U.S. patent application number 12/351741 was filed with the patent office on 2011-02-10 for methods for amplification of nucleic acids using spanning primers.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION, a Delaware Corporation. Invention is credited to Eugene G. SPIER.
Application Number | 20110033845 12/351741 |
Document ID | / |
Family ID | 38119218 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110033845 |
Kind Code |
A1 |
SPIER; Eugene G. |
February 10, 2011 |
Methods For Amplification of Nucleic Acids Using Spanning
Primers
Abstract
The teachings relate to methods and kits for detecting whether
target nucleic acid sequences are present and/or quantitating
target nucleic acid sequences.
Inventors: |
SPIER; Eugene G.; (Palo
Alto, CA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
LIFE TECHNOLOGIES CORPORATION, a
Delaware Corporation
Carlsbad
CA
|
Family ID: |
38119218 |
Appl. No.: |
12/351741 |
Filed: |
January 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11542584 |
Oct 2, 2006 |
7485425 |
|
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12351741 |
|
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60723579 |
Oct 3, 2005 |
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Current U.S.
Class: |
435/6.12 ;
435/91.5 |
Current CPC
Class: |
C12Q 1/6858 20130101;
C12Q 1/6858 20130101; C12Q 1/6858 20130101; C12Q 2561/125 20130101;
C12Q 2561/125 20130101; C12Q 2525/307 20130101; C12Q 2531/113
20130101; C12Q 2525/307 20130101; C12Q 2525/161 20130101 |
Class at
Publication: |
435/6 ;
435/91.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Claims
1. A method for amplifying at least one target nucleic acid
sequence comprising: forming an amplification reaction composition
comprising: a target nucleic acid sequence; a polymerase; a first
primer comprising (i) a sequence complementary to the 5' end of the
target nucleic acid sequence and (ii) a sequence complementary to
the 3' end of the target nucleic acid sequence; and subjecting the
amplification reaction composition to at least one amplification
reaction to form at least one amplification product.
2. The method of claim 1, wherein the amplification reaction
composition further comprises a second primer comprising (i) a
sequence complementary to the 3' end of a complement of the target
nucleic acid sequence and (ii) a sequence complementary to any
portion of the first primer.
3. The method of claim 1, wherein the amplification reaction
composition further comprises a second primer comprising (i) a
sequence complementary to the 3' end of a complement of the target
nucleic acid sequence and (ii) a sequence complementary to the 5'
end of the first primer.
4. The method of claim 2, wherein the second primer comprises a
thymidine between (i) the sequence complementary to the 3' end of a
complement of the target nucleic acid sequence and (ii) the
sequence complementary to any portion of the first primer.
5. The method of claim 3, wherein the second primer comprises a
thymidine between (i) the sequence complementary to the 3' end of a
complement of the target nucleic acid sequence and (ii) the
sequence complementary to the 5' end of the first primer.
6. The method of claim 1, wherein the 3' end of the target nucleic
acid sequence is blocked.
7. The method of claim 1, wherein the amplification reaction
composition further comprises at least one probe.
8. The method of claim 7, further comprising detecting the at least
one amplification product.
9. The method of claim 1, wherein the amplification reaction
comprises an annealing step that takes place at a predetermined
annealing temperature, and wherein the annealing temperature of two
first cycles of amplification is 65.degree. C., and is increased to
at least 70.degree. C. for subsequent cycles of amplification.
10. The method of claim 1, wherein the amplification reaction
comprises an annealing step that takes place at a predetermined
annealing temperature, and wherein the annealing temperature is
70.degree. C. or greater.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/723,579, filed Oct. 3, 2005, and U.S. Utility
application Ser. No. 11/542,584, filed Oct. 2, 2006 which is
incorporated by reference herein for any purpose.
FIELD
[0002] The teachings relate to methods and kits for the
amplification of target nucleic acids.
BACKGROUND
[0003] The detection of the presence or absence of (or quantity of)
one or more target nucleic acid sequences in a sample containing
one or more target nucleic acid sequences is commonly practiced.
For example, the detection of cancer and many infectious diseases,
such as AIDS and hepatitis, routinely includes screening biological
samples for the presence or absence of diagnostic nucleic acid
sequences. Also, detecting the presence or absence of nucleic acid
sequences is often used in forensic science, paternity testing,
genetic counseling, and organ transplantation. Certain
amplification reactions are useful in certain research, diagnostic,
medical, forensic and industrial fields. In certain instances, an
amplification reaction generates amplification products for use in
downstream assays. In certain instances, an amplification reaction
generates reaction products for forensic or diagnostic
purposes.
SUMMARY
[0004] A method for amplifying at least one target nucleic acid
sequence is provided, comprising:
[0005] forming an amplification reaction composition comprising:
[0006] a target nucleic acid sequence; [0007] a polymerase; [0008]
a first primer comprising (i) a sequence complementary to the 5'
end of the target nucleic acid sequence and (ii) a sequence
complementary to the 3' end of the target nucleic acid sequence;
and
[0009] subjecting the amplification reaction composition to at
least one amplification reaction to form at least one amplification
product.
[0010] A method for determining whether at least one target nucleic
acid sequence is present in a sample is provided, comprising:
[0011] forming a ligation reaction composition comprising the
sample, and a ligation probe set for each target nucleic acid
sequence, the probe set comprising (a) a first probe, comprising a
first target-specific portion, and (b) a second probe, comprising a
second target-specific portion, wherein the probes in each set are
suitable for ligation together when hybridized adjacent to one
another on the complementary target nucleic acid sequence;
[0012] forming a first test composition by subjecting the ligation
reaction composition to at least one cycle of ligation, wherein
adjacently hybridizing complementary probes are ligated to one
another to form a ligation product comprising the first probe and
the second probe;
[0013] forming an amplification reaction composition comprising:
[0014] at least some of the first test composition; [0015] a
polymerase; [0016] a first primer comprising (i) a sequence
complementary to the 5' end of the ligation product and (ii) a
sequence complementary to the 3' end of the ligation product;
[0017] forming a second test composition by subjecting the
amplification reaction composition to at least one amplification
reaction, wherein the second test composition comprises at least
one amplification product if a target nucleic acid sequence is
present in the sample; and
[0018] determining whether the at least one target nucleic acid
sequence is present by detecting at least one amplification
product.
[0019] A method for determining whether at least one target nucleic
acid sequence is present in a sample is provided, comprising:
[0020] (a) forming a reaction composition comprising: [0021] the
sample; [0022] a ligation probe set for each target nucleic acid
sequence, the probe set comprising (i) at least one first probe,
comprising a first target-specific portion and (ii) at least one
second probe, comprising a second target-specific portion, [0023]
wherein the probes in each set are suitable for ligation together
to form a ligation product when hybridized adjacent to one another
on a complementary target nucleic acid sequence; [0024] a
polymerase; and [0025] a first primer comprising (i) a sequence
complementary to the 5' end of the ligation product and (ii) a
sequence complementary to the 3' end of the ligation product;
[0026] (b) subjecting the reaction composition to at least one
cycle of ligation, wherein adjacently hybridizing complementary
probes are ligated to one another to form a ligation product
comprising the first probe and the second probe;
[0027] (c) after the at least one cycle of ligation, subjecting the
reaction composition to at least one amplification reaction to form
at least one amplification product if a target nucleic acid
sequence is present in the sample; and
[0028] (d) determining whether the at least one target nucleic acid
sequence is present by detecting at least one amplification
product.
[0029] A kit for amplifying at least one target nucleic acid
sequence is provided, comprising a polymerase and a first primer
comprising (i) a sequence complementary to the 5' end of the target
nucleic acid sequence and (ii) a sequence complementary to the 3'
end of the target nucleic acid sequence.
[0030] A kit for amplifying at least one target nucleic acid
sequence is provided, comprising:
[0031] a ligation probe set for each target nucleic acid sequence,
the probe set comprising (a) a first probe, comprising a first
target-specific portion, and (b) a second probe, comprising a
second target-specific portion, wherein the probes in each set are
suitable for ligation together when hybridized adjacent to one
another on a complementary target sequence, a polymerase, and a
first primer comprising (i) a sequence complementary to the 5' end
of the target nucleic acid sequence and (ii) a sequence
complementary to the 3' end of the target nucleic acid
sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0033] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The figures
are not intended to limit the scope of the teachings in any
way.
[0034] FIG. 1 is a schematic diagram depicting a method of linear
amplification of a target nucleic acid sequence using a spanning
primer according to certain embodiments. A spanning primer (110)
hybridizes to both the 3' and the 5' end of a target nucleic acid
sequence (120). In the presence of a polymerase (130), the spanning
primer is elongated to form a first amplification product
(140).
[0035] FIGS. 2A and 2B are schematic diagrams depicting methods of
exponential amplification of a target nucleic acid sequence using a
first spanning primer and a second spanning primer according to
certain embodiments. In FIG. 2A, a first spanning primer (110)
hybridizes to both the 3' and the 5' ends of a target nucleic acid
sequence (120). In the presence of a polymerase (130), the first
spanning primer is elongated to form a first amplification product
(140). A second spanning primer (210) hybridizes to both the 3' end
of the first amplification product (140) and to any portion of the
first spanning primer. The second spanning primer (210) is
elongated by the polymerase (130) to form a polynucleotide
comprising the target nucleic acid sequence with the addition of
the second spanning primer at its 5' end (220). FIG. 2B depicts the
same method as FIG. 2A, except that the second spanning primer
(210) hybridizes to both the 3' end of the first amplification
product (140) and to the 5' end of the first spanning primer, which
is incorporated into the first amplification product (140).
[0036] FIGS. 3A and 3B show an exemplary use of spanning primers to
favor amplification of ligation products over amplification of
unligated nucleic acids, according to certain embodiments.
[0037] FIG. 4 is a schematic diagram depicting a method of ligation
and amplification using a spanning primer according to certain
embodiments. A first probe (310) and a second probe (320) are
hybridized to a target nucleic acid sequence (120) adjacent to one
another, such that the first probe and the second probe are
suitable for ligation together. After at least one cycle of
ligation, a ligation product (330) is formed, comprising the first
probe (310) and the second probe (320). A spanning primer (110)
hybridizes to both the 3' and the 5' ends of the ligation product
(330). In the presence of a polymerase (130), the spanning primer
(110) is elongated to form a first amplification product (340).
[0038] FIG. 5 shows exemplary components of an exemplary
oligonucleotide ligation assay (OLA), according to certain
embodiments.
[0039] FIGS. 6A and 6B show certain exemplary embodiments of
amplification of a ligation product using a spanning primer.
[0040] FIGS. 7A and 7B show certain exemplary embodiments of
amplifying a ligation product using first and second spanning
primers.
[0041] FIGS. 8A and 8B show certain exemplary embodiments of
amplifying a ligation product using first and second spanning
primers.
[0042] FIGS. 9A and 9B show certain exemplary methods of genotyping
using first and second spanning primers.
[0043] FIG. 10 shows certain exemplary embodiments in which
allele-specific probes are used to distinguish between an
amplification product specific for a first allele ("allele 1") of a
target nucleic acid and an amplification product specific for a
second allele ("allele 2") of the target nucleic acid.
[0044] FIG. 11 shows certain exemplary embodiments in which
allele-specific probes are used to distinguish between an
amplification product specific for a first allele ("allele 1") of a
target nucleic acid and an amplification product specific for a
second allele ("allele 2") of the target nucleic acid.
[0045] FIGS. 12A, 12B, and 12C shows certain exemplary embodiments
in which allele-specific primers are used to distinguish between an
amplification product specific for a first allele ("allele 1") of a
target nucleic acid and an amplification product specific for a
second allele ("allele 2") of the target nucleic acid.
[0046] FIGS. 13A and 13B show certain exemplary embodiments of
detection using type II restriction endonucleases.
DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
[0047] In this application, the use of the singular includes the
plural unless specifically stated otherwise. In this application,
the word "a" or "an" means "at least one" unless specifically
stated otherwise. In this application, the use of "or" means
"and/or" unless stated otherwise. In this application, the meaning
of the phrase "at least one" is equivalent to the meaning of the
phrase "one or more." Furthermore, the use of the term "including",
as well as other forms, such as "includes" and "included", is not
limiting. Also, terms such as "element" or "component" encompass
both elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0048] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents cited in this application,
including but not limited to patents, patent applications,
articles, books, and treatises, are hereby expressly incorporated
by reference in their entirety for any purpose. In the event that
one or more of the documents incorporated by reference defines a
term that contradicts that term's definition in this application,
this application controls.
CERTAIN DEFINITIONS
[0049] The term "nucleotide base", as used herein, refers to a
substituted or unsubstituted aromatic ring or rings. In certain
embodiments, the aromatic ring or rings contain at least one
nitrogen atom. In certain embodiments, the nucleotide base is
capable of forming Watson-Crick and/or Hoogsteen hydrogen bonds
with an appropriately complementary nucleotide base. Exemplary
nucleotide bases and analogs thereof include, but are not limited
to, naturally occurring nucleotide bases, e.g., adenine, guanine,
cytosine, 6 methyl-cytosine, uracil, thymine, and analogs of the
naturally occurring nucleotide bases, e.g., 7-deazaadenine,
7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine,
N6-.DELTA.2-isopentenyladenine (6iA),
N6-.DELTA.2-isopentenyl-2-methylthioadenine (2ms6iA),
N2-dimethylguanine (dmG), 7-methylguanine (7mG), inosine,
nebularine, 2-aminopurine, 2-amino-6-chloropurine,
2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine,
pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine,
7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine,
4-thiouracil, O.sup.6-methylguanine, N.sup.6-methyladenine,
O.sup.4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil,
pyrazolo[3,4-D]pyrimidines (see, e.g., U.S. Pat. Nos. 6,143,877 and
6,127,121 and PCT published application WO 01/38584),
ethenoadenine, indoles such as nitroindole and 4-methylindole, and
pyrroles such as nitropyrrole. Certain exemplary nucleotide bases
can be found, e.g., in Fasman, 1989, Practical Handbook of
Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca
Raton, Fla., and the references cited therein.
[0050] The term "nucleotide", as used herein, refers to a compound
comprising a nucleotide base linked to the C-1' carbon of a sugar,
such as ribose, arabinose, xylose, and pyranose, and sugar analogs
thereof. The term nucleotide also encompasses nucleotide analogs.
The sugar may be substituted or unsubstituted. Substituted ribose
sugars include, but are not limited to, those riboses in which one
or more of the carbon atoms, for example the 2'-carbon atom, is
substituted with one or more of the same or different Cl, F, --R,
--OR, --NR.sub.2 or halogen groups, where each R is independently
H, C.sub.1-C.sub.6 alkyl or C.sub.5-C.sub.14 aryl. Exemplary
riboses include, but are not limited to, 2'-(C1-C6)alkoxyribose,
2'-(C5-C14)aryloxyribose, 2',3'-didehydroribose,
2'-deoxy-3'-haloribose, 2'-deoxy-3'-fluororibose,
2'-deoxy-3'-chlororibose, 2'-deoxy-3'-aminoribose,
2'-deoxy-3'-(C1-C6)alkylribose, 2'-deoxy-3'-(C1-C6)alkoxyribose and
2'-deoxy-3'-(C5-C14)aryloxyribose, ribose, 2'-deoxyribose,
2',3'-dideoxyribose, 2'-haloribose, 2'-fluororibose,
2'-chlororibose, and 2'-alkylribose, e.g., 2'-O-methyl,
4'-.alpha.-anomeric nucleotides, 1'-.alpha.-anomeric nucleotides,
2'-4'- and 3'-4'-linked and other "locked" or "LNA", bicyclic sugar
modifications (see, e.g., PCT published application nos. WO
98/22489, WO 98/39352, and WO 99/14226; and U.S. Pat. Nos.
6,268,490 and 6,794,499). Exemplary LNA sugar analogs within a
polynucleotide include, but are not limited to, the structures:
##STR00001##
where B is any nucleotide base.
[0051] Modifications at the 2'- or 3'-position of ribose include,
but are not limited to, hydrogen, hydroxy, methoxy, ethoxy,
allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy,
phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo.
Nucleotides include, but are not limited to, the natural D optical
isomer, as well as the L optical isomer forms (see, e.g., Garbesi
(1993) Nucl. Acids Res. 21:4159-65; Fujimori (1990) J. Amer. Chem.
Soc. 112:7435; Urata, (1993) Nucleic Acids Symposium Ser. No.
29:69-70). When the nucleotide base is purine, e.g. A or G, the
ribose sugar is attached to the N.sup.9-position of the nucleotide
base. When the nucleotide base is pyrimidine, e.g. C, T or U, the
pentose sugar is attached to the N.sup.1-position of the nucleotide
base, except for pseudouridines, in which the pentose sugar is
attached to the C5 position of the uracil nucleotide base (see,
e.g., Kornberg and Baker, (1992) DNA Replication, 2.sup.nd Ed.,
Freeman, San Francisco, Calif.).
[0052] One or more of the pentose carbons of a nucleotide may be
substituted with a phosphate ester having the formula:
##STR00002##
where .alpha. is an integer from 0 to 4. In certain embodiments,
.alpha. is 2 and the phosphate ester is attached to the 3'- or
5'-carbon of the pentose. In certain embodiments, the nucleotides
are those in which the nucleotide base is a purine, a
7-deazapurine, a pyrimidine, or an analog thereof. "Nucleotide
5'-triphosphate" refers to a nucleotide with a triphosphate ester
group at the 5' position, and are sometimes denoted as "NTP", or
"dNTP" and "ddNTP" to particularly point out the structural
features of the ribose sugar. The triphosphate ester group may
include sulfur substitutions for the various oxygens, e.g.
.alpha.-thio-nucleotide 5'-triphosphates. For a review of
nucleotide chemistry, see: Shabarova, Z. and Bogdanov, A. Advanced
Organic Chemistry of Nucleic Acids, VCH, New York, 1994.
[0053] The term "nucleotide analog", as used herein, refers to
embodiments in which the pentose sugar and/or the nucleotide base
and/or one or more of the phosphate esters of a nucleotide may be
replaced with its respective analog. In certain embodiments,
exemplary pentose sugar analogs are those described above. In
certain embodiments, the nucleotide analogs have a nucleotide base
analog as described above. In certain embodiments, exemplary
phosphate ester analogs include, but are not limited to,
alkylphosphonates, methylphosphonates, phosphoramidates,
phosphotriesters, phosphorothioates, phosphorodithioates,
phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,
phosphoroanilidates, phosphoroamidates, boronophosphates, etc., and
may include associated counterions.
[0054] Also included within the definition of "nucleotide analog"
are nucleotide analog monomers which can be polymerized into
polynucleotide analogs in which the DNA/RNA phosphate ester and/or
sugar phosphate ester backbone is replaced with a different type of
internucleotide linkage. Exemplary polynucleotide analogs include,
but are not limited to, peptide nucleic acids, in which the sugar
phosphate backbone of the polynucleotide is replaced by a peptide
backbone.
[0055] As used herein, the terms "polynucleotide",
"oligonucleotide", and "nucleic acid" are used interchangeably and
mean single-stranded and double-stranded polymers of nucleotide
monomers, including 2'-deoxyribonucleotides (DNA) and
ribonucleotides (RNA) linked by internucleotide phosphodiester bond
linkages, or internucleotide analogs, and associated counter ions,
e.g., H.sup.+, NH.sub.4.sup.+, trialkylammonium, Mg.sup.2+,
Na.sup.+ and the like. A nucleic acid may be composed entirely of
deoxyribonucleotides, entirely of ribonucleotides, or chimeric
mixtures thereof. The nucleotide monomer units may comprise any of
the nucleotides described herein, including, but not limited to,
nucleotides and nucleotide analogs. Nucleic acids typically range
in size from a few monomeric units, e.g. 5-40 when they are
sometimes referred to in the art as oligonucleotides, to several
thousands of monomeric nucleotide units. Unless denoted otherwise,
whenever a nucleic acid sequence is represented, it will be
understood that the nucleotides are in 5' to 3' order from left to
right and that "A" denotes deoxyadenosine or an analog thereof, "C"
denotes deoxycytidine or an analog thereof, "G" denotes
deoxyguanosine or an analog thereof, and "T" denotes thymidine or
an analog thereof, unless otherwise noted.
[0056] Nucleic acids include, but are not limited to, genomic DNA,
cDNA, hnRNA, snRNA, mRNA, rRNA, tRNA, fragmented nucleic acid,
nucleic acid obtained from subcellular organelles such as
mitochondria or chloroplasts, and nucleic acid obtained from
microorganisms or DNA or RNA viruses that may be present on or in a
biological sample.
[0057] Nucleic acids may be composed of a single type of sugar
moiety, e.g., as in the case of RNA and DNA, or mixtures of
different sugar moieties, e.g., as in the case of RNA/DNA chimeras.
In certain embodiments, nucleic acids are ribopolynucleotides and
2'-deoxyribopolynucleotides according to the structural formulae
below:
##STR00003##
wherein each B is independently the base moiety of a nucleotide,
e.g., a purine, a 7-deazapurine, a pyrimidine, or an analog
thereof; each m defines the length of the respective nucleic acid
and can range from zero to thousands, tens of thousands, or even
more; each R is independently selected from the group comprising
hydrogen, hydroxyl, halogen, --R'', --OR'', and --NR''R'', where
each R'' is independently (C.sub.1-C.sub.6) alkyl or
(C.sub.5-C.sub.14) aryl, or two adjacent Rs are taken together to
form a bond such that the ribose sugar is 2',3'-didehydroribose;
and each R' is independently hydroxyl or
##STR00004##
where .alpha. is zero, one or two.
[0058] In certain embodiments of the ribopolynucleotides and
2'-deoxyribopolynucleotides illustrated above, the nucleotide bases
B are covalently attached to the C1' carbon of the sugar moiety as
previously described.
[0059] The terms "nucleic acid", "polynucleotide", and
"oligonucleotide" may also include nucleic acid analogs,
polynucleotide analogs, and oligonucleotide analogs. The terms
"nucleic acid analog", "polynucleotide analog" and "oligonucleotide
analog" are used interchangeably, and refer to a nucleic acid that
contains at least one nucleotide analog and/or at least one
phosphate ester analog and/or at least one pentose sugar analog.
Also included within the definition of nucleic acid analogs are
nucleic acids in which the phosphate ester and/or sugar phosphate
ester linkages are replaced with other types of linkages, such as
N-(2-aminoethyl)-glycine amides and other amides (see, e.g.,
Nielsen et al., 1991, Science 254: 1497-1500; WO 92/20702; U.S.
Pat. No. 5,719,262; U.S. Pat. No. 5,698,685); morpholinos (see,
e.g., U.S. Pat. No. 5,698,685; U.S. Pat. No. 5,378,841; U.S. Pat.
No. 5,185,144); carbamates (see, e.g., Stirchak & Summerton,
1987, J. Org. Chem. 52: 4202); methylene(methylimino) (see, e.g.,
Vasseur et al., 1992, J. Am. Chem. Soc. 114: 4006);
3'-thioformacetals (see, e.g., Jones et al., 1993, J. Org. Chem.
58: 2983); sulfamates (see, e.g., U.S. Pat. No. 5,470,967);
2-aminoethylglycine, commonly referred to as PNA (see, e.g.,
Buchardt, WO 92/20702; Nielsen (1991) Science 254:1497-1500); and
others (see, e.g., U.S. Pat. No. 5,817,781; Frier & Altman,
1997, Nucl. Acids Res. 25:4429 and the references cited therein).
Phosphate ester analogs include, but are not limited to, (i)
C.sub.1-C.sub.4 alkylphosphonate, e.g. methylphosphonate; (ii)
phosphoramidate; (iii) C.sub.1-C.sub.6 alkyl-phosphotriester; (iv)
phosphorothioate; and (v) phosphorodithioate.
[0060] The terms "annealing" and "hybridization" are used
interchangeably and mean the base-pairing interaction of one
nucleic acid with another nucleic acid that results in formation of
a duplex, triplex, or other higher-ordered structure. In certain
embodiments, the primary interaction is base specific, e.g., NT and
G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding. In
certain embodiments, base-stacking and hydrophobic interactions may
also contribute to duplex stability.
[0061] An "enzymatically active mutant or variant thereof," when
used in reference to an enzyme such as a polymerase or a ligase,
means a protein with appropriate enzymatic activity. Thus, for
example, but without limitation, an enzymatically active mutant or
variant of a DNA polymerase is a protein that is able to catalyze
the stepwise addition of appropriate deoxynucleoside triphosphates
into a nascent DNA strand in a template-dependent manner. An
enzymatically active mutant or variant differs from the
"generally-accepted" or consensus sequence for that enzyme by at
least one amino acid, including, but not limited to, substitutions
of one or more amino acids, addition of one or more amino acids,
deletion of one or more amino acids, and alterations to the amino
acids themselves. With the change, however, at least some catalytic
activity is retained. In certain embodiments, the changes involve
conservative amino acid substitutions. Conservative amino acid
substitution may involve replacing one amino acid with another that
has, e.g., similar hydrophobicity, hydrophilicity, charge, or
aromaticity. In certain embodiments, conservative amino acid
substitutions may be made on the basis of similar hydropathic
indices. A hydropathic index takes into account the hydrophobicity
and charge characteristics of an amino acid, and in certain
embodiments, may be used as a guide for selecting conservative
amino acid substitutions. The hydropathic index is discussed, e.g.,
in Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is understood
in the art that conservative amino acid substitutions may be made
on the basis of any of the aforementioned characteristics.
[0062] Alterations to the amino acids may include, but are not
limited to, glycosylation, methylation, phosphorylation,
biotinylation, and any covalent and noncovalent additions to a
protein that do not result in a change in amino acid sequence.
"Amino acid" as used herein refers to any amino acid, natural or
nonnatural, that may be incorporated, either enzymatically or
synthetically, into a polypeptide or protein.
[0063] Fragments, for example, but without limitation, proteolytic
cleavage products, are also encompassed by this term, provided that
at least some enzyme catalytic activity is retained.
[0064] The skilled artisan will readily be able to measure
catalytic activity using an appropriate well-known assay. Thus, an
appropriate assay for polymerase catalytic activity might include,
for example, measuring the ability of a variant to incorporate,
under appropriate conditions, rNTPs or dNTPs into a nascent
polynucleotide strand in a template-dependent manner. Likewise, an
appropriate assay for ligase catalytic activity might include, for
example, the ability to ligate adjacently hybridized
oligonucleotides comprising appropriate reactive groups. Protocols
for such assays may be found, among other places, in Sambrook et
al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press (1989) (hereinafter "Sambrook et al."), Sambrook and Russell,
Molecular Cloning, Third Edition, Cold Spring Harbor Press (2000)
(hereinafter "Sambrook and Russell"), Ausubel et al., Current
Protocols in Molecular Biology (1993) including supplements through
September 2005, John Wiley & Sons (hereinafter "Ausubel et
al.").
[0065] A "target," "target nucleic acid," or "target nucleic acid
sequence" is a nucleic acid sequence in a sample. In certain
embodiments, a target nucleic acid sequence serves as a template
for amplification in a PCR reaction. In certain embodiments, a
target nucleic acid sequence serves as a ligation template. Target
nucleic acid sequences may include both naturally occurring and
synthetic molecules. Exemplary target nucleic acid sequences
include, but are not limited to, genomic DNA, ligation products,
and amplification products.
[0066] A "pivotal nucleotide" is a nucleotide of interest in a
target nucleic acid sequence, and may represent, for example,
without limitation, a single polymorphic nucleotide in a
multiallelic target locus. In certain embodiments, a pivotal
nucleotide is deleted in one or more alleles of a multiallelic
target locus. In certain embodiments, a pivotal nucleotide is added
in one or more alleles of a multiallelic target locus. In certain
embodiments, a target nucleic acid sequence may comprise more than
one pivotal nucleotide.
[0067] A "pivotal complement" or "pivotal complement nucleotide" is
a nucleotide base complementary to a pivotal nucleotide.
[0068] A "buffering agent" is a compound added to an amplification
reaction which modifies the stability, activity, and/or longevity
of one or more components of the amplification reaction by
regulating the pH of the amplification reaction. Certain buffering
agents are well known in the art and include, but are not limited
to, Tris and Tricine.
[0069] The term "sample" refers to any substance comprising nucleic
acid material.
[0070] An additive is a compound added to a composition which
modifies the stability, activity, and/or longevity of one or more
components of the composition. In certain embodiments, the
composition is an amplification reaction composition. In certain
embodiments, an additive inactivates contaminant enzymes,
stabilizes protein folding, and/or decreases aggregation. Exemplary
additives that may be included in an amplification reaction
include, but are not limited to, betaine, formamide, KCl,
CaCl.sub.2, MgOAc, MgCl.sub.2, NaCl, NH.sub.4OAc, NaI,
Na(CO.sub.3).sub.2, LiCl, MnOAc, NMP, trehalose, demethylsulfoxide
("DMSO"), glycerol, ethylene glycol, dithiothreitol ("DTT"),
pyrophosphatase (including, but not limited to Thermoplasma
acidophilum inorganic pyrophosphatase ("TAP")), bovine serum
albumin ("BSA"), propylene glycol, glycinamide, CHES, Percoll,
aurintricarboxylic acid, Tween 20, Tween 21, Tween 40, Tween 60,
Tween 85, Brij 30, NP-40, Triton X-100, CHAPS, CHAPSO, Mackernium,
LDAO, Zwittergent 3-10, Xwittergent 3-14, Xwittergent SB 3-16,
Empigen, NDSB-20, T4G32, E. Coli SSB, RecA, nicking endonucleases,
7-deazaG, dUTP, and UNG, anionic detergents, cationic detergents,
non-ionic detergents, zwittergent, sterol, osmolytes, cations, and
any other chemical, protein, or cofactor that may alter the
efficiency of amplification. In certain embodiments, two or more
additives are included in an amplification reaction.
[0071] The term "probe" comprises a polynucleotide that comprises a
specific portion designed to hybridize in a sequence-specific
manner with a complementary region of a specific nucleic acid
sequence, e.g., a target nucleic acid sequence. In certain
embodiments, the specific portion of the probe may be specific for
a particular sequence, or alternatively, may be degenerate, e.g.,
specific for a set of sequences. In certain embodiments, the probe
is labeled.
[0072] A "ligation probe set" is a group of two or more probes
designed to detect at least one target. As a non-limiting example,
a ligation probe set may comprise two nucleic acid probes designed
to hybridize to a target such that, when the two probes are
hybridized to the target adjacent to one another, they are suitable
for ligation together.
[0073] The statement that two probes are "hybridized adjacent to
each other" on a target nucleic acid encompasses the situation in
which the 3' end of one probe and the 5' end of the other probe
hybridize to contiguous regions of the target nucleic acid. The
statement also encompasses the situation in which there is a gap
between the probes when they are initially hybridized to the target
nucleic acid, and the gap is filled by a gap-filling procedure,
e.g., by extending one of the probes with a polymerase or by
hybridizing an oligonucleotide to the region of the target nucleic
acid that is opposite the gap. Thus, the probes become hybridized
adjacent to each other on the target nucleic acid sequence through
the gap-filling procedure.
[0074] "Suitable for ligation" refers to at least one first
target-specific probe and at least one second target-specific
probe, each comprising an appropriately reactive group. Exemplary
reactive groups include, but are not limited to, a free hydroxyl
group on the 3' end of the first probe and a free phosphate group
on the 5' end of the second probe. Exemplary pairs of reactive
groups include, but are not limited to: phosphorothioate and
tosylate or iodide; esters and hydrazide; RC(O)S.sup.-, haloalkyl,
or RCH.sub.2S and .alpha.-haloacyl; thiophosphoryl and
bromoacetoamido groups. Exemplary reactive groups include, but are
not limited to, S-pivaloyloxymethyl-4-thiothymidine. Additionally,
in certain embodiments, first and second target-specific probes are
hybridized to the target sequence such that the 3' end of the first
target-specific probe and the 5' end of the second target-specific
probe are immediately adjacent to allow ligation.
[0075] The term "addressable portion" refers to an oligonucleotide
sequence designed to hybridize to the complement of the addressable
portion. In certain embodiments, an addressable portion may
comprise a tag, such as an allele-specific tag or a locus-specific
tag.
[0076] The term "signal moiety" as used herein refers to any tag,
label, or identifiable moiety.
[0077] "Detectably different signal" means that detectable signals
from different signal moieties are distinguishable from one another
by at least one detection method.
[0078] The term "detectable signal value" refers to a value of the
signal that is detected from a label. In certain embodiments, the
detectable signal value is the amount or intensity of signal that
is detected from a label. Thus, if there is no detectable signal
value from a label, its detectable signal value is zero (0). In
certain embodiments, the detectable signal value is a
characteristic of the signal other than the amount or intensity of
the signal, such as the spectra, wavelength, color, or lifetime of
the signal.
[0079] "Detectably different signal value" means that one or more
detectable signal values are distinguishable from one another by at
least one detection method.
[0080] The term "labeled probe" refers to a probe that provides a
detectably different signal value depending upon whether a given
nucleic acid sequence is present or absent. In certain embodiments,
a labeled probe provides a detectably different signal value when
the intact labeled probe is hybridized to a given nucleic acid
sequence than when the intact labeled probe is not hybridized to a
given nucleic acid sequence. Thus, if a given nucleic acid sequence
is present, the labeled probe provides a detectably different
signal value than when the given nucleic acid sequence is absent.
In certain embodiments, a labeled probe provides a detectably
different signal value when the probe is intact than when the probe
is not intact. In certain such embodiments, a labeled probe remains
intact unless a given nucleic acid sequence is present. In certain
such embodiments, if a given nucleic acid sequence is present, the
labeled probe is cleaved, which results in a detectably different
signal value than when the probe is intact.
[0081] In certain embodiments, the labeled probe is an "interaction
probe." The term "interaction probe" refers to a probe that
comprises at least two moieties that can interact with one another
to provide a detectably different signal value depending upon
whether a given nucleic acid sequence is present or absent. The
signal value that is detected from the interaction probe is
different depending on whether the two moieties are sufficiently
close to one another or are spaced apart from one another. During
certain methods described herein, the proximity of the two moieties
to one another is different depending upon whether the given
nucleic acid is present or absent.
[0082] In certain embodiments, the two moieties of the interaction
probe are moved further apart if the given nucleic acid sequence is
present. In certain embodiments, the interaction probe comprises
two moieties that are linked together by a link element, and the
two moieties become unlinked during the method if the given nucleic
acid sequence is present. The signal value that is detected from
the interaction probe that includes the two moieties linked
together is different from the signal value that is detected from
the interaction probe when the two moieties are not linked.
[0083] The term "threshold difference between signal values" refers
to a set difference between a first detectable signal value and a
second detectable signal value that results when the target nucleic
acid sequence that is being sought is present in a sample, but that
does not result when the target nucleic acid sequence is absent.
The first detectable signal value of a labeled probe is the
detectable signal value from the probe when it is not exposed to a
given nucleic acid sequence. The second detectable signal value is
detected during and/or after an amplification reaction using a
composition that comprises the labeled probe.
[0084] The term "quantitating," when used in reference to an
amplification product, refers to determining the quantity or amount
of a particular sequence that is representative of a target nucleic
acid sequence in the sample. For example, but without limitation,
one may measure the intensity of the signal from a labeled probe.
The intensity or quantity of the signal is typically related to the
amount of amplification product. The amount of amplification
product generated correlates with the amount of target nucleic acid
sequence present prior to amplification or prior to ligation and
amplification, and thus, in certain embodiments, may indicate the
level of expression for a particular gene.
[0085] The term "amplification product" as used herein refers to
the product of an amplification reaction. Exemplary amplification
reactions include, but are not limited to, primer extension, the
polymerase chain reaction, and the like. Thus, exemplary
amplification products include, but are not limited to, primer
extension products, PCR amplicons, and the like.
[0086] The term "primer" refers to a polynucleotide that anneals to
a template nucleic acid sequence and allows synthesis of a sequence
complementary to the template nucleic acid sequence. Primers
include, but are not limited to, spanning primers. A "PCR primer"
refers to a primer in a set of at least two primers that are
capable of exponentially amplifying a target nucleic acid sequence
in the polymerase chain reaction.
[0087] A "spanning primer" is a primer that anneals to separate
(non-contiguous) portions of the same target nucleic acid sequence.
In certain embodiments, a spanning primer anneals to both the 3'
and 5' ends of the same target nucleic acid sequence. In certain
embodiments, a spanning primer anneals to the 3' end and another
portion of the same target nucleic acid sequence. In certain
embodiments, a spanning primer anneals to the 5' end and another
portion of the same target nucleic acid sequence.
[0088] A "universal primer" is capable of hybridizing to the
primer-specific portion of more than one species of probe, ligation
product, and/or amplification product, as appropriate. A "universal
primer set" comprises a first primer and a second primer that
hybridize with a plurality of species of probes, ligation products,
and/or amplification products, as appropriate.
[0089] In this application, a statement that one sequence is the
same as or is complementary to another sequence encompasses
situations where both of the sequences are completely the same or
complementary to one another, and situations where only a portion
of one of the sequences is the same as, or is complementary to, a
portion or the entirety of the other sequence. Here, the term
"sequence" encompasses, but is not limited to, nucleic acid
sequences, polynucleotides, oligonucleotides, probes, primers,
primer-specific portions, target-specific portions, addressable
portions, and oligonucleotide link elements.
[0090] In this application, a statement that one sequence is
complementary to another sequence encompasses situations in which
the two sequences have mismatches. Here, the term "sequence"
encompasses, but is not limited to, nucleic acid sequences,
polynucleotides, oligonucleotides, probes, primers, primer-specific
portions, target-specific portions, addressable portions, and
oligonucleotide link elements. Despite the mismatches, the two
sequences should selectively hybridize to one another under
appropriate conditions.
[0091] In this application, a statement that a first sequence is
complementary to the 5' end of a second sequence encompasses the
situation in which one or more 5' terminal nucleotides of the
second sequence are mismatched with respect to the first sequence.
In certain embodiments, up to five of the 5' terminal nucleotides
of the second sequence are mismatched with respect to the first
sequence. Despite the mismatches, the two sequences should
selectively hybridize to one another under appropriate
conditions.
[0092] In this application, a statement that a first sequence is
complementary to the 3' end of a second sequence encompasses the
situation in which one or more 3' terminal nucleotides of the
second sequence are mismatched with respect to the first sequence.
In certain embodiments, up to five of the 3' terminal nucleotides
of the second sequence are mismatched with respect to the first
sequence. Despite the mismatches, the two sequences should
selectively hybridize to one another under appropriate
conditions.
[0093] The term "selectively hybridize" means that, for particular
identical sequences, a substantial portion of the particular
identical sequences hybridize to a given desired sequence or
sequences, and a substantial portion of the particular identical
sequences do not hybridize to other undesired sequences. A
"substantial portion of the particular identical sequences" in each
instance refers to a portion of the total number of the particular
identical sequences, and it does not refer to a portion of an
individual particular identical sequence. In certain embodiments,
"a substantial portion of the particular identical sequences" means
at least 70% of the particular identical sequences. In certain
embodiments, "a substantial portion of the particular identical
sequences" means at least 80% of the particular identical
sequences. In certain embodiments, "a substantial portion of the
particular identical sequences" means at least 90% of the
particular identical sequences. In certain embodiments, "a
substantial portion of the particular identical sequences" means at
least 95% of the particular identical sequences.
[0094] In certain embodiments, the number of mismatches that may be
present may vary in view of the complexity of the composition.
Thus, in certain embodiments, the more complex the composition, the
more likely undesired sequences will hybridize. For example, in
certain embodiments, with a given number of mismatches, a probe may
more likely hybridize to undesired sequences in a composition with
the entire genomic DNA than in a composition with fewer DNA
sequences, when the same hybridization conditions are employed for
both compositions. Thus, that given number of mismatches may be
appropriate for the composition with fewer DNA sequences, but fewer
mismatches may be more optimal for the composition with the entire
genomic DNA.
[0095] In certain embodiments, sequences are complementary if they
have no more than 20% mismatched nucleotides. In certain
embodiments, sequences are complementary if they have no more than
15% mismatched nucleotides. In certain embodiments, sequences are
complementary if they have no more than 10% mismatched nucleotides.
In certain embodiments, sequences are complementary if they have no
more than 5% mismatched nucleotides.
[0096] In this application, a statement that one sequence
hybridizes or binds to another sequence encompasses situations
where the entirety of both of the sequences hybridize or bind to
one another, and situations where only a portion of one or both of
the sequences hybridizes or binds to the entire other sequence or
to a portion of the other sequence. Here, the term "sequence"
encompasses, but is not limited to, nucleic acid sequences,
polynucleotides, oligonucleotides, probes, primers, primer-specific
portions, target-specific portions, addressable portions, and
oligonucleotide link elements.
[0097] In certain embodiments, the term "to a measurably lesser
extent" encompasses situations in which the event in question is
reduced at least 10 fold. In certain embodiments, the term "to a
measurably lesser extent" encompasses situations in which the event
in question is reduced at least 100 fold.
[0098] In certain embodiments, a statement that a component may be,
is, or has been "substantially removed" means that at least 90% of
the component may be, is, or has been removed. In certain
embodiments, a statement that a component may be, is, or has been
"substantially removed" means that at least 95% of the component
may be, is, or has been removed.
Certain Exemplary Spanning Primers and Certain Methods of Use
[0099] In certain embodiments, methods are provided for amplifying
a target nucleic acid using a spanning primer. In certain
embodiments, the spanning primer anneals to at least two separate
(non-contiguous) portions of a target nucleic acid. In certain
embodiments, the spanning primer is complementary to 1) a first
region of the target nucleic acid that is at or near the 5' end of
the target nucleic acid and 2) a second region of the target
nucleic acid that is at or near the 3' end of the target nucleic
acid. In certain such embodiments, the first region is within 100,
50, 25, 10, or 5 nucleotides of the 5' end of the target nucleic
acid. In certain such embodiments, the second region is within 100,
50, 25, 10, or 5 nucleotides of the 3' end of the target nucleic
acid. In certain embodiments, the spanning primer is complementary
to the 5' and 3' ends of the target nucleic acid. In certain
embodiments, the spanning primer comprises a 5' tail that does not
hybridize to the target nucleic acid.
[0100] FIG. 1 shows exemplary embodiments of amplification using a
spanning primer. In FIG. 1, a spanning primer (110) hybridizes to
both the 3' and the 5' ends of a target nucleic acid sequence
(120). In the presence of a polymerase (130), the spanning primer
is elongated to form an amplification product (140).
[0101] In certain embodiments, a target nucleic acid is amplified
exponentially using two or more spanning primers, for example, as
depicted in the embodiments shown in FIGS. 2A and 2B. In FIG. 2A, a
first spanning primer (110) hybridizes to both the 3' and the 5'
ends of a target nucleic acid sequence (120). In the presence of a
polymerase (130), the first spanning primer is elongated to form a
first amplification product (140). A second spanning primer (210)
hybridizes to both the 3' end of the first amplification product
(140) and to any portion of the first spanning primer incorporated
into the first amplification product. The second spanning primer
(210) is elongated by the polymerase (130) to form a polynucleotide
comprising the target nucleic acid sequence with the addition of
the second spanning primer at its 5' end (220). FIG. 2B depicts the
same method as FIG. 2A, except that the second spanning primer
(210) hybridizes to both the 3' end of the first amplification
product (140) and to the 5' end of the first spanning primer
incorporated into the first amplification product.
[0102] In certain embodiments, a spanning primer is used to amplify
a ligation product. In certain such embodiments, a spanning primer
amplifies a ligation product with greater efficiency than one or
more non-spanning primers. For example, in certain embodiments, a
first nucleic acid and a second nucleic acid are combined in a
ligation mixture under conditions permissive for ligation of the
first nucleic acid to the second nucleic acid. In certain
instances, ligation may be incomplete, such that the ligation
mixture comprises unligated nucleic acids as well as ligation
products. In certain embodiments, the ligation products may be
amplified from the ligation mixture using, e.g., a non-spanning
primer that anneals to either the first or the second nucleic acid
in the ligation product. Under certain conditions, however, the
non-spanning primer is capable of annealing to the unligated first
or second nucleic acid in the ligation mixture and is extended.
This decreases the efficiency of the amplification of the ligation
products and generates spurious amplification products.
[0103] In certain embodiments, a spanning primer favors
amplification of ligation products over amplification of unligated
nucleic acids in a ligation mixture. FIGS. 3A and 3B illustrates
certain such embodiments. For example, in the embodiments shown in
FIGS. 3A and 3B, a spanning primer anneals to a first region that
is at the 5' end of a ligation product and a second region that is
at the 3' end of the ligation product. In certain such embodiments,
the spanning primer may comprise non-annealing nucleotides disposed
between the nucleotides that anneal to the first region and second
region of the ligation product. In certain such embodiments, the
number of non-annealing nucleotides may be any number of
nucleotides between 0 and 10 nucleotides.
[0104] In certain embodiments, a spanning primer is capable of
annealing to unligated nucleic acids. In certain embodiments, under
certain conditions, however, the spanning primer does not anneal as
efficiently to unligated nucleic acids as it does to the ligation
product. Thus, amplification of the ligation product by the
spanning primer is favored.
[0105] In certain embodiments, a spanning primer does not anneal to
both an unligated first nucleic acid and an unligated second
nucleic acid as efficiently as it does to a ligation product
comprising the first and second nucleic acids. For example, FIG. 3A
shows certain embodiments in which the spanning primer is capable
of annealing to the unligated first and second nucleic acids in a
"three-molecule" interaction (2). This three-molecule interaction,
however, is not as favored kinetically or thermodynamically as the
annealing of the spanning primer to the ligation product, a
"two-molecule" interaction (1).
[0106] In certain embodiments, under certain annealing conditions,
a spanning primer does not anneal to either the unligated first
nucleic acid or the unligated second nucleic acid as efficiently as
it does to the ligation product. For example, in certain
embodiments, the Tm of the hybridization complex between the
spanning primer and the ligation product is higher than the Tm of
the hybridization complex between the spanning primer and either
the first or the second unligated nucleic acid. For example, as
illustrated in FIG. 3B, the region of complementarity between the
spanning primer and the ligation product is greater than the region
of complementarity between the spanning primer and the unligated
first nucleic acid. Thus, the Tm of the hybridization complex
between the spanning primer and the ligation product is higher than
the Tm of the hybridization complex between the spanning primer and
the unligated first nucleic acid. One skilled in the art would
readily understand that, for the same reasons, the Tm of the
hybridization complex between the spanning primer and the ligation
product is higher than the Tm of the hybridization complex between
the spanning primer and the unligated second nucleic acid. In view
of this difference in Tms, one skilled in the art could ascertain
an annealing temperature that favors the formation of the
hybridization complex including the ligation product over the
hybridization complex including the unligated first or second
nucleic acids.
[0107] For example, in certain embodiments, amplification may be
performed using an annealing temperature that is greater than the
Tm of the hybridization complex between the spanning primer and at
least one of the unligated nucleic acids. For example, the
annealing temperature may be at least 5.degree. C., at least
10.degree. C., at least 20.degree. C., or at least 30.degree. C.
greater than the Tm of the hybridization complex between the
spanning primer and an unligated nucleic acid. In certain such
embodiments, the annealing temperature is less than or equal to the
Tm of the hybridization complex between the spanning primer and the
ligation product. For example, the annealing temperature may be any
temperature from 0.degree. C. to at least 10.degree. C. less than
the Tm of the hybridization complex between the spanning primer and
the ligation product. Under certain such exemplary conditions, the
formation of a hybridization complex between the spanning primer
and the ligation product is favored over the formation of a
hybridization complex between the spanning primer and an unligated
nucleic acid.
[0108] In certain embodiments, ligation products are amplified
directly from all or a portion of a ligation mixture using a
spanning primer. For example, in certain embodiments, the ligation
products are not isolated or purified from the ligation mixture
prior to the amplification. In certain embodiments, the ligation
mixture is not diluted prior to the amplification.
[0109] In certain embodiments, spanning primers are used to amplify
ligation products produced in an oligonucleotide ligation assay
(OLA). Certain exemplary methods for performing OLA and amplifying
ligation products are described, e.g., in U.S. patent application
Ser. No. 09/584,905, filed May 30, 2000; U.S. patent application
Ser. No. 10/011,993, filed Dec. 5, 2001, corresponding to U.S.
Patent Application Publication No. US 2003/0119004 A1; Patent
Cooperation Treaty Application No. PCT/US01/17329, filed May 30,
2001, corresponding to PCT International Publication No. WO
01/92579 A2, published Dec. 6, 2001, and U.S. Patent Application
Publication No. US 2003/0190646 A1; Patent Cooperation Treaty
Application No. PCT/US97/45559, filed May 27, 1997; and U.S. Pat.
No. 6,027,889, issued Feb. 22, 2000.
[0110] FIG. 4 shows exemplary embodiments in which a ligation
product resulting from an OLA is amplified using a spanning primer.
In the embodiments shown in FIG. 4, a first probe (310) and a
second probe (320) are hybridized to a target nucleic acid sequence
(120) adjacent to each other, such that the adjacent ends of the
first and second probes are ligated together under suitable
conditions.
[0111] In certain embodiments, there is a gap between the first
probe and the second probe upon hybridization of those probes to
the target nucleic acid sequence. In certain such embodiments, the
gap is filled by a gap-filling procedure. For example, in certain
embodiments, the gap is filled by an oligonucleotide that is
complementary to the target nucleic acid sequence opposite the gap.
In certain embodiments, the gap is filled by extension of the 3'
end of either the first or second probe. Thus, in certain
embodiments, the first and second probes become hybridized adjacent
to one another on the target nucleic acid sequence through the
gap-filling procedure. In certain embodiments, a gap-filling
procedure increases the specificity of the OLA.
[0112] In the embodiments shown in FIG. 4, a ligation product (330)
is formed after at least one cycle of ligation, wherein the
ligation product comprises the first probe (310) and the second
probe (320). A spanning primer (110) hybridizes to both the 3' and
the 5' ends of the ligation product (330). In the presence of a
polymerase (130), the spanning primer (110) is elongated to form a
first amplification product (340).
[0113] In certain embodiments, an OLA may be used for genotyping a
nucleic acid. For example, in certain embodiments, an OLA may be
used to determine the allele present at a polymorphic locus, such
as a locus comprising a "SNP," or "single nucleotide polymorphism."
In an exemplary OLA, illustrated in FIG. 5, a sample comprising a
target nucleic acid that comprises a SNP is combined with a
ligation probe set. The ligation probe set comprises one or more
allele-specific oligonucleotides (ASOs) (also referred to as
"allele specific probes") and a locus-specific oligonucleotide
(LSO) (also referred to as a "locus-specific probe"). The ASO and
the LSO each comprise a target-specific portion (T-SP) that
hybridizes to the target nucleic acid. The target-specific portion
of the ASO comprises a nucleotide called a "pivotal complement"
(PC). That nucleotide is complementary to one of the possible
nucleotides (X), or "pivotal nucleotides," at the polymorphic
nucleotide. When more than one ASO is used, the ASOs may comprise
different pivotal complements to distinguish different alleles. In
certain embodiments, the pivotal complement is located at the 5'
terminal nucleotide of an ASO. In certain embodiments, the pivotal
complement is located at the 3' terminal nucleotide of an ASO. The
ASO and the LSO hybridize adjacent to each other on the target
nucleic acid, such that the 5' end of one of the oligonucleotides
is adjacent to the 3' end of the other. Under conditions permissive
for ligation, an ASO comprising a pivotal complement that is
complementary to the pivotal nucleotide becomes ligated to an
adjacently hybridized LSO, whereas an ASO comprising a pivotal
nucleotide that is not complementary to the pivotal nucleotide does
not substantially ligate to an adjacently hybridized LSO. In
various embodiments, ligation products are detected using methods
comprising amplifying the ligation product and detecting the
amplification product.
[0114] In certain embodiments, a ligation product comprising an ASO
and a LSO is amplified using a first spanning primer that anneals
to a region that is at the 5' end of the ligation product and a
region that is at the 3' end of the ligation product. For example,
in certain embodiments, the first spanning primer comprises a first
portion and a second portion, wherein the first portion is 3' of
the second portion. In certain such embodiments, such as the
embodiments shown in FIG. 6A, the first portion anneals to the 3'
end of the LSO and the second portion anneals to the 5' end of the
ASO in the ligation product. In certain such embodiments, the 3'
end of the LSO is blocked, e.g., by an amine group or by a minor
groove binder-nonfluorescent quencher (MGB-NFQ). In certain such
embodiments, the LSO or a ligation product comprising the LSO is
incapable of priming nucleic acid synthesis using the first
spanning primer as a template.
[0115] Alternatively, in certain embodiments, the first portion of
the first spanning primer anneals to the 3' end of the ASO and the
second portion anneals to the 5' end of the LSO in the ligation
product. In certain such embodiments, the 3' end of the ASO is
blocked, e.g., by an amine group. In certain such embodiments, the
ASO or a ligation product comprising the ASO is incapable of
priming nucleic acid synthesis using the first spanning primer as a
template.
[0116] In certain embodiments, the first portion of the first
spanning primer is about 5, 6, 7, 8, 9, or 10 nucleotides in
length. In certain embodiments, the second portion of the first
spanning primer is about 9, 10, 11, or 12 nucleotides in length. In
certain embodiments, the Tm of the hybridization complex between
the first spanning primer and the ligation product is any
temperature from about 60.degree. C. to 75.degree. C., including
all temperatures within that range.
[0117] In certain embodiments, such as the embodiment shown in FIG.
5, an ASO and/or a LSO comprises a primer-specific portion (P--SP)
that does not hybridize to the target nucleic acid. In certain
embodiments, an ASO comprises an addressable portion, such as a
tag, that does not hybridize to the target nucleic acid. In certain
such embodiments, such as the embodiment shown in FIG. 5, the tag
("TAG") is disposed between the primer-specific portion (P-SP) and
the target-specific portion (T-SP) of the ASO. In certain
embodiments in which more than one ASO is used, ASOs having
different pivotal complements comprise different tag sequences, and
ASOs having the same pivotal complement comprise the same tag
sequence. In certain such embodiments, the tag sequences provide a
mechanism for distinguishing ligation products or amplification
products in an allele-specific manner.
[0118] In certain embodiments, an LSO comprises an addressable
portion, such as a tag, that does not hybridize to the target
nucleic acid. In certain such embodiments, the tag is disposed
between the primer-specific portion and the target-specific portion
of the LSO. In certain embodiments, the tag sequence provides a
mechanism for identifying the locus from which a ligation product
or amplification product is derived. In certain embodiments, the
tag sequence provides a mechanism for separating ligation or
amplification products using methods such as hybridization based
pullout (HBP). See, e.g., WO 01/92579.
[0119] In certain embodiments, illustrated in FIG. 6B, a ligation
product comprising an ASO and a LSO is amplified using a first
spanning primer comprising a first portion (red) that anneals to
the primer-specific portion of the LSO and a second portion (dark
blue) that anneals to the primer-specific portion of the ASO. In
certain embodiments, the sequences of the primer-specific portions
of the ASO and LSO for a particular target nucleic acid (e.g., a
first locus) are the same as the sequences of the primer-specific
portions of an ASO and LSO for a different target nucleic acid
(e.g., a second locus). In certain such embodiments, a first
spanning primer may serve as a universal primer for the
amplification of ligation products corresponding to two different
target nucleic acids (e.g., two different genomic loci), thus
allowing "multiplex" amplification to take place.
[0120] In certain embodiments, such as the embodiments shown in
FIGS. 6A and 6B, a first spanning primer comprises a 5' "tail"
(light blue) that does not anneal to the ligation product. In
certain such embodiments, the 5' tail or its complement comprises a
sequence that may serve as a binding site for a universal
primer.
[0121] In certain embodiments of amplification, a first spanning
primer is extended using a polymerase that lacks 5' to 3'
exonuclease activity. In certain such embodiments, the polymerase
possesses strand displacement activity, as illustrated in FIGS. 7A
and 8A. In certain such embodiments, the amplification product
comprises the complement of the ligation product plus additional
sequence at the 5' end of the amplification product. That
additional sequence corresponds to the 5' end of the first spanning
primer. In certain embodiments, a polymerase that lacks 5' to 3'
exonuclease activity is the Stoffel fragment of Taq DNA polymerase.
In certain such embodiments, the Stoffel fragment adds an adenine
(A) to the 3' end of the amplification product in a
non-template-directed manner. See, e.g., FIGS. 7A and 8A.
[0122] In certain embodiments of amplification, a first spanning
primer is extended using a polymerase that possesses 5' to 3'
exonuclease activity. In certain such embodiments, the polymerase
will extend the first spanning primer using the ligation product as
a template. When the extending polymerase reaches the portion of
the ligation product that is annealed to the second portion of the
first spanning primer, the polymerase will degrade the region of
the first spanning primer that is annealed to the ligation
product.
[0123] In certain embodiments, the amplification product resulting
from extension of the first spanning primer (the first
amplification product) is further amplified using a second primer.
In certain such embodiments, as illustrated in FIG. 7B, the second
primer comprises a portion that anneals to a region at the 3' end
of the first amplification product. In certain embodiments, such as
the embodiments shown in FIG. 8B, the second primer is a second
spanning primer comprising a first portion and a second portion,
wherein the first portion is 3' of the second portion. In certain
such embodiments, the first portion anneals to a region at the 3'
end of the first amplification product, and the second portion
anneals to a region at the 5' end of the first amplification
product. In certain embodiments, such as the embodiments shown in
FIG. 8B, the region at the 5' end of the first amplification
product comprises the 5' tail of the first spanning primer.
[0124] In certain embodiments, the first portion of the second
spanning primer is about 10, 11, 12, 13, 14, or 15 nucleotides in
length. In certain embodiments, the second portion of the second
spanning primer is about 9, 10, 11, 12, 13, or 14 nucleotides in
length. In certain embodiments, the Tm of the hybridization complex
between the second spanning primer and the first amplification
product is any temperature from about 60.degree. C. to 78.degree.
C., including all temperatures within that range.
[0125] In certain embodiments, such as the embodiments shown in
FIGS. 7B and 8B, the second primer comprises a 5' tail (black) that
does not anneal to the first amplification product. In certain such
embodiments, the 5' tail or its complement comprises a sequence
that may serve as a binding site for a universal primer.
[0126] In certain embodiments, such as those shown in FIGS. 7B and
8B, the second primer is extended to form a second amplification
product, i.e, an amplification product that comprises a sequence
complementary to the first amplification product that resulted from
extension of the first spanning primer. In various embodiments, any
of the polymerases discussed above may be used to extend the second
primer. In certain embodiments, such as the embodiments shown in
FIGS. 7B and 8B, the first spanning primer anneals to the second
amplification product and is extended. In this manner, in certain
embodiments, multiple amplification cycles may be carried out using
the first spanning primer and second primer in the same reaction
mixture.
[0127] Certain Exemplary Cycling Conditions in Amplification
Reactions Using Spanning Primers
[0128] In certain embodiments, the annealing temperature for
amplification may be from about 55.degree. C. to 75.degree. C.,
including all temperatures within that range. In certain such
embodiments, the annealing temperature for amplification may be
from about 65.degree. C. to about 75.degree. C. In certain such
embodiments, the annealing temperature is higher than that
typically used in certain PCRs (-55.degree. C.). In certain
embodiments, a higher annealing temperature may minimize
non-specific priming as well as priming by unligated ASOs and/or
LSOs. In certain embodiments, a higher annealing temperature may
also minimize other PCR artifacts such as primer dimers.
[0129] In certain embodiments in which multiple amplification
cycles are performed, different amplification cycles may have
different annealing temperatures. For example, in certain
embodiments, the first 1, 2, 3, 4, or 5 cycles may have a lower
annealing temperature than subsequent cycles. For example, in
certain embodiments, the first 1, 2, 3, 4, or 5 cycles may be
performed using a first annealing temperature of about 65 to about
70.degree. C., including all temperatures within that range. In
certain such embodiments, subsequent cycles may be performed using
a second annealing temperature that is higher than the first
annealing temperature, e.g., at 70.degree. C.-75.degree. C.,
including all temperatures within that range.
[0130] In certain embodiments, the annealing step of an
amplification cycle is from about 10-30 seconds in length,
including all times between those endpoints. In certain
embodiments, annealing and extension may be performed in a single
step. In certain embodiments, a total of about 20 to about 40
cycles are performed, including any number of cycles between those
endpoints.
[0131] Certain Exemplary Methods of Genotyping Using Spanning
Primers
[0132] As discussed above, in certain embodiments, OLA can be used
to determine the allele or alleles present at a polymorphic locus,
such as a locus comprising a SNP. FIGS. 9-13 show certain exemplary
embodiments for detecting the particular allele or alleles present
at a biallelic locus. In those figures, nucleic acid sequences
having the same color are the same or are complementary to one
another. One skilled in the art would understand that various
embodiments may be adapted for the detection of up to four alleles
at any given SNP.
[0133] FIG. 9A shows amplification of a ligation product resulting
from oligonucleotide ligation at a biallelic locus, according to
certain embodiments. In FIG. 9A, the ligation product comprises an
ASO (ASO-1) and a LSO. ASO-1 comprises a pivotal complement (a "T")
that is complementary to the pivotal nucleotide for one of the
alleles ("allele 1") at the biallelic locus. ASO-1 also comprises a
primer-specific portion (dark blue). The LSO comprises a
primer-specific portion (red). If allele 1 is present at the
biallelic locus, a ligation product comprising ASO-1 and LSO is
formed, as shown in FIG. 9A.
[0134] In FIG. 9A, a first spanning primer specific for allele 1 is
used to amplify the ligation product. The first spanning primer
comprises a first portion (red) that anneals to the primer-specific
portion of the LSO and a second portion that anneals to the
primer-specific portion of ASO-1 (dark blue) in the ligation
product. The first spanning primer further comprises a 5' tail
(light blue). Together, the 5' tail (light blue) and the second
portion (dark blue) of the first spanning primer comprise an
allele-specific tag (specific for allele 1), which will be
incorporated into a first amplification product. The term
"allele-specific tag" refers to a nucleic acid sequence or its
complement which indicates whether a particular allele is present
in a target nucleic acid.
[0135] The first amplification product resulting from extension of
the first spanning primer is amplified using a second spanning
primer specific for allele 1. Specifically, the second spanning
primer comprises a first portion (dark blue) that anneals to the
complement of the primer-specific portion of ASO-1 and a second
portion (light blue) that anneals to the 5' tail of the first
spanning primer in the first amplification product. In certain
embodiments, the second spanning primer further comprises a 5' tail
(orange). The second amplification product resulting from extension
of the second spanning primer comprises the complement of the
allele-specific tag. The second amplification product may then be
amplified by the first spanning primer. Multiple amplification
cycles may then be carried out using the first and second spanning
primers.
[0136] In certain embodiments, if allele 1 is not present at the
biallelic locus, then the pivotal complement of ASO-1 will not base
pair with the pivotal nucleotide at the biallelic locus. In certain
such embodiments, no ligation product will form, and thus, there
will be no ligation product for the first spanning primer to
amplify. Thus, in certain such embodiments, the absence of
amplification products from the first and/or second spanning
primers indicates the absence of allele 1 at the biallelic locus.
Conversely, in certain embodiments, if allele 1 is present at the
biallelic locus, then the pivotal complement of ASO-1 will base
pair with the pivotal nucleotide at the biallelic locus. In certain
such embodiments, ligation products will form, and the first
spanning primer will amplify those ligation products. Thus, in
certain such embodiments, the presence of amplification products
from the first and/or second spanning primers indicates the
presence of allele 1 at the biallelic locus.
[0137] In certain embodiments, ligation products comprising ASO-1
and LSO may form even if allele 1 is not present at the biallelic
locus. In certain such embodiments, those "non-specific" ligation
products may be amplified by the first spanning primer. However,
such non-specific ligation occurs to a measurably lesser extent
than "allele-specific" ligation, which occurs when allele 1 is
present at the biallelic locus. In certain embodiments, one can
quantify non-specific ligation by subjecting ASO-1 and LSO to
conditions permissive for ligation in the presence of a target
nucleic acid comprising the biallelic locus in which allele 1 is
not present (e.g., a negative control). In certain embodiments, one
can quantify allele-specific ligation by subjecting ASO-1 and LSO
to conditions permissive for ligation in the presence of a target
nucleic acid comprising the biallelic locus in which allele 1 is
present (e.g., a positive control). In certain embodiments, one can
quantify ligation, e.g., by detecting amplification products from
the first and/or second spanning primers. The quantitative
difference between non-specific and allele-specific ligation can be
used to set an appropriate threshold value, above which it is
considered that allele 1 is present at the biallelic locus.
[0138] FIG. 9B shows amplification of a ligation product resulting
from oligonucleotide ligation at the same biallelic locus shown in
FIG. 9A, according to certain embodiments. In FIG. 9B, the ligation
product comprises an ASO (ASO-2) and a LSO. ASO-2 comprises a
pivotal complement (a "C") that is complementary to the pivotal
nucleotide for the second allele ("allele 2") at the biallelic
locus. ASO-2 also comprises a primer-specific portion (brown). The
LSO comprises a primer-specific portion (red). If allele 2 is
present at the biallelic locus, a ligation product comprising ASO-2
and LSO is formed, as shown in FIG. 9B.
[0139] In FIG. 9B, a first spanning primer specific for allele 2 is
used to amplify the ligation product. The first spanning primer
comprises a first portion (red) that anneals to the primer-specific
portion of the LSO and a second portion (brown) that anneals to the
primer-specific portion of ASO-2 in the ligation product. The first
spanning primer further comprises a 5' tail (pink). Together, the
5' tail (pink) and the second portion (brown) of the first spanning
primer comprise an allele-specific tag (specific for allele 2),
which will be incorporated into a first amplification product.
[0140] In FIG. 9B, the first amplification product resulting from
extension of the first spanning primer is amplified using a second
spanning primer specific for allele 2. Specifically, the second
spanning primer comprises a first portion (brown) that anneals to
the primer-specific portion of ASO-2 and a second portion (pink)
that anneals to the 5' tail of the first spanning primer in the
amplification product. In certain embodiments, the second spanning
primer further comprises a 5' tail (orange). The second
amplification product resulting from extension of the second
spanning primer comprises the complement of the allele-specific
tag. The second amplification product may then be amplified by the
first spanning primer. Multiple amplification cycles may then be
carried out using the first and second spanning primers.
[0141] In certain embodiments, if allele 2 is not present at the
biallelic locus, then the pivotal complement of ASO-2 will not base
pair with the pivotal nucleotide at the biallelic locus. In certain
such embodiments, no ligation product will form, and thus, there
will be no ligation product for the first spanning primer to
amplify. Thus, in certain such embodiments, the absence of
amplification products from the first and/or second spanning
primers indicates the absence of allele 2 at the biallelic locus.
Conversely, in certain embodiments, if allele 2 is present at the
biallelic locus, then the pivotal complement of ASO-2 will base
pair with the pivotal nucleotide at the biallelic locus. In certain
such embodiments, ligation products will form, and the first
spanning primer will amplify those ligation products. Thus, in
certain such embodiments, the presence of amplification products
from the first and/or second spanning primers indicates the
presence of allele 2 at the biallelic locus.
[0142] In certain embodiments, ligation products comprising ASO-2
and LSO may form even if allele 2 is not present at the biallelic
locus. In certain such embodiments, those "non-specific" ligation
products may be amplified by the first spanning primer. However,
such non-specific ligation occurs to a measurably lesser extent
than "allele-specific" ligation, which occurs when allele 2 is
present at the biallelic locus. In certain embodiments, one can
quantify non-specific ligation by subjecting ASO-2 and LSO to
conditions permissive for ligation in the presence of a target
nucleic acid comprising the biallelic locus in which allele 2 is
not present (e.g., a negative control). In certain embodiments, one
can quantify allele-specific ligation by subjecting ASO-2 and LSO
to conditions permissive for ligation in the presence of a target
nucleic acid comprising the biallelic locus in which allele 2 is
present (e.g., a positive control). In certain embodiments, one can
quantify ligation, e.g., by detecting amplification products from
the first and/or second spanning primers. The quantitative
difference between non-specific and allele-specific ligation can be
used to set an appropriate threshold value, above which it is
considered that allele 2 is present at the biallelic locus.
[0143] In certain embodiments, the allele(s) present at a
polymorphic locus are identified by detecting allele-specific
tag(s) present in amplification products specific for that locus.
For example, in certain embodiments, the amplification products of
FIG. 9A and the amplification products of FIG. 9B are detected and
distinguished from each other by detecting their respective
allele-specific tags. In certain such embodiments, the
amplification reactions shown in FIGS. 9A and 9B may be performed
in a single reaction mixture. Thus, in certain embodiments, if only
the allele-specific tag specific for allele 1 is detected in the
amplification mixture, then the biallelic locus is homozygous for
allele 1. In certain embodiments, if only the allele-specific tag
specific for allele 2 is detected in the amplification mixture,
then the biallelic locus is homozygous for allele 2. In certain
embodiments, if both the allele-specific tag specific for allele 1
and the allele-specific tag specific for allele 2 are detected in
the amplification mixture, then the biallelic locus is heterozygous
for allele 1 and allele 2.
[0144] In various embodiments, an allele-specific tag may be
detected using a first spanning primer or a second spanning primer
that comprises an allele-specific label. For example, in certain
embodiments, the second spanning primer in FIG. 9A may be labeled
with a particular label, and the second spanning primer in FIG. 9B
may be labeled with a different label. Thus, in certain
embodiments, detection of a signal from a given label indicates the
presence of a particular allele-specific tag, which in turn
indicates the allele present at the biallelic locus. In certain
such embodiments, the detectable signal value is greater than an
appropriate threshold value.
[0145] In various embodiments, an allele-specific tag may be
detected using a nucleic acid probe that hybridizes to the
allele-specific tag. In certain embodiments, the probe comprises a
label. In certain embodiments, the probe comprises a mobility
modifier. In certain such embodiments, the probe is detected using
a mobility-dependent analysis technique. In certain embodiments,
different allele-specific tags are detected using probes comprising
different labels. In certain embodiments, different allele-specific
tags are detected using probes comprising different mobility
modifiers.
[0146] In certain embodiments, a LSO comprises a tag that does not
hybridize to the target nucleic acid. In certain such embodiments,
the tag is a locus-specific tag. The term "locus-specific tag"
refers to a nucleic acid sequence or its complement which indicates
whether a particular target nucleic acid is present. In certain
embodiments, a locus-specific tag is disposed between the
primer-specific portion and the target-specific portion of the LSO.
In certain such embodiments, the locus-specific tag is used to
identify the target nucleic acid, e.g., the polymorphic locus, to
which the LSO is capable of hybridizing. Thus, in certain
embodiments, detection of the locus-specific tag in an
amplification mixture indicates the presence of an amplification
product corresponding to that target nucleic acid.
[0147] In certain embodiments, locus-specific tags may be used to
distinguish amplification products derived from different target
nucleic acids (e.g., different loci) in a multiplex amplification
reaction. For example, LSOs that hybridize to different polymorphic
loci may comprise different locus-specific tags. The different
locus-specific tags identify the loci to which the different LSOs
hybridizes. Amplification products comprising those locus-specific
tags may thus be distinguished from one another.
[0148] In certain embodiments, an amplification product comprises
both a locus-specific tag (e.g., to identify the locus from which
the amplification product is derived) and an allele-specific tag
(e.g., to identify the particular allele that is present at the
locus). Certain exemplary embodiments are shown in FIGS. 9A and 9B.
The amplification products shown in FIG. 9A comprise a different
allele-specific tag and the same locus-specific tag as the
amplification products shown in FIG. 9B. In certain embodiments, an
allele-specific tag and a locus-specific tag in a given
amplification product are within about 5, 6, 7, 8, 9, or 10
nucleotides of one another. In certain such embodiments, the
allele-specific tag and the locus-specific tag are within about 6-8
nucleotides of one another.
[0149] In various embodiments, the allele-specific tag and/or the
locus-specific tag in an amplification product is detected. In
certain embodiments, the allele-specific tag and/or the
locus-specific tag in an amplification product is detected based on
the mobility of the amplification product. In certain embodiments,
the allele-specific tag and/or the locus-specific tag in an
amplification product is detected using an allele-specific probe
that hybridizes to the allele-specific tag and/or a locus-specific
probe that hybridizes to the locus-specific tag. In certain
embodiments, one can identify the allele present in a target
nucleic acid by detecting the particular allele-specific probe that
hybridizes to an amplification product specific for that target
nucleic acid. In certain embodiments, one can identify the target
nucleic acid from which an amplification product is obtained by
detecting the particular locus-specific probe that hybridizes to
the amplification product. In certain embodiments, because it is
possible to distinguish amplification products in an
allele-specific and a locus-specific manner, one can thus ascertain
the alleles present in multiple target nucleic acids in a single
multiplex reaction mixture.
[0150] For example, in certain embodiments, different probes may be
used to distinguish different alleles at a particular locus.
Referring to the exemplary embodiments in FIGS. 9A and 9B, in
certain embodiments, a first allele-specific probe may hybridize to
the "allele 1" allele-specific tag, while a second allele-specific
probe may hybridize to the "allele 2" allele-specific tag. In
certain embodiments, allele-specific probes that recognize
different allele-specific tags comprise different labels. In
certain embodiments, allele-specific probes that recognize
different allele-specific tags comprise different mobility
modifiers. In certain embodiments, allele-specific probes that
recognize different allele-specific tags comprise the same mobility
modifier but different labels.
[0151] In certain embodiments, the allele-specific tag and the
locus-specific tag in an amplification product may be detected
using a single nucleic acid probe that hybridizes to both the
allele-specific tag and the locus-specific tag. For example,
referring to the exemplary embodiments in FIGS. 9A and 9B, a first
probe may hybridize to both the "allele 1" allele-specific tag and
the locus-specific tag in the first or second amplification product
shown in FIG. 9A, while a second probe may hybridize to both the
"allele 2" allele-specific tag and the locus-specific tag in the
first or second amplification product shown in FIG. 9B. In certain
embodiments, the first probe and the second probe comprise
different labels. In certain embodiments, the first probe and the
second probe comprise the same label but different mobility
modifiers. In certain embodiments, the first probe and the second
probe comprise the same mobility modifier but different labels.
[0152] In certain embodiments, the allele-specific tag and the
locus-specific tag in an amplification product are detected using a
probe specific for the locus-specific tag and a separate probe
specific for the allele-specific tag. For example, in certain
embodiments, an amplification product is exposed to a
locus-specific probe comprising the complement of the
locus-specific tag under hybridization conditions. In certain such
embodiments, the locus-specific probe is immobilized on a solid
support, such as an array or a bead. In certain embodiments, by
detecting whether the amplification product hybridizes to a
particular locus-specific probe, one can identify the particular
target nucleic acid from which the amplification product is
obtained.
[0153] In certain embodiments, the amplification product is also
exposed to one or more allele-specific probes, each allele-specific
probe being capable of hybridizing to a different allele-specific
tag. In certain such embodiments, by detecting which
allele-specific probes hybridize to the amplification product, one
can identify the particular alleles present in a target nucleic
acid.
[0154] FIG. 10 shows certain exemplary embodiments in which
allele-specific probes are used to distinguish between an
amplification product specific for a first allele ("allele 1") of a
target nucleic acid and an amplification product specific for a
second allele ("allele 2") of the target nucleic acid. The
amplification products comprise the same locus-specific tag but
different allele-specific tags. The amplification products are
exposed to three different probes: 1) a first allele-specific probe
that hybridizes to an "allele 1" allele-specific tag; 2) a second
allele-specific probe that hybridizes to an "allele 2"
allele-specific tag; and 3) a common locus-specific probe that
hybridizes to the locus-specific tag in both amplification
products. In the embodiments shown in FIG. 10, the first and second
allele-specific probes comprise different labels. In the
embodiments shown in FIG. 10, the common locus-specific probe
comprises a mobility modifier.
[0155] In the embodiments shown in FIG. 10, the first
allele-specific probe and the common locus-specific probe hybridize
adjacent to each other on the amplification product specific for
allele 1. The second allele-specific probe and the common
locus-specific probe hybridize adjacent to each other on the
amplification product specific for allele 2. The resulting
hybridization complexes are exposed to ligation conditions. Under
such conditions, the first allele-specific probe and common
locus-specific probe are ligated to each other (indicated by an
asterisk), and the second allele-specific probe and common
locus-specific probe are ligated to each other (indicated by an
asterisk). In certain embodiments, the resulting ligation products
are subjected to a mobility-dependent analysis technique, such that
the ligation products have substantially the same mobility but they
have different labels, depending on whether they comprise the first
allele-specific probe or the second allele-specific probe.
[0156] FIG. 11 shows certain exemplary embodiments in which
allele-specific probes are used to distinguish between an
amplification product specific for a first allele ("allele 1") of a
target nucleic acid and an amplification product specific for a
second allele ("allele 2") of the target nucleic acid. The
amplification products comprise the same locus-specific tag but
different allele-specific tags. In the embodiments shown in FIG.
11, the amplification products are exposed to a common
locus-specific probe under hybridization conditions. Multiple
copies of the locus-specific probe may be immobilized on a solid
support, e.g., on a bead or at a particular position on an array.
The amplification products are also exposed to 1) a first
allele-specific probe that hybridizes to an "allele 1"
allele-specific tag, and 2) a second allele-specific probe that
hybridizes to an "allele 2" allele-specific tag. In the embodiments
shown in FIG. 11, the first allele-specific probe and the second
allele-specific probe comprise different labels.
[0157] In the embodiments shown in FIG. 11, the first
allele-specific probe and the locus-specific probe hybridize
adjacent to each other on the amplification product specific for
allele 1. Likewise, the second allele-specific probe and the
locus-specific probe hybridize adjacent to each other on the
amplification product specific for allele 2. Under ligation
conditions, the first allele-specific probe and the locus-specific
probe are ligated to each other (indicated by an asterisk), and the
second allele-specific probe and the locus-specific probe are
ligated to each other (indicated by an asterisk). Thus, in certain
embodiments, the first and second allele-specific probes become
attached to the solid support through the locus-specific probe. The
labels that are detected on the solid support indicate which
alleles are present in the target nucleic acid.
[0158] In certain embodiments, the allele-specific tag and the
locus-specific tag in an amplification product are detected using a
probe specific for the locus-specific tag and a primer specific for
the allele-specific tag. For example, in certain embodiments, an
amplification product is exposed to one or more allele-specific
primers, wherein each primer hybridizes to a different
allele-specific tag. In certain such embodiments, the
allele-specific primer that hybridizes to the amplification product
is extended to form an allele-specific extension product. In
certain embodiments, detection of the allele-specific extension
product indicates the presence of a particular allele in a target
nucleic acid.
[0159] In certain embodiments, an allele-specific extension product
comprises a locus-specific tag. In certain embodiments, a region of
the allele-specific extension product comprising the locus-specific
tag is allowed to hybridize to a locus-specific probe that
comprises the complement of the locus-specific tag. In certain such
embodiments, the locus-specific probe is immobilized on a solid
support, such as an array or a bead. In certain embodiments, by
detecting a hybridization complex between the allele-specific
extension product and the locus-specific probe, one can identify
the particular target nucleic acid from which the extension product
is obtained.
[0160] FIG. 12 shows certain exemplary embodiments in which
allele-specific primers are used to distinguish between an
amplification product specific for a first allele ("allele 1") of a
target nucleic acid and an amplification product specific for a
second allele ("allele 2") of the target nucleic acid. In the
embodiments shown in FIG. 12A, the amplification products comprise
the same locus-specific tag but different allele-specific tags. The
amplification products are exposed to 1) a first allele-specific
primer, which hybridizes to an "allele 1" allele-specific tag, and
2) a second allele-specific primer, which hybridizes to an "allele
2" allele-specific tag. In the embodiments shown in FIG. 12A, the
allele-specific primers comprise different labels. The first
allele-specific primer is extended to form a first allele-specific
extension product (shown in FIGS. 12B and 12C), and the second
allele-specific primer is extended to form a second allele-specific
extension product (shown in FIGS. 12B and 12C).
[0161] In the embodiments shown in FIGS. 12B and 12C, both the
first allele-specific extension product and the second
allele-specific extension product comprise the same locus-specific
tag. Accordingly, as shown in FIG. 12B, the extension products
hybridize to a common locus-specific probe. In the embodiments
shown in FIG. 12B, the common locus-specific probe is immobilized
on a solid support, e.g., on a bead or at a particular position on
an array. The labels detected on the solid support indicate which
alleles are present in the target nucleic acid. For example, the
presence of two labels on the solid support indicates the presence
of two alleles (i.e., heterozygosity) in a given target nucleic
acid, whereas the presence of only one label on the solid support
indicates the presence of a single allele (homozygosity) in a given
target nucleic acid.
[0162] In the embodiments shown in FIG. 12C, the extension products
hybridize to one of two different probes that are both allele- and
locus-specific. In FIG. 12C, the first probe comprises the "allele
1" allele-specific tag and the locus-specific tag, and the second
probe comprises the "allele 2" allele-specific tag and the same
locus-specific tag. In the embodiments shown in FIG. 12C, the first
and second probes are immobilized on a solid support, e.g., on a
bead or at a particular position on an array. Thus, the labels that
are detected on the solid support indicate the alleles that are
present in the target nucleic acid.
[0163] In certain embodiments, the allele-specific tag and the
locus-specific tag in an amplification product are detected using a
type II restriction endonuclease. A type II restriction
endonuclease cleaves a nucleic acid at a site other than its
recognition site. Exemplary type II restriction endonucleases
include, but are not limited to, FokI and BsmFI. In certain
embodiments, an allele-specific tag comprises a recognition site
for a type II restriction endonuclease. In certain embodiments,
different allele-specific tags comprise different recognition sites
in order to distinguish among different alleles at a given locus.
In certain embodiments, a type II endonuclease recognizes a
recognition site in the allele-specific tag, but cleaves within the
locus-specific tag in the amplification product. In certain such
embodiments, the absence or presence of a cleavage product is
detected, and thus, the absence or presence of the amplification
product is detected. In certain such embodiments, the presence of a
cleavage product indicates the presence of the allele corresponding
to the allele-specific tag.
[0164] FIG. 13 shows exemplary embodiments of detection using type
II restriction endonucleases. FIG. 13A shows an amplification
product specific for a first allele ("allele 1"). That
amplification product comprises an "allele 1" allele-specific tag
that is recognized by the type II restriction endonuclease FokI. In
certain embodiments, when FokI recognizes the "allele 1"
allele-specific tag in the amplification product, it cleaves the
amplification product within the locus-specific tag. FIG. 13B shows
an amplification product specific for a second allele ("allele 2").
That amplification product comprises an "allele 2" allele-specific
tag that is recognized by the type II restriction endonuclease
BsmFI. In certain embodiments, when BsmFI recognizes the "allele 2"
allele-specific tag in the amplification product, it cleaves the
amplification product within the locus-specific tag.
[0165] In certain embodiments, to determine whether a single
reaction mixture contains either or both of the amplification
products shown in FIG. 13, an aliquot of the reaction mixture is
exposed to FokI, and a separate aliquot of the reaction mixture is
exposed to BsmFI. The absence or presence of cleavage products in
both reaction mixtures is detected, e.g., by detecting
hybridization of all or a portion of the locus-specific tag to a
complementary nucleic acid sequence.
[0166] One skilled in the art will understand that any of the above
embodiments may be modified to detect the complements of the
allele-specific tags and/or complements of the locus-specific tags.
Certain other variations on any of the above embodiments are within
the skill of one skilled in the art.
[0167] Certain Methods of Nucleic Acid Detection Using Spanning
Primers
[0168] In certain embodiments, methods for detecting target nucleic
acid sequences are present in a sample (or quantitating target
nucleic acid sequences in a sample) are provided. In certain
embodiments, a method for amplifying at least one target nucleic
acid sequence is provided, comprising: forming an amplification
reaction composition comprising: a target nucleic acid sequence; a
polymerase; and a first primer comprising (i) a sequence
complementary to the 5' end of the target nucleic acid sequence and
(ii) a sequence complementary to the 3' end of the target nucleic
acid sequence; and subjecting the amplification reaction
composition to at least one amplification reaction to form at least
one amplification product. In certain embodiments, the 3' end of
the target nucleic acid sequence is blocked. In certain
embodiments, the polymerase lacks exonuclease activity.
[0169] In certain embodiments, the amplification reaction
composition further comprises dNTPs. In certain embodiments, the
amplification reaction composition further comprises a buffering
agent. In certain embodiments, the amplification reaction
composition further comprises an additive.
[0170] In certain embodiments, the amplification reaction
composition further comprises a second primer. In certain
embodiments, the second primer comprises (i) a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence and (ii) a sequence complementary to any portion of
the first primer. In certain such embodiments, the second primer
comprises a thymidine between (i) the sequence complementary to the
3' end of a complement of the target nucleic acid sequence and (ii)
the sequence complementary to any portion of the first primer. In
certain embodiments, a second primer comprises (i) a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence and (ii) a sequence complementary to the 5' end of
the first primer. In certain such embodiments, the second primer
comprises a thymidine between (i) the sequence complementary to the
3' end of a complement of the target nucleic acid sequence and (ii)
the sequence complementary to the 5' end of the first primer.
[0171] In certain embodiments, the first primer is a universal
primer. In certain embodiments, the second primer is a universal
primer. In certain embodiments, both the first and second primers
are universal primers.
[0172] In certain embodiments, the amplification reaction comprises
an annealing step that takes place at a predetermined annealing
temperature. In certain embodiments, the annealing temperature of
the first few cycles of amplification is from 62 to 66.degree. C.,
including all temperatures between those endpoints, and is
increased to at least 70.degree. C. for subsequent cycles of
amplification. In certain embodiments, the annealing temperature of
the first few cycles of amplification is 65.degree. C. and is
increased to at least 70.degree. C. for subsequent cycles of
amplification. In certain embodiments, the first few cycles
comprise two cycles. In certain embodiments, the first few cycles
comprise three, four, or five cycles. In certain embodiments, the
annealing temperature is 70.degree. C. or greater.
[0173] In certain embodiments, the method for amplifying at least
one target nucleic acid sequence further comprises detecting the at
least one amplification product. In certain embodiments, the
amplification reaction composition further comprises at least one
probe. In certain embodiments, the at least one probe is detectably
labeled. In certain embodiments, the at least one probe is specific
for a sequence located within the first primer or a sequence
complementary to a sequence located within the first primer. In
certain embodiments, the at least one probe is specific for a
sequence located within the second primer or a sequence
complementary to a sequence located within the second primer. In
certain embodiments, the at least one probe is specific for a
sequence located within the target nucleic acid sequence or a
sequence complementary to a sequence located within the target
nucleic acid sequence.
[0174] In certain embodiments, methods for detecting whether target
nucleic acid sequences are present in a sample (or quantitating
target nucleic acid sequences in a sample) are provided. In certain
embodiments, a method for determining whether at least one target
nucleic acid sequence is present in a sample is provided,
comprising: forming a ligation reaction composition comprising the
sample and a ligation probe set for each target nucleic acid
sequence, the ligation probe set comprising (a) a first probe,
comprising a first target-specific portion, and (b) a second probe,
comprising a second target-specific portion, wherein the probes in
each set are suitable for ligation together when hybridized
adjacent to one another on the corresponding target nucleic acid
sequence; forming a first test composition by subjecting the
ligation reaction composition to at least one cycle of ligation,
wherein adjacently hybridizing complementary probes are ligated to
one another to form a ligation product comprising the first probe
and the second probe; forming an amplification reaction composition
comprising: at least some of the first test composition; a
polymerase; and a first primer comprising (i) a sequence
complementary to the 5' end of the ligation product and (ii) a
sequence complementary to the 3' end of the ligation product;
forming a second test composition by subjecting the amplification
reaction composition to at least one amplification reaction,
wherein the second test composition comprises at least one
amplification product if a target nucleic acid sequence is present
in the sample, and determining whether the at least one target
nucleic acid sequence is present by detecting at least one
amplification product. In certain embodiments, the 3' end of the
target nucleic acid sequence is blocked. In certain embodiments,
the polymerase lacks exonuclease activity.
[0175] In certain embodiments, the amplification reaction
composition comprises dNTPs. In certain embodiments, the
amplification reaction composition comprises a buffering agent. In
certain embodiments, the amplification reaction composition
comprises an additive.
[0176] In certain embodiments, the ligation and amplification
reactions are performed in separate reaction vessels. In certain
embodiments, the ligation and amplification reactions are performed
in a single reaction vessel. In certain embodiments, the ligation
product is purified prior to amplification. In certain embodiments,
the ligation product is not purified prior to amplification.
[0177] In certain embodiments, the first primer is a universal
primer. In certain embodiments, the second primer is a universal
primer. In certain embodiments, both the first and second primers
are universal primers.
[0178] In certain embodiments, the amplification reaction comprises
an annealing step that takes place at a predetermined annealing
temperature. In certain embodiments, the annealing temperature of
two first cycles of amplification is 65.degree. C. and is increased
to at least 70.degree. C. for subsequent cycles of amplification.
In certain embodiments, the annealing temperature is 70.degree. C.
or greater.
[0179] In certain embodiments, the amplification reaction
composition further comprises a second primer. In certain
embodiments, the second primer comprises (i) a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence and (ii) a sequence complementary to any portion of
the first primer. In certain such embodiments, the second primer
comprises a thymidine between (i) the sequence complementary to the
3' end of a complement of the target nucleic acid sequence and (ii)
the sequence complementary to any portion of the first primer. In
certain embodiments, a second primer comprises (i) a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence and (ii) a sequence complementary to the 5' end of
the first primer. In certain such embodiments, the second primer
comprises a thymidine between (i) the sequence complementary to the
3' end of a complement of the target nucleic acid sequence and (ii)
the sequence complementary to the 5' end of the first primer.
[0180] In certain embodiments, each probe set further comprises a
third probe comprising a third target specific portion, wherein the
third target specific portion differs from the first target
specific portion by at least one nucleotide. In certain
embodiments, the first probe comprises a first addressable specific
portion. In certain embodiments, the third probe comprises a second
addressable specific portion. In certain embodiments, the second
probe comprises a third addressable specific portion.
[0181] In certain embodiments, each target nucleic acid sequence
contains at least one pivotal nucleotide, such that a first allele
of the target nucleic acid sequence comprises a first nucleotide at
the at least one pivotal nucleotide, and a second allele of the
target nucleic acid sequence comprises a second nucleotide at the
at least one pivotal nucleotide, and wherein the first nucleotide
and the second nucleotide are different. In certain such
embodiments, each probe set further comprises a third probe,
comprising a third target specific portion, wherein the third
target specific portion differs from the first target specific
portion by at least one nucleotide, and wherein the first target
specific portion comprises at least one pivotal complement for the
first allele of the target nucleic acid sequence and the third
target specific portion comprises at least one pivotal complement
for the second allele of the target nucleic acid sequence.
[0182] In certain such embodiments, the first probe comprises a
first addressable specific portion and the third probe comprises a
second addressable specific portion, such that the presence of the
first addressable specific portion in at least one amplification
product indicates the presence of the first allele of the target
nucleic acid sequence and the presence of the second addressable
specific portion in at least one amplification product indicates
the presence of the second allele of the target nucleic acid
sequence. In certain such embodiments, the second probe comprises a
third addressable specific portion, such that the presence of the
third addressable specific portion in at least one amplification
product indicates the presence of the pivotal nucleotide in the
target nucleic acid sequence.
[0183] In certain such embodiments, the amplification reaction
composition further comprises a second primer comprising a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence, and the detecting comprises one of the following
methods. In certain embodiments, the detecting comprises: exposing
at least some of the second test composition to (a) a first
detection probe comprising (i) a sequence complementary to the
first addressable specific portion and (ii) a sequence
complementary to the third addressable specific portion; and (b) a
second detection probe comprising (i) a sequence complementary to
the second addressable specific portion and (ii) a sequence
complementary to the third addressable specific portion; and
detecting whether the first detection probe hybridizes to at least
one amplification product to determine whether the first allele of
the target nucleic acid sequence is present and detecting whether
the second detection probe hybridizes to at least one amplification
product to determine whether the second allele of the target
nucleic acid sequence is present.
[0184] In certain embodiments, the detecting comprises: exposing at
least some of the second test composition to a first restriction
endonuclease and a second restriction endonuclease to produce a
third test composition which comprises a cleavage product if at
least one amplification product is present in the second test
composition, wherein the first addressable specific portion has a
recognition site for the first restriction endonuclease, the second
addressable specific portion has a recognition site for the second
restriction endonuclease, and wherein the cleavage site for the
first restriction endonuclease and the second restriction
endonuclease is within the third addressable specific portion of at
least one amplification product; exposing the third test
composition to a first detection probe comprising a sequence
complementary to the first addressable specific portion and to a
second detection probe comprising a sequence complementary to the
second addressable specific portion; separating the hybridized
cleavage product from unhybridized first detection probes and
second detection probes; and detecting the presence or absence of
the cleavage product.
[0185] In certain embodiments, the detecting comprises: forming a
second ligation reaction composition comprising at least some of
the second test composition, a first detection probe comprising a
sequence complementary to the first addressable specific portion
and a first label, a second detection probe comprising a second
label and a sequence complementary to the second addressable
specific portion, and a third detection probe comprising a sequence
complementary to the third addressable specific portion, wherein
the first detection probe and the third detection probe are
suitable for ligation together when hybridized adjacent to one
another on at least one amplification product, and wherein the
second probe and the third probe are suitable for ligation together
when hybridized adjacent to one another on at least one
amplification product; forming a third test composition by
subjecting the second ligation reaction composition to at least one
cycle of ligation, wherein adjacently hybridizing complementary
detection probes are ligated to one another to form a second
ligation product comprising the first detection probe or the second
detection probe and the third detection probe; separating the
second ligation product from unligated first detection probes,
second detection probes, and third detection probes; and detecting
the presence or absence of the first label and the second
label.
[0186] In certain embodiments, the detecting comprises: forming a
second amplification reaction composition comprising at least some
of the second test composition, a detection probe comprising a
sequence complementary to the third addressable specific portion, a
first PCR primer comprising a first label and a sequence
complementary to the first addressable specific portion, a second
PCR primer comprising a second label and a sequence complementary
to the second addressable specific portion, and a polymerase,
wherein the detection probe is attached to a solid support;
subjecting the second amplification reaction composition to at
least one amplification reaction; and detecting the presence or
absence of the first label and the second label.
[0187] In certain embodiments, the detecting comprises: forming a
second ligation reaction composition comprising at least some of
the second test composition, a first detection probe comprising a
first label and a sequence complementary to the first addressable
specific portion, a second detection probe comprising a second
label and a sequence complementary to the second addressable
specific portion, and a third detection probe comprising a sequence
complementary to the third addressable specific portion, wherein
the first detection probe and the third detection probe are
suitable for ligation together when hybridized adjacent to one
another on at least one amplification product, and wherein the
second detection probe and the third detection probe are suitable
for ligation together when hybridized adjacent to one another on at
least one amplification product, and wherein the third detection
probe is attached to a solid support; forming a third test
composition by subjecting the ligation reaction composition to at
least one cycle of ligation, wherein adjacently hybridizing
complementary probes are ligated to one another to form a second
ligation product comprising the first detection probe or the second
detection probe and the third detection probe; separating the
second ligation product from unligated first detection probes and
second detection probes; and detecting the presence or absence of
the first label and the second label.
[0188] In certain embodiments, the detecting comprises: exposing
the second test composition to at least two different
sequence-specific mobility-modifiers, wherein each different
mobility-modifier is capable of sequence-specific binding to a
different addressable specific portion and comprises (a) a tag
complement for specifically binding the addressable specific
portion of at least one amplification product, and (b) a tail which
imparts to each mobility modifier a mobility that is distinctive
relative to the mobilities of one or more other of the at least two
different mobility-modifiers in a mobility-dependent analysis
technique; removing mobility-modifiers that are not
sequence-specifically bound to the amplification reaction products
from mobility-modifiers that are sequence-specifically bound to at
least one amplification product; releasing the
sequence-specifically bound mobility-modifiers from the
amplification reaction products; subjecting the released
mobility-modifiers to a mobility-dependent analysis technique; and
detecting one or more target nucleic acid sequences by detecting
distinctive positions of the mobility-modifiers. In certain such
embodiments, at least one sequence-specific mobility modifier
comprises a label. In certain embodiments, the mobility-dependent
analysis technique is electrophoresis.
[0189] In certain embodiments, at least one amplification product
comprises a first or second addressable specific portion that is
four, five, six, seven, eight, nine, or ten nucleotides from the
third addressable specific portion. In certain such embodiments, at
least one amplification product comprises a first or second
addressable specific portion that is eight nucleotides from the
third addressable specific portion.
[0190] In certain embodiments, a method for determining whether at
least one target nucleic acid sequence is present in a sample is
provided, comprising: (a) forming a reaction composition
comprising: the sample; a ligation probe set for each target
nucleic acid sequence, the probe set comprising (a) at least one
first probe, comprising a first target-specific portion and (b) at
least one second probe, comprising a second target-specific
portion, wherein the probes in each set are suitable for ligation
together to form a ligation product when hybridized adjacent to one
another on a complementary target nucleic acid sequence; a
polymerase; and a first primer comprising (i) a sequence
complementary to the 5' end of the ligation product and (ii) a
sequence complementary to the 3' end of the ligation product; (b)
subjecting the reaction composition to at least one cycle of
ligation, wherein adjacently hybridizing complementary probes are
ligated to one another to form a ligation product comprising the
first probe and the second probe; (c) after the at least one cycle
of ligation, subjecting the reaction composition to at least one
amplification reaction to form at least one amplification product
if a target nucleic acid sequence is present in the sample; and (d)
determining whether the at least one target nucleic acid sequence
is present by detecting at least one amplification product. In
certain such embodiments, the quantity of target nucleic acid
sequences in the sample is determined. In certain embodiments, the
3' end of the target nucleic acid sequence is blocked. In certain
embodiments, the polymerase lacks exonuclease activity.
[0191] In certain embodiments, the amplification reaction
composition comprises dNTPs. In certain embodiments, the
amplification reaction composition comprises a buffering agent. In
certain embodiments, the amplification reaction composition
comprises an additive.
[0192] In certain embodiments, the ligation product is purified
prior to amplification. In certain embodiments, the ligation
product is not purified prior to amplification.
[0193] In certain embodiments, the amplification reaction comprises
an annealing step that takes place at a predetermined annealing
temperature. In certain embodiments, the annealing temperature of
the first few cycles of amplification is from 62 to 66.degree. C.,
including all temperatures between those endpoints, and is
increased to at least 70.degree. C. for subsequent cycles of
amplification. In certain embodiments, the annealing temperature of
the first few cycles of amplification is 65.degree. C. and is
increased to at least 70.degree. C. for subsequent cycles of
amplification. In certain embodiments, the first few cycles
comprise two cycles. In certain embodiments, the first few cycles
comprise three, four, or five cycles.
[0194] In certain embodiments, the amplification reaction
composition further comprises a second primer. In certain
embodiments, the second primer comprises (i) a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence and (ii) a sequence complementary to any portion of
the first primer. In certain such embodiments, the second primer
comprises a thymidine between (i) the sequence complementary to the
3' end of a complement of the target nucleic acid sequence and (ii)
the sequence complementary to any portion of the first primer. In
certain embodiments, the second primer comprises (i) a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence and (ii) a sequence complementary to the 5' end of
the first primer. In certain such embodiments, the second primer
comprises a thymidine between (i) the sequence complementary to the
3' end of a complement of the target nucleic acid sequence and (ii)
the sequence complementary to the 5' end of the first primer.
[0195] In certain embodiments, each probe set further comprises a
third probe comprising a third target specific portion, wherein the
third target specific portion differs from the first target
specific portion by at least one nucleotide. In certain
embodiments, the first probe comprises a first addressable specific
portion. In certain embodiments, the third probe comprises a second
addressable specific portion. In certain embodiments, the second
probe comprises a third addressable specific portion.
[0196] In certain embodiments, each target nucleic acid sequence
contains at least one pivotal nucleotide, such that a first allele
of the target nucleic acid sequence comprises a first nucleotide at
the at least one pivotal nucleotide, and a second allele of the
target nucleic acid sequence comprises a second nucleotide at the
at least one pivotal nucleotide, and wherein the first nucleotide
and the second nucleotide are different. In certain such
embodiments, each probe set further comprises a third probe,
comprising a third target specific portion, wherein the third
target specific portion differs from the first target specific
portion by at least one nucleotide, and wherein the first target
specific portion comprises at least one pivotal complement for the
first allele of the target nucleic acid sequence and the third
target specific portion comprises at least one pivotal complement
for the second allele of the target nucleic acid sequence.
[0197] In certain such embodiments, the first probe comprises a
first addressable specific portion and the third probe comprises a
second addressable specific portion, such that the presence of the
first addressable specific portion in at least one amplification
product indicates the presence of the first allele of the target
nucleic acid sequence and the presence of the second addressable
specific portion in at least one amplification product indicates
the presence of the second allele of the target nucleic acid
sequence. In certain such embodiments, the second probe comprises a
third addressable specific portion, such that the presence of the
third addressable specific portion in at least one amplification
product indicates the presence of the pivotal nucleotide in the
target nucleic acid sequence.
[0198] In certain such embodiments, the amplification reaction
composition further comprises a second primer comprising a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence, and the detecting comprises one of the following
methods. In certain embodiments, the detecting comprises: exposing
at least some of the second test composition to (a) a first
detection probe comprising (i) a sequence complementary to the
first addressable specific portion and (ii) a sequence
complementary to the third addressable specific portion; and (b) a
second detection probe comprising (i) a sequence complementary to
the second addressable specific portion and (ii) a sequence
complementary to the third addressable specific portion; and
detecting whether the first detection probe hybridizes to at least
one amplification product to determine whether the first allele of
the target nucleic acid sequence is present and detecting whether
the second detection probe hybridizes to at least one amplification
product to determine whether the second allele of the target
nucleic acid sequence is present.
[0199] In certain embodiments, the detecting comprises: exposing at
least some of the second test composition to a first restriction
endonuclease and a second restriction endonuclease to produce a
third test composition which comprises a cleavage product if at
least one amplification product is present in the second test
composition, wherein the first addressable specific portion has a
recognition site for the first restriction endonuclease, the second
addressable specific portion has a recognition site for the second
restriction endonuclease, and wherein the cleavage site for the
first restriction endonuclease and the second restriction
endonuclease is within the third addressable specific portion of at
least one amplification product; exposing the third test
composition to a first detection probe comprising a sequence
complementary to the first addressable specific portion and to a
second detection probe comprising a sequence complementary to the
second addressable specific portion; separating the hybridized
cleavage product from unhybridized first detection probes and
second detection probes; and detecting the presence or absence of
the cleavage product.
[0200] In certain embodiments, the detecting comprises: forming a
second ligation reaction composition comprising at least some of
the second test composition, a first detection probe comprising a
sequence complementary to the first addressable specific portion
and a first label, a second detection probe comprising a second
label and a sequence complementary to the second addressable
specific portion, and a third detection probe comprising a sequence
complementary to the third addressable specific portion, wherein
the first detection probe and the third detection probe are
suitable for ligation together when hybridized adjacent to one
another on at least one amplification product, and wherein the
second probe and the third probe are suitable for ligation together
when hybridized adjacent to one another on at least one
amplification product; forming a third test composition by
subjecting the second ligation reaction composition to at least one
cycle of ligation, wherein adjacently hybridizing complementary
detection probes are ligated to one another to form a second
ligation product comprising the first detection probe or the second
detection probe and the third detection probe; separating the
second ligation product from unligated first detection probes,
second detection probes, and third detection probes; and detecting
the presence or absence of the first label and the second
label.
[0201] In certain embodiments, the detecting comprises: forming a
second amplification reaction composition comprising at least some
of the second test composition, a detection probe comprising a
sequence complementary to the third addressable specific portion, a
first PCR primer comprising a first label and a sequence
complementary to the first addressable specific portion, a second
PCR primer comprising a second label and a sequence complementary
to the second addressable specific portion, and a polymerase,
wherein the detection probe is attached to a solid support;
subjecting the second amplification reaction composition to at
least one amplification reaction; and detecting the presence or
absence of the first label and the second label.
[0202] In certain embodiments, the detecting comprises: forming a
second ligation reaction composition comprising at least some of
the second test composition, a first detection probe comprising a
first label and a sequence complementary to the first addressable
specific portion, a second detection probe comprising a second
label and a sequence complementary to the second addressable
specific portion, and a third detection probe comprising a sequence
complementary to the third addressable specific portion, wherein
the first detection probe and the third detection probe are
suitable for ligation together when hybridized adjacent to one
another on at least one amplification product, and wherein the
second detection probe and the third detection probe are suitable
for ligation together when hybridized adjacent to one another on at
least one amplification product, and wherein the third detection
probe is attached to a solid support; forming a third test
composition by subjecting the ligation reaction composition to at
least one cycle of ligation, wherein adjacently hybridizing
complementary probes are ligated to one another to form a second
ligation product comprising the first detection probe or the second
detection probe and the third detection probe; separating the
second ligation product from unligated first detection probes and
second detection probes; and detecting the presence or absence of
the first label and the second label.
[0203] In certain embodiments, the detecting comprises: exposing
the second test composition to at least two different
sequence-specific mobility-modifiers, wherein each different
mobility-modifier is capable of sequence-specific binding to a
different addressable specific portion and comprises (a) a tag
complement for specifically binding the addressable specific
portion of at least one amplification product, and (b) a tail which
imparts to each mobility modifier a mobility that is distinctive
relative to the mobilities of one or more other of the at least two
different mobility-modifiers in a mobility-dependent analysis
technique; removing mobility-modifiers that are not
sequence-specifically bound to the amplification reaction products
from mobility-modifiers that are sequence-specifically bound to at
least one amplification product; releasing the
sequence-specifically bound mobility-modifiers from the
amplification reaction products; subjecting the released
mobility-modifiers to a mobility-dependent analysis technique; and
detecting one or more target nucleic acid sequences by detecting
distinctive positions of the mobility-modifiers. In certain such
embodiments, at least one sequence-specific mobility modifier
comprises a label. In certain embodiments, the mobility-dependent
analysis technique is electrophoresis.
[0204] In certain embodiments, at least one amplification product
comprises a first or second addressable specific portion that is
four, five, six, seven, eight, nine, or ten nucleotides from the
third addressable specific portion. In certain such embodiments, at
least one amplification product comprises a first or second
addressable specific portion that is eight nucleotides from the
third addressable specific portion.
Certain Exemplary Kits
[0205] In certain embodiments, kits are provided that are designed
to expedite performing certain methods. In certain embodiments,
kits serve to expedite the performance of the methods of interest
by assembling two or more components used in carrying out the
methods. In certain embodiments, kits may contain components in
pre-measured unit amounts to minimize the need for measurements by
end-users. In certain embodiments, kits may include instructions
for performing one or more methods. In certain embodiments, the kit
components are optimized to operate in conjunction with one
another.
[0206] In certain embodiments, a kit for amplifying at least one
target nucleic acid sequence is provided. In certain embodiments, a
kit comprises a polymerase and a first primer comprising (i) a
sequence complementary to the 5' end of the target nucleic acid
sequence and (ii) a sequence complementary to the 3' end of the
target nucleic acid sequence. In certain embodiments, the kit
further comprises a second primer. In certain embodiments, the
second primer comprises (i) a sequence complementary to the 3' end
of a complement of the target nucleic acid sequence and (ii) a
sequence complementary to the 5' end of the first primer. In
certain embodiments, a second primer comprises (i) a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence and (ii) a sequence complementary to any portion of
the first primer. In certain embodiments, the kit comprises dNTPs.
In certain embodiments, the kit comprises one or more buffering
agents. In certain embodiments, the kit comprises one or more
additives. In certain embodiments, the kit further comprises
instructions for use.
[0207] In certain embodiments, a kit comprises a ligation probe set
for each target nucleic acid sequence, wherein the probe set
comprises (a) a first probe, comprising a first target-specific
portion, and (b) a second probe, comprising a second
target-specific portion, wherein the probes in each set are
suitable for ligation together when hybridized adjacent to one
another on a complementary target sequence. In certain embodiments,
a kit further comprises a polymerase and a first primer comprising
(i) a sequence complementary to the 5' end of the target nucleic
acid sequence and (ii) a sequence complementary to the 3' end of
the target nucleic acid sequence. In certain embodiments, a kit
further comprises a second primer. In certain embodiments the
second primer comprises (i) a sequence complementary to the 3' end
of a complement of the target nucleic acid sequence and (ii) a
sequence complementary to the 5' end of the first primer. In
certain embodiments, the second primer comprises (i) a sequence
complementary to the 3' end of a complement of the target nucleic
acid sequence and (ii) a sequence complementary to any portion of
the first primer. In certain embodiments, the kit comprises a
ligase. In certain embodiments, the kit comprises dNTPs. In certain
embodiments, the kit comprises one or more buffering agents. In
certain embodiments, the kit comprises one or more additives. In
certain embodiments, the kit comprises instructions for use.
Certain Components for Ligation
[0208] Certain Target Nucleic Acids
[0209] Exemplary target nucleic acid sequences include, but are not
limited to, RNA and DNA. Exemplary RNA target sequences include,
but are not limited to, mRNA, snRNA, rRNA, tRNA, viral RNA, and
variants of RNA, such as splicing variants. Exemplary DNA target
sequences include, but are not limited to, genomic DNA, plasmid
DNA, phage DNA, nucleolar DNA, mitochondrial DNA, and chloroplast
DNA.
[0210] Exemplary target nucleic acid sequences include, but are not
limited to, cDNA, yeast artificial chromosomes (YAC's), bacterial
artificial chromosomes (BAC's), other extrachromosomal DNA, and
nucleic acid analogs. Exemplary nucleic acid analogs include, but
are not limited to, LNAs, PNAs, PPGs (e.g., pyrazolopyrimidine G),
and other nucleic acid analogs.
[0211] A variety of methods are available for obtaining a target
nucleic acid sequence for use with certain compositions and methods
of the present teachings. When the nucleic acid target is obtained
through isolation from a biological matrix, certain isolation
techniques include, but are not limited to, (1) organic extraction
followed by ethanol precipitation, e.g., using a phenol/chloroform
organic reagent (e.g., Ausubel et al., eds., Current Protocols in
Molecular Biology Volume 1, Chapter 2, Section I, John Wiley &
Sons, New York (1993)), in certain embodiments, using an automated
DNA extractor, e.g., the Model 341 DNA Extractor available from
Applied Biosystems (Foster City, Calif.); (2) stationary phase
adsorption methods (e.g., Boom et al., U.S. Pat. No. 5,234,809;
Walsh et al., Biotechniques 10(4): 506-513 (1991)); and (3)
salt-induced DNA precipitation methods (e.g., Miller et al.,
Nucleic Acids Research, 16(3): 9-10 (1988)), such precipitation
methods being typically referred to as "salting-out" methods. In
certain embodiments, the above isolation methods may be preceded by
an enzyme digestion step to help eliminate unwanted protein from
the sample, e.g., digestion with proteinase K, or other like
proteases. See, e.g., Published U.S. Patent Application No.
2005/0009045.
[0212] In certain embodiments, a target nucleic acid sequence may
be derived from any living, or once living, organism, including but
not limited to prokaryote, eukaryote, plant, animal, and virus. In
certain embodiments, the target nucleic acid sequence may originate
from a nucleus of a cell, e.g., genomic DNA, or may be extranuclear
nucleic acid, e.g., plasmid, mitochondrial nucleic acid, various
RNAs, and the like. In certain embodiments, if the sequence from
the organism is RNA, it may be reverse-transcribed into a cDNA
target nucleic acid sequence. Furthermore, in certain embodiments,
the target nucleic acid sequence may be present in a double
stranded or single stranded form.
[0213] Exemplary target nucleic acid sequences include, but are not
limited to, amplification products, ligation products, reverse
transcription products, primer extension products, methylated DNA,
and cleavage products. Exemplary amplification products include,
but are not limited to, PCR and products of certain isothermal
amplification techniques.
[0214] In certain embodiments, nucleic acids in a sample may be
subjected to a cleavage or fragmentation procedure. In certain
embodiments, such cleavage products and/or fragments may comprise
targets.
[0215] Different target nucleic acid sequences may be different
portions of a single contiguous nucleic acid or may be on different
nucleic acids. Different portions of a single contiguous nucleic
acid may or may not overlap.
[0216] The person of ordinary skill will appreciate that while a
target nucleic acid sequence is typically described as a
single-stranded molecule, the opposing strand of a double-stranded
molecule comprises a complementary sequence that may also be used
as a target sequence.
[0217] Certain Ligation Probe Sets
[0218] A ligation probe set, according to certain embodiments,
comprises two or more probes that comprise a target-specific
portion that is designed to hybridize in a sequence-specific manner
with a complementary region on a specific target nucleic acid
sequence (see, e.g., FIG. 5). In certain embodiments, a probe of a
ligation probe set may further comprise a primer-specific portion,
and/or an addressable portion, or a combination of these additional
components. In certain embodiments, any of the probe's components
may overlap any other probe component(s). For example, but without
limitation, the target-specific portion may overlap the
primer-specific portion. Also, without limitation, the addressable
portion may overlap with the target-specific portion or the primer
specific-portion, or both.
[0219] In certain embodiments, at least one probe of a ligation
probe set comprises the addressable portion located between the
target-specific portion and the primer-specific portion. In certain
embodiments, the probe's addressable portion may comprise a
sequence that is the same as, or is complementary to, at least a
portion of a labeled probe. In certain embodiments, the probe's
primer-specific portion may comprise a sequence that is the same
as, or is complementary to, at least a portion of a labeled probe.
In certain embodiments, the probe's addressable portion is not
complementary with target sequences, primer sequences, or probe
sequences other than complementary portions of labeled probes.
[0220] The sequence-specific portions of probes are of sufficient
length to permit specific annealing to complementary sequences in
primers, addressable portions, and/or targets as appropriate. In
certain embodiments, the length of the addressable portions and
target-specific portion are any number of nucleotides from 6 to 35.
In certain embodiments, the length of the addressable portions and
target-specific portion is greater than 35. Detailed descriptions
of probe design that provide for sequence-specific annealing can be
found, among other places, in Dieffenbach and Dveksler, PCR Primer,
A Laboratory Manual, Cold Spring Harbor Press (1995), and Kwok et
al., Nucl. Acids Res. 18:999-1005 (1990).
[0221] Under appropriate conditions, adjacently hybridized probes
may be ligated together to form a ligation product, provided that
they comprise appropriate reactive groups, for example, without
limitation, a free 3'-hydroxyl and 5'-phosphate group.
[0222] According to certain embodiments, some ligation probe sets
may comprise more than one first probe or more than one second
probe to allow sequence discrimination between target sequences
that differ by one or more nucleotides.
[0223] The skilled artisan will appreciate that, in various
embodiments, a pivotal nucleotide(s) may be located anywhere in the
target sequence and that likewise, a pivotal complement(s) may be
located anywhere within a target-specific portion of the probe(s).
For example, according to various embodiments, the pivotal
complement may be located at the 3' end of a probe, at the 5' end
of a probe, or anywhere between the 3' end and the 5' end of a
probe.
[0224] In certain embodiments, certain mechanisms may be employed
to avoid ligation of probes that do not include the correct
complementary nucleotide at the pivotal complement. For example, in
certain embodiments, conditions may be employed such that a probe
of a ligation probe set will hybridize to the target sequence to a
measurably lesser extent if there is a mismatch at the pivotal
nucleotide. Thus, in such embodiments, such non-hybridized probes
will not be ligated to the other probe in the probe set.
[0225] In certain embodiments, the first probes and second probes
in a ligation probe set are designed with similar melting
temperatures (T.sub.m). Where a probe includes a pivotal
complement, in certain embodiments, the T.sub.m for the probe(s)
comprising the pivotal complement(s) of the target pivotal
nucleotide sought will be approximately 4-15.degree. C. lower than
the other probe(s) that do not contain the pivotal complement in
the probe set. In certain such embodiments, the probe comprising
the pivotal complement(s) will also be designed with a T.sub.m near
the ligation temperature. Thus, a probe with a mismatched
nucleotide will more readily dissociate from the target at the
ligation temperature. The ligation temperature, therefore, in
certain embodiments provides another way to discriminate between,
for example, multiple potential alleles in the target.
[0226] Further, in certain embodiments, ligation probe sets do not
comprise a pivotal complement at the terminus of the first or the
second probe (e.g., at the 3' end or the 5' end of the first or
second probe). Rather, the pivotal complement is located somewhere
between the 5' end and the 3' end of the first or second probe. In
certain such embodiments, probes with target-specific portions that
are fully complementary with their respective target regions will
hybridize under high stringency conditions. Probes with one or more
mismatched bases in the target-specific portion, by contrast, will
hybridize to their respective target region to a measurably lesser
extent. Both the first probe and the second probe must be
hybridized to the target for a ligation product to be
generated.
[0227] In certain embodiments, highly related sequences that differ
by as little as a single nucleotide can be distinguished. For
example, according to certain embodiments, one can distinguish the
two potential alleles in a biallelic locus as follows. One can
combine a ligation probe set comprising two first probes, differing
in their addressable portions and their pivotal complement, one
second probe, and the sample containing the target. All three
probes will hybridize with the target sequence under appropriate
conditions. Only the first probe with the hybridized pivotal
complement, however, will be ligated efficiently to the hybridized
second probe. Thus, if only one allele is present in the sample,
only one ligation product for that target will be generated. Both
ligation products would be formed in a sample from a heterozygous
individual. In certain embodiments, ligation of probes with a
pivotal complement that is not complementary to the pivotal
nucleotide may occur, but such ligation occurs to a measurably
lesser extent than ligation of probes with a pivotal complement
that is complementary to the pivotal nucleotide.
Certain Components for Detection
[0228] Many different signal moieties may be used in various
embodiments. For example, exemplary signal moieties include, but
are not limited to, fluorophores, radioisotopes, chromogens,
enzymes, antigens, heavy metals, dyes, phosphorescence groups,
chemiluminescent groups, and electrochemical detection moieties.
Exemplary fluorophores that may be used as signal moieties include,
but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy
5), fluorescein, Vic.TM., Liz.TM., Tamra.TM., 5-Fam.TM., 6-Fam.TM.,
and Texas Red (Molecular Probes). (Vic.TM., Liz.TM., Tamra.TM.,
5-Fam.TM., and 6-Fam.TM. (all available from Applied Biosystems,
Foster City, Calif.) Exemplary radioisotopes include, but are not
limited to, .sup.32P, .sup.33P, and .sup.35S. Signal moieties
include elements of multi-element indirect reporter systems, e.g.,
biotin/avidin, antibody/antigen, ligand/receptor, enzyme/substrate,
and the like, in which the element interacts with other elements of
the system in order to effect a detectable signal. Certain
exemplary multi-element systems include, but are not limited to, a
biotin reporter group attached to a probe and an avidin conjugated
with a fluorescent label. Certain detailed protocols for methods of
attaching signal moieties to oligonucleotides can be found in,
among other places, G. T. Hermanson, Bioconjugate Techniques,
Academic Press, San Diego, Calif. (1996) and S. L. Beaucage et al.,
Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons,
New York, N.Y. (2000).
[0229] As discussed above, the term "interaction probe" refers to a
probe that comprises at least two moieties that can interact with
one another to provide a detectably different signal value
depending upon whether a given nucleic acid sequence is present or
absent. In certain embodiments, one of the moieties is a signal
moiety and the other moiety is a quencher moiety. The signal value
that is detected from the signal moiety is different depending on
whether the quencher moiety is sufficiently close to the signal
moiety or is spaced apart from the signal moiety. In certain
embodiments, the quencher moiety decreases the detectable signal
value from the signal moiety when the quencher moiety is
sufficiently close to the signal moiety. In certain embodiments,
the quencher moiety decreases the detectable signal value to zero
or close to zero when the quencher moiety is sufficiently close to
the signal moiety.
[0230] In certain embodiments, one of the moieties of the
interaction probe is a signal moiety and the other moiety is a
donor moiety. The signal value that is detected from the signal
moiety is different depending on whether the donor moiety is
sufficiently close to the signal moiety or is spaced apart from the
signal moiety. In certain embodiments, the donor moiety increases
the detectable signal value from the signal moiety when the donor
moiety is sufficiently close to the signal moiety. In certain
embodiments, the detectable signal value is zero or close to zero
when the donor moiety is not sufficiently close to the signal
moiety.
[0231] In certain embodiments employing a donor moiety and signal
moiety, one may use certain energy-transfer fluorescent dyes.
Certain nonlimiting exemplary pairs of donors (donor moieties) and
acceptors (signal moieties) are illustrated, e.g., in U.S. Pat.
Nos. 5,863,727; 5,800,996; and 5,945,526. Use of certain such
combinations of a donor and an acceptor have also been called FRET
(Fluorescent Resonance Energy Transfer).
[0232] In certain embodiments, the moieties of the interaction
probe are linked to one another by a link element such as, but not
limited to, an oligonucleotide. In certain such embodiments, the
presence of a sequence that hybridizes to an interaction probe
impacts the proximity of the moieties to one another during the
methods described herein. In various embodiments, the moieties may
be attached to the link element in various ways known in the art.
For example, certain nonlimiting protocols for attaching moieties
to oligonucleotides are found in, among other places, G. T.
Hermanson, Bioconjugate Techniques, Academic Press, San Diego,
Calif. (1996) and S. L. Beaucage et al., Current Protocols in
Nucleic Acid Chemistry, John Wiley & Sons, New York, N.Y.
(2000). In certain embodiments, an interaction probe comprises more
than one signal moiety. In certain embodiments, an interaction
probe comprises more than one quencher moiety. In certain
embodiments, an interaction probe comprises more than one donor
moiety.
[0233] According to certain embodiments, the interaction probe may
be a "5'-nuclease probe," which comprises a signal moiety linked to
a quencher moiety or a donor moiety through a short oligonucleotide
link element. When the 5'-nuclease probe is intact, the quencher
moiety or the donor moiety influences the detectable signal from
the signal moiety. According to certain embodiments, the
5'-nuclease probe binds to a specific nucleic acid sequence, and is
cleaved by a polymerase or other polypeptide having 5' nuclease
activity when the probe is replaced by a newly polymerized strand
during an amplification reaction such as PCR or some other strand
displacement protocol.
[0234] When the oligonucleotide link element of the 5'-nuclease
probe is cleaved, the detectable signal from the signal moiety
changes when the signal moiety becomes further separated from the
quencher moiety or the donor moiety. In certain such embodiments
that employ a quencher moiety, the signal value increases when the
signal moiety becomes further separated from the quencher moiety.
In certain such embodiments that employ a donor moiety, the signal
value decreases when the signal moiety becomes further separated
from the donor moiety.
[0235] In certain embodiments, the 5'-nuclease probe is a
5'-nuclease fluorescent probe, in which the signal moiety is a
fluorescent moiety and the quencher moiety is a fluorescence
quencher moiety. When the probe is cleaved during a strand
displacement protocol, the fluorescent moiety emits a detectable
fluorescent signal. In certain embodiments, a 5'-nuclease
fluorescent probe may emit a given level of signal when it is
hybridized to a complementary sequence prior to cleavage, and the
level of the signal is increased with cleavage. Certain exemplary
embodiments of 5'-nuclease fluorescent probes are described, e.g.,
in U.S. Pat. No. 5,538,848, and exemplified by the TaqMan.RTM.
probe molecule, which is part of the TaqMan.RTM. assay system
(available from Applied Biosystems, Foster City, Calif.).
[0236] According to certain embodiments, the interaction probe may
be a "hybridization dependent probe," which comprises a signal
moiety linked to a quencher moiety or a donor moiety through an
oligonucleotide link element. When the hybridization dependent
probe is not bound to a given nucleic acid sequence, and is thus
single stranded, the oligonucleotide link element can bend
flexibly, and the quencher moiety or the donor moiety is
sufficiently close to the signal moiety to influence the detectable
signal from the signal moiety. In certain embodiments, the
oligonucleotide link element of a hybridization dependent probe is
designed such that when it is not hybridized to a given nucleic
acid sequence, it folds back and hybridizes to itself, e.g., a
molecular beacon probe. See, e.g., U.S. Pat. Nos. 5,118,801;
5,312,728; and 5,925,517. In certain embodiments, the
oligonucleotide link element of a hybridization dependent probe
does not hybridize to itself when it is not hybridized to the given
nucleic acid sequence.
[0237] When a hybridization dependent probe is bound to a given
nucleic acid as double stranded nucleic acid, the quencher moiety
or the donor moiety is spaced apart from the signal moiety such
that the detectable signal is changed. In certain such embodiments
that employ a quencher moiety, the signal value increases when the
signal moiety becomes further separated from the quencher moiety.
In certain such embodiments that employ a donor moiety, the signal
value decreases when the signal moiety becomes further separated
from the donor moiety.
[0238] In certain embodiments of hybridization dependent probes,
the signal moiety is a fluorescent moiety and the quencher moiety
is a fluorescence quencher moiety. When the probe is hybridized to
a specific nucleic acid sequence, the fluorescent moiety emits a
detectable fluorescent signal. When the probe is not hybridized to
a nucleic acid sequence and is intact, quenching occurs and little
or no fluorescence is detected.
[0239] Certain exemplary embodiments of hybridization dependent
probes are described, e.g., in U.S. Pat. No. 5,723,591.
[0240] In certain embodiments, one employs nucleic acids in the
hybridization dependent probes such that a substantial portion of
the hybridization dependent probes are not cleaved by an enzyme
during an amplification reaction. A "substantial portion of the
hybridization dependent probes are not cleaved" refers to a portion
of the total number of hybridization dependent probes that are
designed to hybridize to a given nucleic sequence that is being
amplified, and it does not refer to a portion of an individual
probe. In certain embodiments, "a substantial portion of
hybridization dependent probes that are not cleaved" means that at
least 90% of the hybridization dependent probes are not cleaved. In
certain embodiments, at least 95% of the hybridization dependent
probes are not cleaved. In certain embodiments, one employs PNA for
some or all of the nucleic acids of a hybridization dependent
probe.
[0241] In certain embodiments, one employs hybridization dependent
probes in which a substantial portion of the hybridization
dependent probes do not hybridize to an addressable portion or a
complement of the addressable portion during an extension reaction.
A "substantial portion of the hybridization dependent probes do not
hybridize" here refers to a portion of the total number of
hybridization dependent probes that are designed to hybridize to a
given nucleic sequence that is being amplified, and it does not
refer to a portion of an individual probe. In certain embodiments,
"a substantial portion of hybridization dependent probes that do
not hybridize" means that at least 90% of the hybridization
dependent probes do not hybridize. In certain embodiments, at least
95% of the hybridization dependent probes do not hybridize.
[0242] According to certain embodiments, the interaction probe may
comprise two oligonucleotides that hybridize to a given nucleic
acid sequence adjacent to one another. In certain embodiments, one
of the oligonucleotides comprises a signal moiety and one of the
oligonucleotides comprises a quencher moiety or a donor moiety.
When both oligonucleotides are hybridized to the given nucleic acid
sequence, the quencher moiety or the donor moiety is sufficiently
close to the signal moiety to influence the detectable signal from
the signal moiety.
[0243] In certain such embodiments that employ a donor moiety, the
signal value increases when the two oligonucleotides are hybridized
to the given nucleic acid sequence. In certain such embodiments
that employ a quencher moiety, the signal value decreases when the
two oligonucleotides are hybridized to the given nucleic acid
sequence. In certain embodiments, the signal moiety is a
fluorescent moiety.
[0244] Other examples of suitable interaction probes according to
various embodiments are i-probes, scorpion probes, eclipse probes,
and others. Exemplary, but nonlimiting, probes are discussed, for
example, in Whitcombe et al., Nat. Biotechnol., 17(8):804-807
(1999) (includes scorpion probes); Thelwell et al., Nucleic Acids
Res., 28(19):3752-3761 (2000) (includes scorpion probes); Afonina
et al., Biotechniques, 32(4):940-944, 946-949 (2002) (includes
eclipse probes); Li et al., Nuc. Acids Res., 30(2):E5 (2002);
Kandimall et al., Bioorg. Med. Chem., 8(8):1911-1916 (2000);
Isacsson et al., Mol. Cell. Probes, 14(5):321-328 (2000); French et
al, Mol. Cell. Probes, 15(6):363-374 (2001); and Nurmi et al., Nuc.
Acids Res., 28(8), E28 (2000). Exemplary quencher moieties
according to certain embodiments may be those available from Epoch
Biosciences, Bothell, Wash.
[0245] In certain embodiments, one may use a labeled probe and a
threshold difference between first and second detectable signal
values to detect the presence or absence of a target nucleic acid
in a sample. In such embodiments, if the difference between the
first and second detectable signal values is the same as or greater
than the threshold difference, i.e., there is a threshold
difference, one concludes that the target nucleic acid is present.
If the difference between the first and second detectable signal
values is less than the threshold difference, i.e., there is no
threshold difference, one concludes that the target nucleic acid is
absent.
[0246] Certain nonlimiting examples of how one may set a threshold
difference according to certain embodiments follow.
[0247] First, in certain embodiments, a labeled probe that is not
hybridized to a complementary sequence may have a first detectable
signal value of zero. In certain embodiments, when one forms an
amplification reaction composition comprising the labeled probe,
and any unligated ligation probes and ligation products that
include complementary addressable portions, before amplification,
the detectable signal value may increase to 0.4. In certain such
embodiments, when such an amplification reaction composition does
not include any ligation products comprising the complementary
addressable portion, the detectable signal value may remain at 0.4
during and/or after an amplification reaction. (In other words, the
second detectable signal value is 0.4.) In certain such
embodiments, when such an amplification reaction composition,
however, includes a ligation product comprising a complementary
addressable portion, the detectable signal value may increase to 2
during and/or after an amplification reaction. (In other words, the
second detectable signal value is 2.)
[0248] Thus, in certain such embodiments, one may set a threshold
difference between first and second detectable signal values at a
value somewhere between a value just above 0.4 to about 2. For
example, one may set the threshold difference at somewhere between
0.5 to 2.
[0249] Second, in certain embodiments, a labeled probe that is not
hybridized to a complementary sequence may have a first detectable
signal value of zero. In certain embodiments, when one forms an
amplification reaction composition comprising the labeled probe,
and any unligated ligation probes and ligation products that
include complementary addressable portions, before amplification,
the detectable signal value may increase to 0.4. In certain such
embodiments, when such an amplification reaction composition does
not include any ligation products comprising the complementary
addressable portion, the detectable signal value may increase to
0.7 during and/or after an amplification reaction. (In other words,
the second detectable signal value is 0.7.) In certain such
embodiments, when such an amplification reaction composition,
however, includes a ligation product comprising a complementary
addressable portion, the detectable signal value may increase to 2
during and/or after an amplification reaction. (In other words, the
second detectable signal value is 2.)
[0250] Thus, in certain such embodiments, one may set a threshold
difference between first and second detectable signal values at a
value somewhere between a value just above 0.7 to about 2. For
example, one may set the threshold difference at somewhere between
0.8 to 2.
[0251] Third, in certain embodiments, a labeled probe that is not
hybridized to a complementary sequence may have a first detectable
signal value of zero. In certain embodiments, when one forms an
amplification reaction composition comprising the labeled probe,
and any unligated ligation probes and ligation products that
include complementary addressable portions, before amplification,
the detectable signal value may increase to 0.4. In certain such
embodiments, when such an amplification reaction composition does
not include any ligation products comprising the complementary
addressable portion, the detectable signal value may increase
linearly during and/or after an amplification reaction. (In other
words, the second detectable signal value is linearly increased
from the first detectable signal value.) In certain such
embodiments, when such an amplification reaction composition,
however, includes a ligation product comprising a complementary
addressable portion, the detectable signal value may increase
exponentially during and/or after an amplification reaction. (In
other words, the second detectable signal value is exponentially
increased from the first detectable signal value.)
[0252] Thus, in certain such embodiments, one may measure
detectable signal values at two or more points during
amplification, and at the end of the amplification reaction, to
determine if the increase in detectable signal value is linear or
exponential. In certain embodiments, one may measure detectable
signal values at three or more points during amplification to
determine if the increase in detectable signal value is linear or
exponential. In certain embodiments, if the increase is
exponential, there is a threshold difference between the first and
second detectable signal values.
[0253] In certain embodiments, one may employ different labeled
probes that are specific to different addressable portions. In
certain such embodiments, one may employ different labeled probes
that comprise different sequences and detectably different signal
moieties. Detectably different signal moieties include, but are not
limited to, moieties that emit light of different wavelengths,
moieties that absorb light of different wavelengths, moieties that
have different fluorescent decay lifetimes, moieties that have
different spectral signatures, and moieties that have different
radioactive decay properties.
[0254] In certain embodiments, one may employ a labeled probe that
remains intact unless a particular nucleic acid sequence is
present. A label is attached to the probe. If the particular
nucleic acid is present, the probe will be cleaved. Certain
examples, of such probes include, but are not limited to, probes
that are cleaved by 5' nuclease activity during an extension
reaction and probes that are cleaved by RNase H or another agent
with similar activity.
[0255] In certain such embodiments, the cleaved portion of the
probe with the label can be separated from intact probes in view of
different migration rates of the cleaved portion of the probe and
the intact probe using a method such as a "mobility-dependent
analysis technique." A "mobility-dependent analysis technique"
refers to any analysis based on different rates of migration
between different analytes. Exemplary mobility-dependent analysis
techniques include, but are not limited to, electrophoresis, mass
spectroscopy, chromatography, sedimentation, gradient
centrifugation, field-flow fractionation, and multi-stage
extraction techniques. Thus, in such embodiments, one may determine
the presence or absence of (or quantitate) a particular nucleic
acid sequence in a sample by detecting the presence of (or
quantitating) labeled cleaved portions of the labeled probe.
[0256] In certain embodiments, one may employ a mobility modifier
to separate different cleaved portions of labeled probes from one
another. For example, in certain such embodiments, different
labeled probes with the same label could be used for different loci
if the labeled probes for each different loci had a different
mobility modifier. In certain embodiments, mobility modifiers may
be oligonucleotides of different lengths effecting different
mobilities. In certain embodiments, mobility modifiers may be
non-nucleotide polymers, such as a polyethylene oxide (PEO),
polyglycolic acid, polyurethane polymers, polypeptides, or
oligosaccharides, as non-limiting examples. In certain embodiments,
mobility modifiers may work by adding size to a polynucleotide, or
by increasing the "drag" of the molecule during migration through a
medium without substantially adding to the size. Certain mobility
modifiers such as PEO's have been described, e.g., in U.S. Pat.
Nos. 5,470,705; 5,580,732; 5,624,800; and 5,989,871.
Certain Primers
[0257] A primer set according to certain embodiments comprises at
least one primer capable of hybridizing with the primer-specific
portion of at least one probe of a ligation probe set. In certain
embodiments, a primer set comprises at least one first primer and
at least one second primer, wherein the at least one first primer
specifically hybridizes with one probe of a ligation probe set (or
a complement of such a probe) and the at least one second primer of
the primer set specifically hybridizes with a second probe of the
same ligation probe set (or a complement of such a probe). In
certain embodiments, the first and second primers of a primer set
have different hybridization temperatures, to permit
temperature-based asymmetric PCR reactions. In certain embodiments,
the primer set comprises at least one spanning primer.
[0258] The skilled artisan will appreciate that while probes and
primers may be described in the singular form, a plurality of
probes or primers may be encompassed by the singular term, as will
be apparent from the context. Thus, for example, in certain
embodiments, a ligation probe set typically comprises a plurality
of first probes and a plurality of second probes.
[0259] The criteria for designing certain sequence-specific primers
and probes are well known to persons of ordinary skill in the art.
Detailed descriptions of certain primer design that provide for
sequence-specific annealing can be found, among other places, in
Dieffenbach and Dveksler, PCR Primer, A Laboratory Manual, Cold
Spring Harbor Press (1995), and Kwok et al., Nucl. Acids Res.
18:999-1005 (1990). The sequence-specific portions of the primers
are of sufficient length to permit specific annealing to
complementary sequences in ligation products and amplification
products, as appropriate.
[0260] According to certain embodiments, a primer set comprises at
least one first primer. In certain embodiments, the first primer in
that primer set is designed to hybridize with both a 3'
primer-specific portion of a ligation or amplification product and
a 5' primer-specific portion of the same ligation or amplification
product in a sequence-specific manner. In certain embodiments, the
primer set further comprises at least one second primer. In certain
embodiments, the second primer of a primer set is designed to
hybridize with both the complement of the 5' primer-specific
portion of that same ligation or amplification product and the
complement of the 3' primer-specific portion of that same ligation
or amplification product in a sequence-specific manner.
[0261] A universal primer or primer set may be employed according
to certain embodiments. In certain embodiments, a universal primer
or a universal primer set hybridizes with two or more of the
probes, ligation products, and/or amplification products in a
reaction, as appropriate. In certain embodiments, a universal
primer or primer set comprises at least one spanning primer. When
universal primer sets are used in certain amplification reactions,
such as, but not limited to, PCR, qualitative or quantitative
results may be obtained for a broad range of template
concentrations.
[0262] In certain embodiments involving a ligation reaction and an
amplification reaction, one may employ at least one probe and/or at
least one primer that includes a minor groove binder attached to
it. Certain exemplary minor groove binders and certain exemplary
methods of attaching minor groove binders to oligonucleotides are
discussed, e.g., in U.S. Pat. Nos. 5,801,155 and 6,084,102. Certain
exemplary minor groove binders are those available from Epoch
Biosciences, Bothell, Wash. According to certain embodiments, a
minor groove binder may be attached to at least one moiety selected
from: at least one probe of a ligation probe set; at least one
primer of a primer set; and at least one labeled probe.
[0263] According to certain embodiments, a minor groove binder is
attached to a probe that includes a 3' primer-specific portion. In
certain such embodiments, the presence of the minor groove binder
facilitates use of a short primer that hybridizes to the 3'
primer-specific portion in an amplification reaction. For example,
in certain embodiments, the short primer, or segment of the primer
that hybridizes to the primer-specific portion or its complement,
may have a length of anywhere between 8 and 15 nucleotides.
[0264] In certain embodiments, a minor groove binder is attached to
at least one of a forward primer and a reverse primer to be used in
an amplification reaction. In certain such embodiments, a primer
with a minor groove binder attached to it may be a short primer.
For example, in certain embodiments, the short primer, or segment
of the primer that hybridizes to the primer-specific portion or its
complement, may have a length of anywhere between 8 and 15
nucleotides. In certain embodiments, both the forward and reverse
primers may have minor groove binders attached to them.
[0265] In certain embodiments, one may use minor groove binders as
follows in methods that employ a ligation probe set comprising: a
first probe comprising a 5' primer specific portion; and a second
probe comprising a 3' primer-specific portion. A minor groove
binder is attached to the 3' end of the second probe, and a minor
groove binder is attached to a primer that hybridizes to the
complement of the 5' primer-specific portion of the first probe. In
certain such embodiments, the presence of the minor groove binders
facilitates use of short forward and reverse primers in an
amplification reaction. For example, in certain embodiments, the
short primer, or segment of the primer that hybridizes to the
primer-specific portion or its complement, may have a length of
anywhere between 8 and 15 nucleotides.
[0266] One may use any of the arrangements involving minor groove
binders discussed above with various methods employing ligation
probes with addressable portions as discussed herein. In certain
embodiments, one may use such arrangements with different types of
ligation and amplification methods. For example, one may use at
least one probe and/or at least one primer with an attached minor
groove binder in any of a variety of methods employing ligation and
amplification reactions. Exemplary methods include, but are not
limited to, those discussed in U.S. Pat. No. 6,027,889, PCT
Published Patent Application No. WO 01/92579, and U.S. patent
application Ser. Nos. 09/584,905 and 10/011,993.
[0267] In certain embodiments, one may employ non-natural
nucleotides other than the naturally occurring nucleotides A, G, C,
T, and U. For example, in certain embodiments, one may employ
primer-specific portions and primers and/or addressable portions
and labeled probes that comprise pairs of non-natural nucleotides
that specifically hybridize to one another and not to naturally
occurring nucleotides. Exemplary, but nonlimiting, non-natural
nucleotides are discussed, e.g., in Wu et al. J. Am. Chem. Soc.
(2000) 122: 7621-32; Berger et al. Nuc. Acids Res. (2000) 28:
2911-14, Ogawa et al. J. Am. Chem. Soc. (2000) 122: 3274-87
[0268] The skilled artisan will appreciate that the complement of
the disclosed probe, target, and primer sequences, or combinations
thereof, may be employed in certain embodiments. For example,
without limitation, a genomic DNA sample may comprise both the
target sequence and its complement. Thus, in certain embodiments,
when a genomic sample is denatured, both the target sequence and
its complement are present in the sample as single-stranded
sequences. In certain embodiments, ligation probes may be designed
to specifically hybridize to an appropriate sequence, either the
target sequence or its complement.
Certain Exemplary Ligation Methods
[0269] In various embodiments, ligation comprises any enzymatic or
chemical process wherein an internucleotide linkage is formed
between the opposing ends of nucleic acid sequences. In certain
embodiments, the nucleic acid sequences are adjacently hybridized
to a template such that their opposing ends are proximal.
Additionally, the opposing ends of the annealed nucleic acid
sequences are ligated together under suitable conditions. The
internucleotide linkage may include, but is not limited to,
phosphodiester bond formation. Such bond formation may include,
without limitation, those created enzymatically by a DNA or RNA
ligase, such as bacteriophage T4 DNA ligase, T4 RNA ligase, T7 DNA
ligase, Thermus thermophilus (Tth) ligase, Thermus aquaticus (Taq)
ligase, Tsp AK16D ligase, or Pyrococcus furiosus (Pfu) ligase.
Other internucleotide linkages include, without limitation,
covalent bond formation between appropriate reactive groups such as
between an .alpha.-haloacyl group and a phosphothioate group to
form a thiophosphorylacetylamino group; and between a
phosphorothioate and a tosylate or iodide group to form a
5'-phosphorothioester or pyrophosphate linkage.
[0270] In certain embodiments, chemical ligation may, under
appropriate conditions, occur spontaneously such as by
autoligation. Alternatively, in certain embodiments, "activating,"
condensing, or reducing agents may be used. Examples of activating
agents, condensing agents, and reducing agents include, but are not
limited to, carbodiimide, cyanogen bromide (BrCN), imidazole,
1-methylimidazole/carbodiimide/cystamine, N-cyanoimidazole, and
dithiothreitol (DTT) (see, e.g., Xu et al., Nucl. Acids Res.
27:875-81, 1999; Gryaznov and Letsinger, Nucl. Acids Res. 21:
1403-08, 1993; Gryaznov et al., Nucleic Acid Res. 22:2366-69, 1994;
Kanaya and Yanagawa, Biochemistry 25:7423-30, 1986; Luebke and
Dervan, Nucl. Acids Res. 20:3005-09, 1992; Sievers and von
Kiedrowski, Nature 369:221-24, 1994; Liu and Taylor, Nucl. Acids
res. 26:3300-04, 1999; Wang and Kool, Nucl. Acids Res. 22:2326-33,
1994; Purmal et al., Nucl. Acids Res. 20:3713-19, 1992; Ashley and
Kushlan, Biochemistry 30:2927-33, 1991; Chu and Orgel, Nucl. Acids
Res. 16:3671-91, 1988; Sokolova et al., FEBS Letters 232:153-55,
1988; Naylor and Gilham, Biochemistry 5:2722-28, 1966; Hames and
Ellington, Chem. & Biol. 4:595-605, 1997; and U.S. Pat. No.
5,476,930). Non-enzymatic ligation according to certain embodiments
may utilize specific reactive groups on the respective 3' and 5'
ends of the aligned probes. In certain embodiments, chemical
ligation may occur by photoligation. Photoligation includes, but is
not limited to: probes comprising nucleotide analogs, including but
not limited to, 4-thiothymidine (s4T), 5-vinyluracil and its
derivatives, or combination thereof; light in the UV-A range (about
320 nm to about 400 nm); light in the UV-B range (about 290 nm to
about 320 nm); combinations of light in the UV-A and UV-B range;
light with a wavelength between about 300 nm and about 375 nm;
light with a wavelength of about 360 nm to about 370 nm; light with
a wavelength of about 364 nm to about 368 nm; and light with a
wavelength of about 366 nm. In certain embodiments, photoligation
is reversible. Descriptions of photoligation can be found in, for
example, Fujimoto et al., Nucl. Acid Symp. Ser. 42:39-40, 1999;
Fujimoto et al., Nucl. Acid Res. Suppl. 1: 185-86, 2001; Fujimoto
et al., Nucl. Acid. Suppl. 2: 155-56, 2002; and Liu and Taylor,
Nucl. Acid Res. 26: 3300-04, 1998.
[0271] In certain embodiments, ligation generally comprises at
least one cycle of ligation, for example, the sequential procedures
of: hybridizing the target-specific portions of a first probe and a
second probe, which are suitable for ligation, to their respective
complementary regions on a target nucleic acid sequence; ligating
the 3' end of the first probe with the 5' end of the second probe
to form a ligation product; and denaturing the nucleic acid duplex
to separate the ligation product from the target nucleic acid
sequence. The cycle may or may not be repeated. For example,
without limitation in certain embodiments, thermocycling the
ligation reaction may be employed to linearly increase the amount
of ligation product.
[0272] According to certain embodiments, one may use ligation
techniques such as gap-filling ligation, including, without
limitation, gap-filling OLA and LCR, bridging oligonucleotide
ligation, and correction ligation. Descriptions of these techniques
can be found, among other places, in U.S. Pat. No. 5,185,243,
published European Patent Applications EP 320308 and EP 439182,
published PCT Patent Application WO 90/01069, published PCT Patent
Application WO 02/02823, and U.S. Pat. No. 6,511,810.
[0273] In certain embodiments, ligation comprises at least one
gap-filling procedure in situations where there is a gap between
two probes of a ligation probe set when the probes are initially
hybridized to a target nucleic acid. In certain embodiments, a DNA
polymerase is used to extend the 3'-end of the first probe by one
or more nucleotides to fill the gap. Thus, the probes become
hybridized adjacent to each other on the target nucleic acid. In
certain embodiments, a `gap oligonucleotide` is hybridized in the
gap between the ends of the two probes. In certain such
embodiments, the 3'-end of the first probe can be ligated to the
5'-end of the gap oligonucleotide and the 3'-end of the gap
oligonucleotide can be ligated to the 5'-end of the second probe.
Thus, the probes become hybridized adjacent to each other on the
target nucleic acid through the gap oligonucleotide. In certain
embodiments, the use of a gap oligonucleotide increases the
specificity of an OLA.
[0274] In certain embodiments, one may employ
poly-deoxy-inosinic-deoxy-cytidylic acid (Poly [d(I-C)]) (Available
in Roche Applied Science catalog, 2002) in a ligation reaction. In
certain embodiments, one uses any number between 15 to 80 ng/.mu.L
of Poly [d(I-C)] in a ligation reaction. In certain embodiments,
one uses 30 ng/.mu.L of Poly [d(I-C)] in a ligation reaction.
[0275] In certain embodiments, one may use Poly [d(I-C)] in a
ligation reaction with various methods employing ligation probes.
In certain embodiments, one may use Poly [d(I-C)] with different
types of ligation methods. For example, in certain embodiments, one
may use Poly [d(I-C)] in a method employing ligation reactions.
Exemplary methods include, but are not limited to, those discussed
in U.S. Pat. No. 6,027,889, PCT Published Patent Application No. WO
01/92579, and U.S. Patent Application Publication 2004-0121371.
[0276] In certain embodiments, in a ligation reaction, one may
employ unrelated double-stranded nucleic acid that does not include
a sequence that is the same as or is similar to the target nucleic
acid sequence that is sought. In certain such embodiments, such
double-stranded nucleic acid also will not include a sequence that
is the same as or is similar to the sequences of the
primer-specific portions of the ligation probes. In certain such
embodiments, such double-stranded nucleic acid also will not
include a sequence that is the same as or is similar to the
sequences of the target-specific portions of the ligation probes.
In certain embodiments, one may employ double-stranded poly A and
poly T nucleic acid. In certain embodiments, one may employ
double-stranded poly G and poly C nucleic acid. In certain such
embodiments, one may employ nucleic acid from an organism unrelated
to the organism from which the target nucleic acid sequence is
derived. In certain embodiments, one may employ bacterial nucleic
acid. In certain embodiments, one may employ viral DNA. In certain
embodiments, one may employ plasmid DNA. In certain embodiments,
the double-stranded nucleic acid assists in reducing the amount of
ligation that may occur between ligation probes when the sought
target nucleic acid sequence is not present.
[0277] In certain embodiments, one uses any number between 15 to 80
ng/.mu.L of unrelated double-stranded nucleic acid in a ligation
reaction. In certain embodiments, one uses 30 ng/.mu.L of unrelated
double-stranded nucleic acid in a ligation reaction.
[0278] In certain embodiments, one may use unrelated
double-stranded nucleic acid in a ligation reaction employing
ligation probes. In certain embodiments, one may use unrelated
double-stranded nucleic acid with different types of ligation
methods. For example, in certain embodiments, one may use unrelated
double-stranded nucleic acid in a method employing ligation
reactions. Exemplary methods include, but are not limited to, those
discussed in U.S. Pat. No. 6,027,889, PCT Published Patent
Application No. WO 01/92579, and U.S. Patent Application
Publication No. 2004-0121371.
[0279] Exemplary, but nonlimiting ligation reaction conditions may
be as follows. In certain embodiments, the ligation reaction
temperature may range anywhere from about 45.degree. C. to about
55.degree. C. for anywhere from two to 10 minutes. In certain
embodiments, any number from 2 to 100 cycles of ligation are
performed. In certain embodiments, 60 cycles of ligation are
performed. In certain embodiments, allele specific ligation probes
(a probe of a probe set that is specific to a particular allele at
a given locus) are in a concentration anywhere from 2 to 100 nM. In
certain embodiments, allele specific ligation probes are in a
concentration of 50 nM. In certain embodiments, allele specific
ligation probes are in a concentration anywhere from 1 to 7 nM. In
certain embodiments, locus specific ligation probes (a probe of a
probe set that is not specific to a particular allele, but is
specific for a given locus) are in a concentration anywhere from 2
to 200 nM. In certain embodiments, locus specific ligation probes
are in a concentration of 100 nM. In certain embodiments,
fragmented genomic DNA is in a concentration anywhere from 5
ng/.mu.l to 200 ng/.mu.l in the ligation reaction. In certain
embodiments, fragmented genomic DNA is in a concentration of 130
ng/.mu.l in the ligation reaction. In certain embodiments, the pH
for the ligation reaction is anywhere from 7 to 8. In certain
embodiments, the Mg.sup.2+ concentration is anywhere from 2 to 22
nM. In certain embodiments, the ligase concentration is anywhere
from 0.04 to 0.16 U/.mu.l. In certain embodiments, the ligase
concentration is anywhere from 0.02 to 0.12 U/.mu.l. In certain
embodiments, the K.sup.+ concentration is anywhere from 0 to 70 mM.
In certain embodiments, the K.sup.+ concentration is anywhere from
0 to 20 mM. In certain embodiments, the Poly [d(I-C)] concentration
is anywhere from 0 to 30 ng/.mu.l. In certain embodiments, the Poly
[d(I-C)] concentration is anywhere from 0 to 20 ng/.mu.l. In
certain embodiments, the NAD+ concentration is anywhere from 0.25
to 2.25 mM. In certain embodiments, the ATP concentration is
anywhere from 0.1 to 10 mM.
[0280] In certain embodiments, one forms a test composition for a
subsequent amplification reaction by subjecting a ligation reaction
composition to at least one cycle of ligation. In certain
embodiments, after ligation, the test composition may be used
directly in the subsequent amplification reaction. In certain
embodiments, prior to the amplification reaction, the test
composition may be subjected to a purification technique that
results in a test composition that includes less than all of the
components that may have been present after the at least one cycle
of ligation. For example, in certain embodiments, one may purify
the ligation product.
[0281] Purifying the ligation product according to certain
embodiments comprises any process that removes at least some
unligated probes, target nucleic acid sequences, enzymes, and/or
accessory agents from the ligation reaction composition following
at least one cycle of ligation. Exemplary processes include, but
are not limited to, molecular weight/size exclusion processes,
e.g., gel filtration chromatography or dialysis; sequence-specific
hybridization-based pullout methods; affinity capture techniques;
precipitation; adsorption; and other nucleic acid purification
techniques. The skilled artisan will appreciate that purifying the
ligation product prior to amplification in certain embodiments
reduces the quantity of primers needed to amplify the ligation
product, thus reducing the cost of detecting a target sequence.
Also, in certain embodiments, purifying the ligation product prior
to amplification may decrease possible side reactions during
amplification and may reduce competition from unligated probes
during hybridization.
[0282] Hybridization-based pullout (HBP) according to certain
embodiments comprises a process wherein a nucleotide sequence
complementary to at least a portion of one probe (or its
complement), for example, the primer-specific portion, is bound or
immobilized to a solid or particulate pullout support (see, e.g.,
U.S. Pat. No. 6,124,092). In certain embodiments, a composition
comprising ligation product, target sequences, and unligated probes
is exposed to the pullout support. The ligation product, under
appropriate conditions, hybridizes with the support-bound
sequences. The unbound components of the composition are removed,
purifying the ligation products from those ligation reaction
composition components that do not contain sequences complementary
to the sequence on the pullout support. One subsequently removes
the purified ligation products from the support and combines them
with at least one primer set to form a first amplification reaction
composition. The skilled artisan will appreciate that, in certain
embodiments, additional cycles of HBP using different complementary
sequences on the pullout support may remove all or substantially
all of the unligated probes, further purifying the ligation
product.
Certain Exemplary Amplification Methods
[0283] Amplification according to various embodiments, encompasses
a broad range of techniques for amplifying nucleic acid sequences,
either linearly or exponentially. Exemplary amplification
techniques include, but are not limited to, PCR or any other method
employing a primer extension step. Other nonlimiting examples of
amplification include, but are not limited to, ligase detection
reaction (LDR) and ligase chain reaction (LCR). Amplification
methods may comprise thermal-cycling or may be performed
isothermally. In various embodiments, the term "amplification
product" includes products from any number of cycles of
amplification reactions.
[0284] In certain embodiments, amplification methods comprise at
least one cycle of amplification, for example, but not limited to,
the sequential procedures of: hybridizing primers to
primer-specific portions of the ligation product or amplification
products from any number of cycles of an amplification reaction;
synthesizing a strand of nucleotides in a template-dependent manner
using a polymerase; and denaturing the newly-formed nucleic acid
duplex to separate the strands. The cycle may or may not be
repeated.
[0285] Descriptions of certain amplification techniques can be
found, among other places, in H. Ehrlich et al., Science,
252:1643-50 (1991), M. Innis et al., PCR Protocols: A Guide to
Methods and Applications, Academic Press, New York, N.Y. (1990), R.
Favis et al., Nature Biotechnology 18:561-64 (2000), and H. F.
Rabenau et al., Infection 28:97-102 (2000); Sambrook and Russell,
and Ausubel et al., supra.
[0286] Primer extension is an amplification process comprising
elongating a primer that is annealed to a template in the 5' to 3'
direction using a template-dependent polymerase. In certain
embodiments, the primer that is extended is a spanning primer.
According to certain embodiments, with appropriate buffers, salts,
pH, temperature, and nucleotide triphosphates, including analogs
and derivatives thereof, a template dependent polymerase
incorporates nucleotides complementary to the template strand
starting at the 3'-end of an annealed primer, to generate a
complementary strand. Detailed descriptions of primer extension
according to certain embodiments can be found, among other places,
in Sambrook et al., Sambrook and Russell, and Ausubel et al,
supra.
[0287] Certain embodiments of amplification may employ multiplex
PCR, in which multiple target sequences are simultaneously
amplified (see, e.g., H. Geada et al., Forensic Sci. Int. 108:31-37
(2000) and D. G. Wang et al., Science 280:1077-82 (1998)).
[0288] In certain embodiments, one employs asymmetric PCR.
According to certain embodiments, asymmetric PCR comprises an
amplification reaction composition comprising (i) at least one
primer set in which there is an excess of one primer (relative to
the other primer in the primer set); (ii) at least one primer set
that comprises only a first primer or only a second primer; (iii)
at least one primer set that, during given amplification
conditions, comprises a primer that results in amplification of one
strand and comprises another primer that is disabled; or (iv) at
least one primer set that meets the description of both (i) and
(iii) above. Consequently, when the ligation product is amplified,
an excess of one strand of the amplification product (relative to
its complement) is generated.
[0289] In certain embodiments, one may use at least one primer set
wherein the melting temperature (Tm.sub.50) of one of the primers
is higher than the Tm.sub.50 of the other primer. Such embodiments
have been called asynchronous PCR (A-PCR). See, e.g., U.S. Pat. No.
6,887,664. In certain embodiments, the Tm.sub.50 of the first
primer is at least 4-15.degree. C. different from the Tm.sub.50 of
the second primer. In certain embodiments, the Tm.sub.50 of the
first primer is at least 8-15.degree. C. different from the
Tm.sub.50 of the second primer. In certain embodiments, the
Tm.sub.50 of the first primer is at least 10-15.degree. C.
different from the Tm.sub.50 of the second primer. In certain
embodiments, the Tm.sub.50 of the first primer is at least
10-12.degree. C. different from the Tm.sub.50 of the second primer.
In certain embodiments, in at least one primer set, the Tm.sub.50
of the at least one first primer differs from the melting
temperature of the at least one second primer by at least about
4.degree. C., by at least about 8.degree. C., by at least about
10.degree. C., or by at least about 12.degree. C.
[0290] In certain embodiments of A-PCR, in addition to the
difference in Tm.sub.50 of the primers in a primer set, there is
also an excess of one primer relative to the other primer in the
primer set. In certain embodiments, there is a five to twenty-fold
excess of one primer relative to the other primer in the primer
set. In certain embodiments of A-PCR, the primer concentration is
at least 50 mM.
[0291] In A-PCR according to certain embodiments, one may use
conventional PCR in the first cycles such that both primers anneal
and both strands are amplified. By raising the temperature in
subsequent cycles, however, one may disable the primer with the
lower Tm such that only one strand is amplified. Thus, the
subsequent cycles of A-PCR in which the primer with the lower Tm is
disabled result in asymmetric amplification. Consequently, when the
ligation product is amplified, an excess of one strand of the
amplification product (relative to its complement) is
generated.
[0292] According to certain embodiments of A-PCR, the level of
amplification can be controlled by changing the number of cycles
during the first phase of conventional PCR cycling. In such
embodiments, by changing the number of initial conventional cycles,
one may vary the amount of the double strands that are subjected to
the subsequent cycles of PCR at the higher temperature in which the
primer with the lower Tm is disabled.
[0293] In certain embodiments, an A-PCR protocol may comprise use
of a pair of primers, each of which has a concentration of at least
50 mM. In certain embodiments, conventional PCR, in which both
primers result in amplification, is performed for the first 20-30
cycles. In certain embodiments, after 20-30 cycles of conventional
PCR, the annealing temperature increases to 66-70.degree. C., and
PCR is performed for 5 to 40 cycles at the higher annealing
temperature. In such embodiments, the lower Tm primer is disabled
during such 5 to 40 cycles at higher annealing temperature. In such
embodiments, asymmetric amplification occurs during the second
phase of PCR cycles at a higher annealing temperature.
[0294] In certain embodiments, one employs asymmetric
reamplification. According to certain embodiments, asymmetric
reamplification comprises generating single-stranded amplification
product in a second amplification process. In certain embodiments,
the double-stranded amplification product of a first amplification
process serves as the amplification target in the asymmetric
reamplification process. In certain embodiments, one may achieve
asymmetric reamplification using asynchronous PCR in which initial
cycles of PCR conventionally amplify two strands and subsequent
cycles are performed at a higher annealing temperature that
disables one of the primers of a primer set as discussed above. In
certain embodiments, the second amplification reaction composition
comprises at least one primer set which comprises the at least one
first primer, or the at least one second primer of a primer set,
but typically not both. The skilled artisan understands that, in
certain embodiments, asymmetric reamplification will also
eventually occur if the primers in the primer set are not present
in an equimolar ratio. In certain asymmetric reamplification
methods, typically only single-stranded amplicons are generated
since the second amplification reaction composition comprises only
first or second primers from each primer set or a non-equimolar
ratio of first and second primers from a primer set.
[0295] In certain embodiments, additional polymerase may also be a
component of the second amplification reaction composition. In
certain embodiments, there may be sufficient residual polymerase
from the first amplification composition to synthesize the second
amplification product.
[0296] Certain methods of optimizing amplification reactions are
known to those skilled in the art. For example, it is known that
PCR may be optimized by altering times and temperatures for
annealing, polymerization, and denaturing, as well as changing the
buffers, salts, and other reagents in the reaction composition.
Optimization may also be affected by the design of the
amplification primers used. For example, the length of the primers,
as well as the G-C:A-T ratio may alter the efficiency of primer
annealing, thus altering the amplification reaction. See James G.
Wetmur, "Nucleic Acid Hybrids, Formation and Structure," in
Molecular Biology and Biotechnology, pp. 605-8, (Robert A. Meyers
ed., 1995).
[0297] In certain amplification reactions, one may use dUTP and
uracil-N-glucosidase (UNG). Discussion of use of dUTP and UNG may
be found, for example, in Kwok et al., Nature, 339:237-238 (1989);
and Longo et al., Gene, 93:125-128 (1990).
[0298] To detect whether a particular sequence is present, in
certain embodiments, a labeled probe is included in the
amplification reaction. According to certain embodiments, the
labeled probe indicates the presence or absence (or amount) of a
specific nucleic acid sequence in the reaction. These include, but
are not limited to, 5'-nuclease probes, cleavage RNA probes, and
hybridization dependent probes. In certain embodiments, the labeled
probe comprises a fluorescing dye connected to a quenching molecule
through a link element, e.g., through a specific oligonucleotide.
Examples of such systems are described, e.g., in U.S. Pat. Nos.
5,538,848 and 5,723,591.
[0299] In certain embodiments, the amount of labeled probe that
gives a fluorescent signal in response to an emitted light
typically relates to the amount of nucleic acid produced in the
amplification reaction. Thus, in certain embodiments, the amount of
fluorescent signal is related to the amount of product created in
the amplification reaction. In such embodiments, one can therefore
measure the amount of amplification product by measuring the
intensity of the fluorescent signal from the fluorescent indicator.
According to certain embodiments, one can employ an internal
standard to quantify the amplification product indicated by the
fluorescent signal. See, e.g., U.S. Pat. No. 5,736,333.
[0300] Devices have been developed that can perform a thermal
cycling reaction with compositions containing a fluorescent
indicator, emit a light beam of a specified wavelength, read the
intensity of the fluorescent dye, and display the intensity of
fluorescence after each cycle. Devices comprising a thermal cycler,
light beam emitter, and a fluorescent signal detector, have been
described, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; and
6,174,670, and include, but are not limited to the ABI Prism.RTM.
7700 Sequence Detection System (Applied Biosystems, Foster City,
Calif.) and the ABI GeneAmp.RTM. 5700 Sequence Detection System
(Applied Biosystems, Foster City, Calif.).
[0301] In certain embodiments, each of these functions may be
performed by separate devices. For example, if one employs a Q-beta
replicase reaction for amplification, the reaction may not take
place in a thermal cycler, but could include a light beam emitted
at a specific wavelength, detection of the fluorescent signal, and
calculation and display of the amount of amplification product.
[0302] In certain embodiments, combined thermal cycling and
fluorescence detecting devices can be used for precise
quantification of target nucleic acid sequences in samples. In
certain embodiments, fluorescent signals can be detected and
displayed during and/or after one or more thermal cycles, thus
permitting monitoring of amplification products as the reactions
occur in "real time." In certain embodiments, one can use the
amount of amplification product and number of amplification cycles
to calculate how much of the target nucleic acid sequence was in
the sample prior to amplification.
[0303] According to certain embodiments, one could simply monitor
the amount of amplification product after a predetermined number of
cycles sufficient to indicate the presence of the target nucleic
acid sequence in the sample. In various embodiments, one skilled in
the art can easily determine, for any given sample type, primer
sequence, and reaction condition, how many cycles are sufficient to
determine the presence of a given target polynucleotide.
[0304] According to certain embodiments, the amplification products
can be scored as positive or negative as soon as a given number of
cycles is complete. In certain embodiments, the results may be
transmitted electronically directly to a database and tabulated.
Thus, in certain embodiments, large numbers of samples may be
processed and analyzed with less time and labor required.
[0305] According to certain embodiments, different labeled probes
may distinguish between different target nucleic acid sequences. A
non-limiting example of such a probe is a 5'-nuclease fluorescent
probe, such as a TaqMan.RTM. probe molecule, wherein a fluorescent
molecule is attached to a fluorescence-quenching molecule through
an oligonucleotide link element. In certain embodiments, the
oligonucleotide link element of the 5'-nuclease fluorescent probe
binds to a specific sequence of an addressable portion or its
complement. In certain embodiments, different 5'-nuclease
fluorescent probes, each fluorescing at different wavelengths, can
distinguish between different amplification products within the
same amplification reaction.
[0306] For example, in certain embodiments, one could use two
different 5'-nuclease fluorescent probes that fluoresce at two
different wavelengths (WL.sub.A and WL.sub.B) and that are specific
to two different addressable portions of two different ligation
products (A' and B', respectively). Ligation product A' is formed
if target nucleic acid sequence A is in the sample, and ligation
product B' is formed if target nucleic acid sequence B is in the
sample. In certain embodiments, ligation product A' and/or B' may
form even if the appropriate target nucleic acid sequence is not in
the sample, but such ligation occurs to a measurably lesser extent
than when the appropriate target nucleic acid sequence is in the
sample. After amplification, one can determine which specific
target nucleic acid sequences are present in the sample based on
the wavelength of signal detected. Thus, if an appropriate
detectable signal value of only wavelength WL.sub.A is detected,
one would know that the sample includes target nucleic acid
sequence A, but not target nucleic acid sequence B. If an
appropriate detectable signal value of both wavelengths WL.sub.A
and WL.sub.B are detected, one would know that the sample includes
both target nucleic acid sequence A and target nucleic acid
sequence B.
[0307] The following examples are intended for illustration
purposes only, and should not be construed as limiting the scope of
the teachings in any way.
EXAMPLES
Example 1
Exemplary Spanning Primer Sequences
[0308] Exemplary spanning primer sequences are provided below (SEQ.
ID NOS. 1-4 are disclosed respectively in order of appearance from
top to bottom):
##STR00005##
Example 2
Linear Amplification Using Spanning Primers
[0309] A target nucleic acid sequence is amplified linearly using a
spanning primer. One ng of target nucleic acid sequence is combined
with 5 mM MgCl.sub.2, 0.5 .mu.M of a spanning primer complementary
to both the 3' end and the 5' end of the target nucleic acid
sequence, 0.2 mM of each of dATP, dCTP, dGTP, and dTTP, 1.times.
amplification buffer (50 mM KCl, 10 mM Tris-Cl, pH 8.4, 0.1 mg/mL
gelatin), and 2.5 U Taq DNA polymerase in a final volume of 100
.mu.L. The target nucleic acid sequence is amplified in an
automated thermal cycler using the following protocol: 94.degree.
C. for 90 seconds (denaturation), 60.degree. C. for 2 minutes
(initial annealing), 72.degree. C. for 3 minutes (extension),
followed by 40 cycles of 94.degree. C. for 90 seconds,
60-70.degree. C. for 2 minutes, and 72.degree. C. for 3 minutes.
The amplification product is detected.
Example 3
Exponential Amplification Using Spanning Primers
[0310] A target nucleic acid sequence is amplified exponentially
using two spanning primers. One ng of target nucleic acid sequence
is combined with 5 mM MgCl.sub.2, 0.5 .mu.M of a first spanning
primer complementary to both the 3' end and the 5' end of the
target nucleic acid sequence, 0.2 mM of each of dATP, dCTP, dGTP,
and dTTP, 1.times. amplification buffer (50 mM KCl, 10 mM Tris-Cl,
pH 8.4, 0.1 mg/mL gelatin), and 2.5 U Taq DNA polymerase in a final
volume of 100 .mu.L. Also included in the reaction is 0.5 .mu.M of
a second spanning primer. In certain embodiments, the second
spanning primer comprises a sequence complementary to both the 3'
end of a complement of the target nucleic acid sequence and to the
5' end of the first spanning primer. In certain embodiments, the
second spanning primer comprises a sequence complementary to both
the 3' end of a complement of the target nucleic acid sequence and
to any portion of the first spanning primer. The target nucleic
acid sequence is amplified in an automated thermal cycler using the
following protocol: 94.degree. C. for 90 seconds (denaturation),
55-60.degree. C. for 2 minutes (initial annealing), 65-72.degree.
C. for 3 minutes (extension), followed by 30-40 cycles of
94.degree. C. for 10-90 seconds, 60-70.degree. C. for 2 minutes,
and 72.degree. C. for 3 minutes. The amplification product is
detected.
Example 4
Detection of Single Nucleotide Polymorphisms Using Ligation and
Linear Amplification
[0311] The presence of a single nucleotide polymorphism (SNP) in a
target nucleic acid sequence is detected using ligation and linear
amplification with a spanning primer. A target nucleic acid
sequence comprising a SNP is incubated with a ligation probe set
comprising (a) a first probe, comprising a first target-specific
portion, and (b) a second probe, comprising a second
target-specific portion, wherein the probes in each set are
suitable for ligation together when hybridized adjacent to one
another on the target nucleic acid sequence. One .mu.g of target
nucleic acid sequence is combined with 1 .mu.g of the first probe
and 1 .mu.g of the second probe, 10 mM Tris-Cl, pH 7.5, 10 mM
MgCl.sub.2, 10 mM dithiothreitol, and 20 U T4 DNA ligase in a final
volume of 20 .mu.L. The reaction is incubated at 15.degree. C. for
12 hours. The resulting ligation product serves as a second target
nucleic acid sequence for subsequent amplification.
[0312] One ng of ligation product is combined with 5 mM MgCl.sub.2,
0.5 .mu.M of a spanning primer complementary to both the 3' end and
the 5' end of the ligation product, 0.2 mM of each of dATP, dCTP,
dGTP, and dTTP, 1.times. amplification buffer (50 mM KCl, 10 mM
Tris-Cl, pH 8.4, 0.1 mg/mL gelatin), and 2.5 U Taq DNA polymerase
in a final volume of 100 .mu.L. The ligation product is amplified
in an automated thermal cycler using the following protocol:
94.degree. C. for 90 seconds (denaturation), 55-60.degree. C. for 2
minutes (initial annealing), 72.degree. C. for 3 minutes
(extension), followed by 29-40 cycles of 94.degree. C. for 10-90
seconds, 60-70.degree. C. for 2 minutes, and 72.degree. C. for 3
minutes. The presence of the SNP is detected by detecting the
presence of the amplification product.
Example 5
Detection of Single Nucleotide Polymorphisms Using Ligation and
Exponential Amplification
[0313] The presence of a single nucleotide polymorphism (SNP) in a
target nucleic acid sequence is detected using ligation and
exponential amplification with a spanning primer. A target nucleic
acid sequence comprising a SNP is incubated with a ligation probe
set comprising (a) a first probe, comprising a first
target-specific portion, and (b) a second probe, comprising a
second target-specific portion, wherein the probes in each set are
suitable for ligation together when hybridized adjacent to one
another on the target nucleic acid sequence. One .mu.g of target
nucleic acid sequence is combined with 1 .mu.g of the first probe
and 1 .mu.g of the second probe, 10 mM Tris-Cl, pH 7.5, 10 mM
MgCl.sub.2, 10 mM dithiothreitol, and 20 U T4 DNA ligase in a final
volume of 20 .mu.L. The reaction is incubated at 15.degree. C. for
12 hours. The resulting ligation product serves as a second target
nucleic acid sequence for subsequent amplification.
[0314] The ligation product is amplified exponentially using two
spanning primers. One ng of ligation product is combined with 5 mM
MgCl.sub.2, 0.5 .mu.M of a first spanning primer complementary to
both the 3' end and the 5' end of the ligation product, 0.2 mM of
each of dATP, dCTP, dGTP, and dTTP, 1.times. amplification buffer
(50 mM KCl, 10 mM Tris-Cl, pH 8.4, 0.1 mg/mL gelatin), and 2.5 U
Taq DNA polymerase in a final volume of 100 .mu.L. Also included in
the reaction is 0.5 .mu.M of a second spanning primer. In certain
embodiments, the second spanning primer comprises a sequence
complementary to both the 3' end of a complement of the ligation
product and to the 5' end of the first spanning primer. In certain
embodiments, the second spanning primer comprises a sequence
complementary to both the 3' end of a complement of the ligation
product and to any portion of the first spanning primer. The
ligation product is amplified in an automated thermal cycler using
the following protocol: 94.degree. C. for 90 seconds
(denaturation), 55-60.degree. C. for 2 minutes (initial annealing),
72.degree. C. for 3 minutes (extension), followed by 29-40 cycles
of 94.degree. C. for 90 seconds, 60-70.degree. C. for 2 minutes,
and 72.degree. C. for 3 minutes. The presence of the SNP is
detected by detecting the presence of the amplification
product.
[0315] The foregoing examples are not intended to limit the scope
of the teachings herein.
Sequence CWU 1
1
4123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1tcgtacgtgg tggtgcgggc ctg 23231DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2aggccaggtg caggcccgca ccaccaccgc t 31326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3aaccacgtac gatcgcacca ccaccg 26439DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
4agcggtggtg gtgcgatcgt acgtggtggt gcgggcctg 39
* * * * *