated from at least said second
amplification primer, wherein said extension product incorporates
said second non-natural nucleotides. In some embodiments, the first
and second amplification primers are selected to hybridize to a
portion of said target nucleic acid to define an amplification
region, wherein said amplification region is selected to minimize
the presence of T nucleotides in said amplification region.
[0008] In some embodiments, the present invention provides improved
methods of performing primer-directed amplification reactions, such
as PCR. In some embodiments, the present invention provides methods
and compositions for reducing signal from background amplification
products, particularly primer-dimer amplification products. In some
embodiments, the reduction of signal from primer-dimer products
comprises use of primers having a tag sequence. When each of the
target-specific primers for PCR amplification comprises the same
tag sequence, each of the strands of the resulting amplicons will
comprise self-complementary portion (i.e., the tag from the primer
at or near the 5' end, and the complement of the tag sequence at or
near the 3' end. For long products, single-stranded portion of the
adapters at each end of the ssDNA fragment may form either a
self-annealing "panhandle-like" structure (amplification
suppressive structure) or a DNA/primer "hybrid" structure
(amplification permissive structure). The relative ratio of
formation of the two structures using the subject method during PCR
cycling depends on a number of factors, including the differences
between the melting temperatures of the suppressive and permissive
structures, the position of the complementary primer binding site
within the adapter sequence, and the size of the DNA fragment to be
amplified. These factors can be manipulated to achieve the desired
suppression of non-target DNA during PCR amplification. In some
embodiments, the formation of panhandle structures in primer-dimer
products separates interactive labels, so as to reduce or eliminate
detection of primer-dimer products formed in the reaction.
[0009] In some embodiments the present invention comprises a method
for detecting a target nucleic acid, comprising: a) contacting the
sample with a polymerase; a first oligonucleotide primer comprising
a 3' region complementary to a first portion of the target nucleic
acid and a 5' region comprising a tag sequence; a second
oligonucleotide primer comprising a 3' region comprising a sequence
complementary to a second portion of the target nucleic acid, a 5'
region comprising the tag sequence, and a 5' terminal region
comprising a non-natural base; b) conducting a polymerase chain
reaction to produce an amplified sample under conditions wherein
the target nucleic acid, if present in the sample, is amplified
using the first and second oligonucleotide primers to generate
amplification products comprising a target amplification product
having (i) a double-stranded region and (ii) a single-stranded
region that comprises the non-natural base, c) contacting said
amplified sample with a reporter comprising a label and a
non-natural base that is complementary to the non-natural base of
the single-stranded region; d) incorporating the reporter into the
amplification product opposite the non-natural base of the
single-stranded region; and e) detecting the incorporation of the
reporter; wherein the detection of the incorporation of the
reporter correlates with presence of the target nucleic acid in the
sample. In some embodiments, the method further comprises the step
e) of treating the amplified sample under conditions wherein
non-target amplification product consisting essentially of
primer-dimer product is treated to separate the strands, wherein
each separated strand forms a duplex region comprising a tag
sequence and a region that is complementary to the tag
sequence.
[0010] In some embodiments, each of the second oligonucleotide
primer and said reporter comprise interactive labels. In some
preferred embodiments, the interactive labels comprise a
fluorophore. In some embodiments, said interactive labels comprise
a quencher.
[0011] In some embodiments, the step of contacting the sample with
a reporter comprises contacting the sample with a reporter
comprising a label and a nucleoside triphosphate of a non-natural
base that is complementary to the non-natural base of the
single-stranded region. In some embodiments, the step of contacting
the sample with a reporter comprises contacting the sample with a
reporter consisting essentially of a label and a nucleoside
triphosphate of a non-natural base that is complementary to the
non-natural base of the single-stranded region.
[0012] In some embodiments, the incorporating step comprises
incorporating the reporter into the amplification product opposite
the non-natural base of the single-stranded region using a nucleic
acid polymerase. In other embodiments, the incorporating step
comprises incorporating the reporter into the amplification product
opposite the non-natural base of the single-stranded region using a
ligase.
[0013] In some embodiments, the present invention comprises a kit
comprising: a first oligonucleotide primer comprising a 3' region
complementary to a first portion of the target nucleic acid and a
5' region comprising a tag sequence, a second oligonucleotide
primer comprising a 3' region comprising a sequence complementary
to a second portion of the target nucleic acid, a 5' region
comprising the tag sequence, and a 5' terminal region comprising a
non-natural base; and a reporter comprising a label and a
non-natural base that is complementary to the non-natural base in
said second oligonucleotide primer. In some embodiments the kit
further comprises a third oligonucleotide primer comprising a 3'
portion consisting essentially of said tag sequence.
[0014] In some embodiments of the kits of the present invention,
the reporter comprises an oligonucleotide comprising the
non-natural base. In some embodiments, the reporter does not
include any base other than the non-natural base.
[0015] In some embodiments, the second region of the second
oligonucleotide primer further comprises a label and the labels of
the reporter and the second region of the second oligonucleotide
primer comprise a pair of fluorophores where the emission of one of
the fluorophores stimulates the emission of the other
fluorophore.
[0016] In some embodiments, the second region of the second
oligonucleotide primer further comprises a label and the labels of
the reporter and the second region of the second oligonucleotide
primer comprise a signal generating element and a signal quenching
element.
DEFINITIONS
[0017] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0018] As used herein, the terms "subject" and "patient" refer to
any organism, including a plant, a microorganism or an animal
(e.g., a mammal such as a dog, cat, livestock, or human, or a
non-mammal, such as a bird, an amphibian or a fish).
[0019] As used herein, the term "kit" refers to any delivery system
for delivering materials. In the context of reaction assays, such
delivery systems include systems that allow for the storage,
transport, or delivery of reaction reagents (e.g.,
oligonucleotides, enzymes, etc. in the appropriate containers)
and/or supporting materials (e.g., buffers, written instructions
for performing the assay etc.) from one location to another. For
example, kits include one or more enclosures (e.g., boxes)
containing the relevant reaction reagents and/or supporting
materials. As used herein, the term "fragmented kit" refers to
delivery systems comprising two or more separate containers that
each contains a subportion of the total kit components. The
containers may be delivered to the intended recipient together or
separately. For example, a first container may contain an enzyme
for use in an assay, while a second container contains
oligonucleotides. The term "fragmented kit" is intended to
encompass kits containing Analyte specific reagents (ASR's)
regulated under section 520(e) of the Federal Food, Drug, and
Cosmetic Act, but are not limited thereto. Indeed, any delivery
system comprising two or more separate containers that each
contains a subportion of the total kit components are included in
the term "fragmented kit." In contrast, a "combined kit" refers to
a delivery system containing all of the components of a reaction
assay in a single container (e.g., in a single box housing each of
the desired components). The term "kit" includes both fragmented
and combined kits.
[0020] The term "label" as used herein refers to any atom or
molecule that can be used to provide a detectable (preferably
quantifiable) effect, and that can be attached to a nucleic acid or
protein. Labels include but are not limited to dyes; radiolabels
such as .sup.32P; binding moieties such as biotin; haptens such as
digoxygenin; luminogenic, phosphorescent or fluorogenic moieties;
mass tags; and fluorescent dyes alone or in combination with
moieties that can suppress or shift emission spectra by
fluorescence resonance energy transfer (FRET). Labels may provide
signals detectable by fluorescence, radioactivity, colorimetry,
gravimetry, X-ray diffraction or absorption, magnetism, enzymatic
activity, characteristics of mass or behavior affected by mass
(e.g., MALDI time-of-flight mass spectrometry), and the like. A
label may be a charged moiety (positive or negative charge) or
alternatively, may be charge neutral. Labels can include or consist
of nucleic acid or protein sequence, so long as the sequence
comprising the label is detectable.
[0021] In some situations, a label comprises two or more
interactive labels, either on a single oligonucleotide, or on
different strands of an nucleic acid duplex or other complex (e.g.,
triplex or quartet). One type of interactive label pair is a
quencher-dye pair. Preferably, the quencher-dye pair comprises a
fluorophore and a quencher. Suitable fluorophores include, for
example, fluorescein, cascade blue, hexachloro-fluorescein,
tetrachloro-fluorescein, TAMRA, ROX, Cy3, Cy3.5, Cy5, Cy5.5,
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid,
4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid,
4,4-difluoro-5-styryl-4-bora-3a,4-adiaz-a-S-indacene-propionic
acid, 6-carboxy-X-rhodamine,
N,N,N',N'-tetramethyl-6-carboxyrhodamine, Texas Red, Eosin,
fluorescein,
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid, 4,4-difluoro-5,p-ethoxyphenyl-4-bora-3a,4a-diaza-s-indacene
3-propionic acid and
4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-5-indacene-propionic acid.
Suitable quenchers include, for example, Dabcyl, QSY7 (Molecular
Probes, Eugene, Oreg.) and the like. In some embodiments, dyes
(e.g., fluorphores or chromophores) can also be used as a quencher
(e.g., if they absorb the emitted light of another dye).
[0022] As used herein the term "interactive label" refers to a
label having two or more components that interact so as to produce
a detectable effect. The interaction is not limited to any
particular nature of interaction. The interaction of the label
components may be via direct contact, e.g., a covalent or
non-covalent contact between two moieties (e.g., a protein-protein
contact, or collisional energy transfer between proximal moieties);
it may comprise resonance energy transfer (e.g., between one or
more dyes, or between a dye and a quencher moieties); it may
comprise a diffusion effect, e.g., wherein the product from a
reaction occurring at the site of one label diffuses to the site of
another label to create a detectable effect. The components of an
interactive label may be the same (e.g., two or more of the same
molecule or atom) or they may be different.
[0023] As used herein, the term "distinct" in reference to signals
refers to signals that can be differentiated one from another,
e.g., by spectral properties such as fluorescence emission
wavelength, color, absorbance, mass, size, fluorescence
polarization properties, charge, etc., or by capability of
interaction with another moiety, such as with a chemical reagent,
an enzyme, an antibody, etc.
[0024] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides such as an oligonucleotide or a target
nucleic acid) related by the base-pairing rules. For example, for
the sequence "5'-A-G-T-3'," is complementary to the sequence
"3'-T-C-A-5'." Complementarity may be "partial," in which only some
of the nucleic acids' bases are matched according to the base
pairing rules. Or, there may be "complete" or "total"
complementarity between the nucleic acids. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods that depend
upon binding between nucleic acids. Either term may also be used in
reference to individual nucleotides, especially within the context
of polynucleotides. For example, a particular nucleotide within an
oligonucleotide may be noted for its complementarity, or lack
thereof, to a nucleotide within another nucleic acid strand, in
contrast or comparison to the complementarity between the rest of
the oligonucleotide and the nucleic acid strand.
[0025] The term "homology" and "homologous" refers to a degree of
identity. There may be partial homology or complete homology. A
partially homologous sequence is one that is less than 100%
identical to another sequence.
[0026] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is influenced by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, and the T.sub.m of the
formed hybrid. "Hybridization" methods involve the annealing of one
nucleic acid to another, complementary nucleic acid, i.e., a
nucleic acid having a complementary nucleotide sequence. The
ability of two polymers of nucleic acid containing complementary
sequences to find each other and anneal through base pairing
interaction is a well-recognized phenomenon. The initial
observations of the "hybridization" process by Marmur and Lane,
Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc.
Natl. Acad. Sci. USA 46:461 (1960) have been followed by the
refinement of this process into an essential tool of modern
biology.
[0027] The complement of a nucleic acid sequence as used herein
refers to an oligonucleotide which, when aligned with the nucleic
acid sequence such that the 5' end of one sequence is paired with
the 3' end of the other, is in "antiparallel association." Certain
bases not commonly found in natural nucleic acids may be included
in the nucleic acids of the present invention and include, for
example, inosine and 7-deazaguanine. Complementarity need not be
perfect; stable duplexes may contain mismatched base pairs or
unmatched bases. Those skilled in the art of nucleic acid
technology can determine duplex stability empirically considering a
number of variables including, for example, the length of the
oligonucleotide, base composition and sequence of the
oligonucleotide, ionic strength and incidence of mismatched base
pairs.
[0028] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. Several
equations for calculating the T.sub.m of nucleic acids are well
known in the art. As indicated by standard references, a simple
estimate of the T.sub.m value may be calculated by the equation:
T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization (1985). Other
references (e.g., Allawi, H. T. & SantaLucia, J., Jr.
Thermodynamics and NMR of internal G. T mismatches in DNA.
Biochemistry 36, 10581-94 (1997) include more sophisticated
computations which take structural and environmental, as well as
sequence characteristics into account for the calculation of
T.sub.m.
[0029] The term "gene" refers to a DNA sequence that comprises
control and coding sequences necessary for the production of an RNA
having a non-coding function (e.g., a ribosomal or transfer RNA), a
polypeptide or a precursor. The RNA or polypeptide can be encoded
by a full length coding sequence or by any portion of the coding
sequence so long as the desired activity or function is
retained.
[0030] The term "wild-type" refers to a gene or a gene product that
has the characteristics of that gene or gene product when isolated
from a naturally occurring source. A wild-type gene is that which
is most frequently observed in a population and is thus arbitrarily
designated the "normal" or "wild-type" form of the gene. In
contrast, the term "modified", "mutant" or "polymorphic" refers to
a gene or gene product which displays modifications in sequence and
or functional properties (i.e., altered characteristics) when
compared to the wild-type gene or gene product. It is noted that
naturally-occurring mutants can be isolated; these are identified
by the fact that they have altered characteristics when compared to
the wild-type gene or gene product.
[0031] The term "recombinant DNA vector" as used herein refers to
DNA sequences containing a desired heterologous sequence. For
example, although the term is not limited to the use of expressed
sequences or sequences that encode an expression product, in some
embodiments, the heterologous sequence is a coding sequence and
appropriate DNA sequences necessary for either the replication of
the coding sequence in a host organism, or the expression of the
operably linked coding sequence in a particular host organism. DNA
sequences necessary for expression in prokaryotes include a
promoter, optionally an operator sequence, a ribosome binding site
and possibly other sequences. Eukaryotic cells are known to utilize
promoters, polyadenlyation signals and enhancers.
[0032] The term "oligonucleotide" as used herein is defined as a
molecule comprising two or more deoxyribonucleotides or
ribonucleotides, preferably at least 5 nucleotides, more preferably
at least about 10-15 nucleotides and more preferably at least about
15 to 30 nucleotides. The exact size will depend on many factors,
which in turn depend on the ultimate function or use of the
oligonucleotide. The oligonucleotide may be generated in any
manner, including chemical synthesis, DNA replication, reverse
transcription, PCR, or a combination thereof.
[0033] Because mononucleotides are reacted to make oligonucleotides
in a manner such that the 5' phosphate of one mononucleotide
pentose ring is attached to the 3' oxygen of its neighbor in one
direction via a phosphodiester linkage, an end of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring and
as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of
a subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5' and 3' ends. A first region along a nucleic
acid strand is said to be upstream of another region if the 3' end
of the first region is before the 5' end of the second region when
moving along a strand of nucleic acid in a 5' to 3' direction.
[0034] When two different, non-overlapping oligonucleotides anneal
to different regions of the same linear complementary nucleic acid
sequence, and the 3' end of one oligonucleotide points towards the
5' end of the other, the former may be called the "upstream"
oligonucleotide and the latter the "downstream" oligonucleotide.
Similarly, when two overlapping oligonucleotides are hybridized to
the same linear complementary nucleic acid sequence, with the first
oligonucleotide positioned such that its 5' end is upstream of the
5' end of the second oligonucleotide, and the 3' end of the first
oligonucleotide is upstream of the 3' end of the second
oligonucleotide, the first oligonucleotide may be called the
"upstream" oligonucleotide and the second oligonucleotide may be
called the "downstream" oligonucleotide.
[0035] The term "primer" refers to an oligonucleotide that is
capable of acting as a point of initiation of synthesis when placed
under conditions in which primer extension is initiated. An
oligonucleotide "primer" may occur naturally, as in a purified
restriction digest or may be produced synthetically.
[0036] A primer is selected to be "substantially" complementary to
a strand of specific sequence of the template. A primer must be
sufficiently complementary to hybridize with a template strand for
primer elongation to occur. A primer sequence need not reflect the
exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer,
with the remainder of the primer sequence being substantially
complementary to the strand. Non-complementary bases or longer
sequences can be interspersed into the primer, provided that the
primer sequence has sufficient complementarity with the sequence of
the template to hybridize and thereby form a template primer
complex for synthesis of the extension product of the primer.
[0037] The term "thermostable" when used in reference to an enzyme,
such as a 5' nuclease, indicates that the enzyme is functional or
active (i.e., can perform catalysis) at an elevated temperature,
i.e., at about 55.degree. C. or higher.
[0038] The term "target nucleic acid" refers to a nucleic acid
molecule containing a sequence that has at least partial
complementarity with at least a probe or primer sequence. The
target nucleic acid may comprise single- or double-stranded DNA or
RNA.
[0039] The term "substantially single-stranded" when used in
reference to a nucleic acid substrate means that the substrate
molecule exists primarily as a single strand of nucleic acid in
contrast to a double-stranded substrate which exists as two strands
of nucleic acid which are held together by inter-strand base
pairing interactions.
[0040] The term "sequence variation" as used herein refers to
differences in nucleic acid sequence between two nucleic acids. For
example, a wild-type structural gene and a mutant form of this
wild-type structural gene may vary in sequence by the presence of
single base substitutions and/or deletions or insertions of one or
more nucleotides. These two forms of the structural gene are said
to vary in sequence from one another. A second mutant form of the
structural gene may exist. This second mutant form is said to vary
in sequence from both the wild-type gene and the first mutant form
of the gene.
[0041] The term "K.sub.m" as used herein refers to the
Michaelis-Menten constant for an enzyme and is defined as the
concentration of the specific substrate at which a given enzyme
yields half its maximum velocity in an enzyme catalyzed
reaction.
[0042] The term "natural" as used herein to describe nucleotides
and bases refers to the most common components of DNA and RNA
nucleic acid, i.e., A, C, G, T and U nucleotides.
[0043] The term "nucleotide analog", "non-natural", or
"non-naturally occurring" as used herein refers to nucleotides
other than the natural nucleotides and bases. Such analogs and
non-natural bases and nucleotides include modified natural
nucleotides and non-naturally occurring nucleotides, including but
not limited to analogs that have altered stacking interactions such
as 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP); base
analogs with alternative hydrogen bonding configurations (e.g.,
such as iso-C and iso-G and other non-standard base pairs described
in U.S. Pat. No. 6,001,983 to S. Benner, and the selectively
binding base analogs described in U.S. Pat. No. 5,912,340 to Igor
V. Kutyavin, et al.); non-hydrogen bonding analogs (e.g.,
non-polar, aromatic nucleoside analogs such as 2,4-difluorotoluene,
described by B. A. Schweitzer and E. T. Kool, J. Org. Chem., 1994,
59, 7238-7242, B. A. Schweitzer and E. T. Kool, J. Am. Chem. Soc.,
1995, 117, 1863-1872); "universal" bases such as 5-nitroindole and
3-nitropyrrole; and universal purines and pyrimidines (such as "K"
and "P" nucleotides, respectively; P. Kong, et al., Nucleic Acids
Res., 1989, 17, 10373-10383, P. Kong et al., Nucleic Acids Res.,
1992, 20, 5149-5152). Nucleotide analogs include modified forms of
deoxyribonucleotides as well as ribonucleotides. "Non-natural" and
"non-naturally occurring" bases and nucleotides are specifically
not limited to such bases as are never found in nature. Natural
processes such as nucleic acid damage can give rise to "natural"
occurrence of bases that are nonetheless not generally considered
to be part of the set of "natural" nucleotides as defined herein.
For example, iso-G can be found in oxidatively damaged DNA. Such
non-natural bases and their behaviors in replication and other
nucleic acid syntheses have been extensively studied in contexts
such as DNA damage studies, although the compounds are sometimes
described using different nomenclature. For example, the
ribonucleoside comprising the isoguanosine base has been referred
to in the literature variously as: iG; isoG; iso-G; isoguanosine;
2-hydroxyadenine; 2-oxoadenine; 2-hydroxy A; and 2-OH-A. The
deoxyribonucleoside comprising the isoguanosine base has been
referred to variously as: iG; isoG; iso dG; deoxyiso-G;
deoxyisoguanosine; 2-hydroxydeoxyadenosine; 2-hydroxy dA; and
2-OH-Ade.
[0044] The prefix "dideoxy" as use herein to describe a nucleotide
(e.g., dideoxyisoguanosine triphosphate, dideoxy G), refers to a
nucleotide lacking hydroxyl group at both the 2' and 3' positions
of the sugar moiety (e.g., the ribose in a natural nucleotide). The
prefix is not intended to indicate the absence of any other
particular group at such positions, and the sugar may comprise
other moieties at one or both positions.
[0045] The term "polymorphic locus" is a locus present in a
population that shows variation between members of the population
(e.g., the most common allele has a frequency of less than 0.95).
In contrast, a "monomorphic locus" is a genetic locus at little or
no variations seen between members of the population (generally
taken to be a locus at which the most common allele exceeds a
frequency of 0.95 in the gene pool of the population).
[0046] The term "microorganism" as used herein means an organism
too small to be observed with the unaided eye and includes, but is
not limited to bacteria, virus, protozoans, fungi, and
ciliates.
[0047] The term "microbial gene sequences" refers to gene sequences
derived from a microorganism.
[0048] The term "bacteria" refers to any bacterial species
including eubacterial and archaebacterial species.
[0049] The term "virus" refers to obligate, ultramicroscopic,
intracellular parasites incapable of autonomous replication (i.e.,
replication requires the use of the host cell's machinery).
[0050] The term "multi-drug resistant" or multiple-drug resistant"
refers to a microorganism that is resistant to more than one of the
antibiotics or antimicrobial agents used in the treatment of said
microorganism.
[0051] The term "sample" in the present specification and claims is
used in its broadest sense. On the one hand it is meant to include
a specimen or culture (e.g., microbiological cultures). On the
other hand, it is meant to include both biological and
environmental samples. A sample may include a specimen of synthetic
origin.
[0052] Biological samples may be animal, including human, fluid,
solid (e.g., stool) or tissue, as well as liquid and solid food and
feed products and ingredients such as dairy items, vegetables, meat
and meat by-products, and waste. Biological samples may be obtained
from all of the various families of domestic animals, as well as
feral or wild animals, including, but not limited to, such animals
as ungulates, bear, fish, lagamorphs, rodents, etc.
[0053] Environmental samples include environmental material such as
surface matter, soil, water and industrial samples, as well as
samples obtained from food and dairy processing instruments,
apparatus, equipment, utensils, disposable and non-disposable
items. These examples are not to be construed as limiting the
sample types applicable to the present invention.
[0054] The term "source of target nucleic acid" refers to any
sample that contains nucleic acids (RNA or DNA). Particularly
preferred sources of target nucleic acids are biological samples
including, but not limited to blood, saliva, cerebral spinal fluid,
pleural fluid, milk, lymph, sputum and semen.
[0055] The term "nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin
which may be single or double stranded, and represent the sense or
antisense strand. Similarly, "amino acid sequence" as used herein
refers to peptide or protein sequence.
[0056] As used herein, the terms "purified" or "substantially
purified" refer to molecules, either nucleic or amino acid
sequences, that are removed from their natural environment,
isolated or separated, and are at least 60% free, preferably 75%
free, and most preferably 90% free from other components with which
they are naturally associated. An "isolated polynucleotide" or
"isolated oligonucleotide" is therefore a substantially purified
polynucleotide.
[0057] The term "continuous strand of nucleic acid" as used herein
is means a strand of nucleic acid that has a continuous, covalently
linked, backbone structure, without nicks or other disruptions. The
disposition of the base portion of each nucleotide, whether
base-paired, single-stranded or mismatched, is not an element in
the definition of a continuous strand. The backbone of the
continuous strand is not limited to the ribose-phosphate or
deoxyribose-phosphate compositions that are found in naturally
occurring, unmodified nucleic acids. A nucleic acid of the present
invention may comprise modifications in the structure of the
backbone, including but not limited to phosphorothioate residues,
phosphonate residues, 2' substituted ribose residues (e.g.,
2'-O-methyl ribose) and alternative sugar (e.g., arabinose)
containing residues.
[0058] The term "continuous duplex" as used herein refers to a
region of double stranded nucleic acid in which there is no
disruption in the progression of basepairs within the duplex (i.e.,
the base pairs along the duplex are not distorted to accommodate a
gap, bulge or mismatch with the confines of the region of
continuous duplex). As used herein the term refers only to the
arrangement of the basepairs within the duplex, without implication
of continuity in the backbone portion of the nucleic acid strand.
Duplex nucleic acids with uninterrupted basepairing, but with nicks
in one or both strands are within the definition of a continuous
duplex.
[0059] The term "duplex" refers to the state of nucleic acids in
which the base portions of the nucleotides on one strand are bound
through hydrogen bonding the their complementary bases arrayed on a
second strand. The condition of being in a duplex form reflects on
the state of the bases of a nucleic acid. By virtue of base
pairing, the strands of nucleic acid also generally assume the
tertiary structure of a double helix, having a major and a minor
groove. The assumption of the helical form is implicit in the act
of becoming duplexed.
[0060] The term "template" refers to a strand of nucleic acid on
which a complementary copy is built from nucleoside triphosphates
through the activity of a template-dependent nucleic acid
polymerase. Within a duplex the template strand is, by convention,
depicted and described as the "bottom" strand. Similarly, the
non-template strand is often depicted and described as the "top"
strand.
DESCRIPTION OF DRAWINGS
[0061] FIG. 1 shows a schematic diagram of one embodiment of the
present invention.
[0062] FIG. 2 shows a schematic diagram of one embodiment of the
present invention.
[0063] FIG. 3 shows a schematic diagram of one embodiment of the
present invention.
[0064] FIG. 4 shows a schematic diagram of one embodiment of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The present invention relates to improved methods and
composition for nucleic acid analysis. In particular, the present
invention provides improved methods and compositions for carrying
out nucleic acid analysis using modified nucleotides.
[0066] In some embodiments, the present invention provides
extension-disabled non-naturally occurring nucleotides for use in
nucleic acid analysis technologies. "Extension-disabled" refers to
nucleotides that are modified to substantially reduce or eliminate
the ability of a polymerase to extend a growing nucleic acid chain
once the extension-disabled nucleotide has been added. In some
embodiments, the extension disabled non-naturally occurring
nucleotide is a dideoxy non-naturally occurring nucleotide.
[0067] Nucleic acid methods, such as the GENECODE methods of Eragen
Corporation employ complementary non-naturally occurring
nucleotides such as iso-G and iso-C to expand the alphabet of the
genetic code for nucleic acid analysis. In these methods, a
non-naturally occurring nucleotide is present in a primer. When the
primer is extended in, for example, a polymerase chain reaction,
the non-natural base becomes incorporated into one strand of the
amplification products. When this strand is used as a template in a
subsequent amplification step, Eragen reports that the natural
bases will not incorporate across from the non-natural base. This
failure to incorporate is used as a point of discrimination in some
GENECODE methods (e.g., by allowing only labeled, complementary
non-natural bases to be added).
[0068] In some embodiments, a nucleoside triphosphate comprising a
second non-naturally occurring base is included in amplifications
in which a first non-naturally occurring base is used in a primer.
The second non-naturally occurring base is selected to be
complementary to the first non-naturally occurring base. When the
strand comprising the primer is used as a template in a subsequent
amplification step, the second non-naturally occurring base is
incorporated into the new strand only where the first non-naturally
occurring base is present in the template. This selective
incorporation is used as a point of discrimination in some GENECODE
methods (e.g., by allowing labeled, complementary non-natural bases
to be added only where a template strand comprises a non-natural
base).
[0069] However, the GENECODE literature does not appreciate a
problem with such methods. Non-natural bases such as iso-G, which
are used in the GENECODE technologies, are capable of forming base
pairs with natural bases. For example, iso-G will basepair with T
and U. At low temperatures, e.g., 2.degree. C., basepairs between
iso-G and T have been shown to be predominantly are in the wobble
form. As the temperature of a reaction is increased (i.e., as
occurs in a polymerase chain reaction), another form or iso-G, the
enol form, becomes significantly populated. This form of iso-G
pairs with T in a Watson-Crick configuration to a significant
extent at physiological temperature (37.degree. C.). (H. Robinson,
et al., Biochemistry 1998 Aug. 4; 37(31):10897-905). Consequently,
iso-G and T can serve as complementary bases in DNA replication
reactions. For example, a number of DNA polymerases have been shown
to catalyze the template-directed formation of a base pair between
(iso-G and T). In a template, iso-G directs the incorporation of
both iso-C and T when Klenow fragment is the catalyst. Some
polymerases will preferentially incorporate a natural base as a
complement of iso-G. For example, T7 RNA polymerase will
incorporate only U as a complement to iso-G in RNA synthesis, even
when iso-CTP is present in the reaction. (C. Switzer, et al.,
Biochemistry 1993 Oct. 5; 32(39):10489-96).
[0070] Where labeled iso-G bases are inadvertently incorporated
(e.g., as a complement to T or U residues in a template strand)
background signal in methods such as the GENECODE method increases.
The present invention provides compositions and methods for
avoiding this problem.
[0071] In one preferred method, an extension-disabled non-naturally
occurring nucleotides is used in the reaction. In such methods, the
only complete amplicons created in a chain extension reaction
(e.g., a GENECODE reaction) will be those that avoid the undesired
incorporation of the non-naturally occurring nucleotides.
[0072] In some embodiments, the accumulation of PCR product may be
monitored by quenching the signal of a label on the second primer
by site-specific incorporation of a nucleotide triphosphate across
the DNA duplex at a position near the label of the second primer.
The labeled nucleoside triphosphate is incorporated into the
elongating first primer during PCR extension. The label on the
labeled nucleoside triphosphate is capable of quenching the label
on the second primer. In an alternative embodiment, fluorescence
energy transfer (FRET) can be observed between the label of the
second primer (donor dye) and a label on the nucleotide (acceptor
dye). Detection of PCR product can be observed by exciting the
donor dye and reading the emission of the incorporated acceptor
dye.
[0073] In design of these systems it is preferred that the labeled
nucleoside triphosphate is complementary to a nucleotide near the
label of the primer. When using a naturally occurring nucleotide
base that is labeled, the ability to selectively incorporate such a
complementary base only near the label of the primer is possible
only in a limited number of cases. This is because all four
naturally occurring nucleotide bases are likely to be incorporated
at other positions during amplification of a target sequence. By
using labeled non-natural bases, e.g., labeled iso-G and iso-C, the
likelihood of incorporating a labeled base only opposite a
complimentary non-natural base is increased. However, as described
above, some non-naturally occurring bases basepair not only to a
non-naturally occurring complement (e.g., iso-G to iso-C), but also
basepair and are incorporated opposite one or more natural bases
(e.g., iso-G and T). In some embodiments, it is desirable to
control further extension of strands after the incorporation of
such a labeled base, e.g., by using a labeled, extension-disabled
non-naturally occurring nucleotide. Thus, when a labeled
non-natural base is incorporated opposite a natural base, the chain
is terminated. Such termination products can be later distinguished
from the full-length intended products by virtue of size, or of
differences in fluorescence characteristics (i.e., the incorporated
label will be at a different distance from the primer label and
will have different characteristics of quenching and/or energy
transfer).
[0074] In some embodiments, PCR amplification products containing
fluorophores quenched by site-specific incorporation of a quenching
compound are subjected to melt curve analysis, e.g., in an
instrument that can monitor fluorescence differences during
temperature changes. The change in fluorescence is monitored while
gradually increasing the temperature of the PCR reaction products
(e.g., at a rate of 0.1.degree. C. per second). The Tm of the
intended product (quencher-incorporated PCR product) as well as
that of any nonspecific product (e.g., quencher-incorporated
products that have been chain-terminated prematurely using the
extension-disabled non-natural nucleotides of the present
invention, or quencher-incorporated primer/dimers) may thus be
determined. If the Tm of the intended product is selected to be
substantially higher than the Tm of primer/dimers and the
terminated products, the signal generated by the intended product
may be specifically observed (and thus the presence or absence, or
the quantity of the initial target material may be determined) by
taking the fluorescent measurement of the reaction at a temperature
above the Tm of the nonspecific products.
[0075] In some embodiments, incorporation of a non-natural base
opposite a natural base is reduced by selection of a target region
of particular sequence. For example, in some embodiments, the
region that is amplified is selected to reduce or eliminate the
presence of a natural base pair to the non-naturally occurring base
(e.g., T-containing region in the target are avoided or reduced if
labeled iso-GTP is used in the amplification reaction). This
technique can be used alone, or in combination with the extension
disabled non-naturally occurring nucleotides methods described
above.
[0076] In some such embodiments, the selection of target sequences
is carried out by "eye"--i.e., a user manually scans the target
sequence to select a region with the desired sequence (e.g., one
that contains no "T"s or is low in "T"s or has "T"s positioned such
that they are less detrimental to the signal detection). In other
embodiments, a processor is used to identify preferred target
sequence by, for example, assessing the presence of, amount or,
and/or location of bases to be avoided. The processor may further
take into account other factors in selecting target regions. For
example, the presence of undesired bases may be assessed in
combination with hybridization characteristics, secondary
structure, intellectual property considerations (e.g., the
avoidance of particular sequences that are patented by others)
etc.
[0077] Thus, the present invention provides systems and methods for
applying target sequence information to a processor, parsing the
sequence information with one or more algorithms, and generating
one or more candidate target sequences. In some embodiments, the
processor provides a report that provides estimated performance
profiles of the assay to permit a user to select the appropriate
target region or regions. In some embodiments, the algorithm
identifies target regions by assessing one or more of: the presence
of undesired bases, the location of undesired bases, the number of
undesired bases, and the distance of undesired bases from the
position of a detection moiety to be used in an assay (e.g., to
reduce or eliminate undesired quenching or lack of quenching in a
FRET assay by the undesired incorporation of a labeled non-natural
nucleotide across from the undesired base).
[0078] Using the methods of the present invention, one is able to
conduct techniques such as the GENECODE methods with longer
amplicons (e.g., greater than 50 nucleotides, greater than 100,
greater than 500, greater than 1000, greater than 2000, etc.),
without incurring detrimental background signal.
Primer Dimer Suppression
[0079] In some embodiments, the present invention provides improved
methods of suppressing background signal from "primer-dimer"
formation in polymerase chain reactions. As described above, in
some embodiments, the accumulation of PCR product may be monitored
by quenching the signal of a label on the second primer by
site-specific incorporation of a nucleotide triphosphate across the
DNA duplex at a position near the label of the second primer. In
some embodiments, labeled nucleoside triphosphate is incorporated
into the elongating first primer during PCR extension. The label on
the labeled nucleoside triphosphate is capable of quenching the
label on the second oligonucleotide primer (see, e.g., FIGS. 1 and
2). In an alternative embodiment, fluorescence energy transfer
(FRET) can be observed between the label of the second
oligonucleotide primer (donor dye) and a label on the nucleotide
(acceptor dye) (see, e.g., FIGS. 3 and 4). Detection of PCR product
can be observed by exciting the donor dye and reading the emission
of the incorporated acceptor dye. In yet other alternatives, the
second primer may be labeled with an acceptor dye and the label on
the nucleotide to be incorporated may be a donor dye. In any of
these embodiments, non-specific amplification products also result
in the incorporation of a labeled non-natural nucleotide as the
complement to the non-natural nucleotide in the second primer.
Thus, non-specific amplification products can result in label
quenching or energy transfer that can be difficult to distinguish
from the intended signal. This problem can be especially acute with
non-specific products termed "primer-dimer" products, wherein the
PCR primers create a small amplicon that consists essentially of
the two primer sequences. These small amplification products tend
to amplify very efficiently and can even become the dominant
product in a reaction under certain conditions.
[0080] The present invention provides methods and compositions to
improve the sensitivity of PCR detection using labeled non-natural
nucleotides by both suppressing formation of primer-dimer and other
non-specific amplification products, and by reducing the signal
effect of any such products that do form during a reaction. In
preferred embodiments, the present invention provides primers that
provide an inhibitory effect on the formation of primer-dimer and
other small amplification products. It has been shown that when
primers have regions of homology that are incorporated into a
duplex fragment via PCR, each resulting strand of the PCR duplex
comprises a pair of complementary regions that can, if the PCR
strands are separated, anneal to each other to form a stem and loop
or "panhandle" structure. Because the complementary regions are
introduced by the primers, e.g., in the form of tails comprising
"tag" sequences, the formation of the panhandle structure involves
annealing of the primer-binding sites, and thus it inhibits further
amplification by blocking the annealing of new primers. The local
concentration of the complementary sequence provided from the
opposite end of a stand is much higher than the concentration of
the complementary primers and thus the other end of the stand can
occupy the primer binding site before a primer has a chance to
anneal. For a very short duplex such as a primer-dimer product, the
panhandle structure formation can dramatically reduce amplification
of the primer-dimer product.
[0081] In a standard labeling system (i.e., using non-interactive
labels), any primer-dimer product that does form can be detected,
even if the signal is significantly reduced. Using the interactive
labels described above, however, the panhandle formation is useful
not just in suppressing further amplification of the primer-dimer
product, but also in reducing detectable signal from any such
amplicon that forms. One embodiment is diagramed in FIG. 1. The
left side of this figure shows one embodiment of the detection
method described above, wherein a non-natural nucleotide comprising
a quencher moiety is incorporated into a PCR product opposite a
non-natural nucleotide having a fluorescent label. In this
embodiment, the PCR primers comprise a 5' tail region that is not
complementary to the target nucleic acid, and each primer has the
same 5' tail sequence. Upon amplification, the 5' tail region of
each primer serves as template for the formation of the
complementary sequence on the 3' end of each completed strand. When
the intended target is amplified, the product is long enough that
the complementary regions on the ends of each strand provide
insignificant interference with subsequent primer binding.
[0082] The right side of FIG. 1 diagrams the formation of a
primer-dimer product using the same primers. The short product
having complementary ends easily forms a panhandle structure that
inhibits further amplification. Nonetheless, any amplification that
does occur does incorporate the label-quencher pair. However, upon
heating and cooling, these product again form the panhandle
structure. Formation of this structure effectively separates the
dye and quencher moieties, thus making the panhandle products
invisible to the detection system that detects only their
proximity.
[0083] In some embodiments, initial target specific priming occurs
using primers that each comprise a non-target tail comprising a
"tag" sequence, wherein the tag sequence is incorporated into
amplification products. Further amplification is carried out using
tag primers. The sequence of the tag primer is conveniently
identical to the sequence of the tag sequence in the tails of the
initial primers. The tag primer preferably comprises a sequence
capable of hybridization to all tag sequences. All tag sequences
are preferably identical. It will be appreciated that minor changes
may be made to the sequence of the tag primer without affecting its
performance to any significant extent.
[0084] Tailed primers can be used to prevent the formation of
primer-dimers and other inter-primer artifacts. While not being
limited to any particular mechanism, it is believed that the
formation of primer dimers is dependent on some degree of homology
between primers and their use at high concentrations. It may be
possible to reduce the formation of primer dimers by careful primer
design. However where many primers are used at high concentrations,
for example in PCR multiplexes, this becomes more difficult. With
the use of tag primers, the target-specific diagnostic primers may
be used at concentrations that allow satisfactory priming on their
genomic template(s) but do not allow significant PCR amplification
or primer-dimer formation. In addition, the presence of
complementary sequences on the ends of the short primer-dimer
strands that do form cause these strands to preferentially fold on
themselves to inhibit further amplification. See, e.g., U.S. Pat.
No. 5,565,340 to Chenchik, et al., U.S. Pat. No. 6,270,967 to
Whitcombe, et al., and Brownie, et al., Nucleic Acids Res. 25(16):
3235-41 (1997), each of which is incorporated herein by reference.
Tag primers are easily adapted for use with the non-natural
nucleotide compositions and methods of the present invention.
[0085] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention that are obvious to those skilled in the relevant fields
are intended to be within the scope of the following claims.
* * * * *