FIELD
[0003] The methods and compositions described herein relate generally to the
area of detecting or detem1ining nucleotide sequences.
BACKGROUND
[0004] A wide variety of nucleic acid amplification methods are available, and
15 many have been employed in the implementation of sensitive genotyping and
diagnostic assays based on nucleic acid detection. Polymerase chain reaction (PCR)
remains the most widely used DNA amplification and qnantitation method. Nested
PCR, a two-stage PCR, is used to increase the specificity and sensitivity of the PCR
(U.S. Patent No. 4,683,195). Nucleic acid amplification is also used in so-called
20 "next-generation" nucleic acid sequencing methods.
[0005] Modified DNA bases have been developed that do not base-pair
efficiently with one another. Examples are described in U.S. Patent No. 5,912,340
(issued June 15, 1999 to Kutyavin et al.) and in Woo et aL (1996) "'G/C-modified
oligodeoxynucleotides with selective complernentarity: synthesis and hybridization
25 properties," Nucleic Acids Research 24(13):2470-2475, both of which are
incorporated by reference for this description. These modified bases have been
termed '"pseudo-complementary" (see, e.g., Lahoud et aL (2008) Nucleic Acids
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Research 36(1 0):3409-3419), and pairs ofthese bases have been referred to as selfavoiding
molecular recognition systems (SAMRS; see, e.g., U.S. Patent No.
8,871,469, issued October 28, 2014 to Benner et at).
SUJ\-IMARY
5 [0006] Various embodiments contemplated herein may include, but need not
be limited to, one or more of the following:
[0007] Embodiment 1 : A method of detem1ining whether a nucleotide
sequence is present in a target nucleic acid sequence in a sample, wherein the target
nucleic acid sequence includes a polymorphic site, wherein the polymorphic site is
10 characterized by a first nucleotide sequence and a second nucleotide sequence,
wherein the first and second nucleotide sequences differ by at least one nucleotide or
ribonucleotide, the method including:
[0008] contacting nucleic acid of: or derived from, the sarnple with forward
and reverse primers capable of amplifying the target nucleic acid sequence, wherein
15 said contacting is in the presence of a blocker oligonucleotide that is complementary
to the first nucleotide sequence to form a reaction mixture, wherein:
20
[0009] ifthe target nucleic acid sequence includes the first nucleotide
sequence, the blocker oligonucleotide anneals to the first nucleotide sequence and
inhibits amplification; or
[0010] if the target nucleic acid sequence includes the second
nucleotide sequence, the blocker oligonucleotide does not anneal to the second
nucleotide sequence and does not inhibit amplification;
[0011] conducting an amplification reaction in the reaction mixture;
[0012] after the amplification reaction, contacting the reaction mixture, or
25 nucleic acids from the reaction mixture, with a capture oligonucleotide that is
complementary to the second nucleotide sequence under conditions suitable for
specitic hybridization; and
[0013] detecting any specific hybridization to the capture oligonucleotide,
wherein the presence of specific hybridization to the capture oligonucleotide indicates
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that the second nucleotide sequence is present in the target nucleic acid sequence,
wherein:
[0014] the blocker oligonucleotide includes one or more first modified
bases and the capture oligonucleotide includes one or more second modified bases, at
5 least one of which is complementary to one of the first modified bases, wherein the
modified bases preferentially pair with unmodified forms of their complementmy
bases, as compared to pairing between modified, complementary bases; and
[0015] the presence of the one or more modified bases in the blocker
oligonucleotide and in the capture oligonucleotide destabilizes hybridization between
10 the blocker oligonucleotide and the capture oligonucleotide.
15
[0016] Embodiment 2: The method of embodiment 1, wherein at least one of
the first modified bases and at least one of the second, complementaty modified bases
in the capture oligonucleotide are bases that do not differ between the first and second
nucleotide sequence.
10017] Embodiment 3: The method of embodiment 1 or embodiment 2,
wherein the first nucleotide sequence includes one allele of a gene, and the second
nucleotide sequence includes another allele of a gene.
[0018] Embodiment 4: The method of embodiment 1 or embodiment 2,
wherein the first nucleotide sequence includes a wild-type sequence, and the second
20 nucleotide sequence includes a mutant sequence.
[0019] Embodiment 5: The method of any one of embodiments 1 to 4,
wherein the polymorphic site is a single nucleotide polymorphism.
[0020] Embodiment 6: The method of any one of embodiments 1-5, wherein
the amplification includes polymerase chain reaction.
[0021] Embodiment 7: The method of any one of embodiments 1-6, wherein
the method includes quantifying any specific hybridization to the capture
oligonucleotide.
[0022] Embodiment 8: The method of any one of embodiments 1-7, wherein
the sample consists of nucleic acids from a single cell.
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[0023]
[00241
Embodiment 9: An oligonucleotide set including:
forward and reverse primers capable of amplifying a target nucleic acid
sequence, wherein the target nucleic acid sequence includes a polymorphic site,
wherein the polymorphic site is characterized by a first nucleotide sequence and a
5 second nucleotide sequence, wherein the first and second nucleotide sequences differ
by at least one nucleotide or ribonucleotide;
[00251 a blocker oligonucleotide that is complementary to the first nucleotide
sequence; and
[0026] a capture oligonucleotide that is complementary to the second
l 0 nucleotide sequence, wherein the blocker oligonucleotide includes one or more first
modified bases and the capture oligonucleotide includes one or more second modified
bases, at least one of which is complementmy to one of the first modified bases,
wherein the modified bases preferentially pair with umnodified fom1s of their
complementary bases, as compared to pairing between modified, complementary
15 bases; and the presence ofthe one or more modified bases in the blocker
oligonucleotide and in the capture oligonucleotide destabilizes hybridization between
the blocker oligonucleotide and the capture oligonucleotide.
[0027] Embodiment 10: The method or oligonucleotide set of any one ofthe
preceding embodiments, wherein the capture oligonucleotide is attached to a support.
20 10028] Embodiment 11 : The method or oligonucleotide set of embodiment
10, wherein the support includes a microbead.
[0029] Embodiment 12: A method of simphfying preparations for nucleic
acid sequencing, the method including:
[0030] adding DNA sequencing adaptors to nucleic acid fragments to produce
25 sequencing templates;
IOO~H]
and
amplifying sequencing templates to produce amplified DNA templates;
[0032] contacting the amplified DNA templates with capture oligonucleotides
attached to a support under conditions suitable for hybridization, wherein the DNA
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sequencing adaptors and the capture oligonucleotides comprise complementary
nucleotide sequences, wherein:
[0033] the DNA sequencing adaptors each comprise one or more first
modified bases in their complementary nucleotide sequence, and the capture
5 oligonucleotides comprise one or more second modified bases in their complementary
nucleotide sequence, wherein at least one of the first and second modi tied bases are
complementary, wherein the modified bases preferentially pair with unmodified fom1s
of their cornplementary bases, as compared to pairing between modified,
complementary bases; and
10 [0034] hybridization of amplified DNA templates to the capture
oligonucleotide is favored over hybridization of free adaptors to the capture
oligonucleotides, eliminating a need to separate amplified DNA templates from free
adaptors before turther DNA sequencing steps.
[0035] Embodiment 13: A combination of components for simplifying
15 nucleic acid sequencing, the combination including:
[0036] DNA sequencing adaptors;
[0037] and capture oligonucleotides attached to, or adapted to be attached to, a
support, wherein the DNA sequencing adaptors and the capture oligonucleotides
comprise complementary nucleotide sequences, wherein:
20 [0038] the DNA sequencing adaptors each comprise one or more tlrst
modified bases in their complementary nucleotide sequence, and the capture
oligonucleotides comprise one or more second modified bases in their complementary
nucleotide sequence, wherein at least one of the tirst and second modi tied bases are
complementary, wherein the modified bases preferentially pair with unmodified fom1s
25 of their complementary bases, as compared to pairing between modified,
complementary bases; and
[0039] hybridization of amplified DNA templates to the capture
oligonucleotide is favored over hybridization of free adaptors to the capture
oligonucleotides, eliminating a need to separate amplified DNA templates from free
30 adaptors.
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5
10
[0040] Embodiment 14: The method of embodiment 12 or the cornbination of
components of embodiment 13, wherein the DNA sequencing adaptors comprise a
nucleotide sequence that is a binding site for a DNA sequencing primer and a barcode
nucleotide sequence.
[0041] Embodiment 15: The method of embodiment 12, wherein the method
additionally includes producing the nucleic acid fragments from genomic DNA, or the
combination of components of embodiment 13, wherein the combination additionally
includes one or rnore reagents that produce the nucleic acid fragments from genomic
DNA.
[0042] Embodiment 16: The method of embodiment 12, wherein said adding
of DNA sequencing adaptors includes ligating the DNA sequencing adaptors to the
nucleic acid fragments, or the combination of components of embodiment 13, wherein
the combination additionally includes a ligase.
[0043] Embodiment 17: The method or combination of components of any
15 one of embodiments 12-16, wherein the method employs, or the combination
includes, a DNA polymerase for amplification.
[0044] Embodiment 18: The method or combination of components of any
one of embodiments 12-17, wherein the method employs, or the combination
includes, a reverse transcriptase for reverse-transcribing nucleic acid fragment that are
20 RNA.
25
[0045] Embodiment 19: The method or combination of components of any
one of embodiments 12-18, wherein the method additionally includes sequencing the
amplified DNA templates or the combination additionally includes additional reagents
for sequencing DNA.
10046] Embodiment 20: The method, oligonucleotide set, or combination of
components of any one of the preceding embodiments, wherein modified
complementary bases form fewer hydrogen bonds with each other than with
unmodified complementary bases.
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[0047] Embodiment 21: The method, oligonucleotide set, or combination of
components of embodiment 20, wherein the Tm of a base pair formed between
modified complementary bases less than 40 "C.
[0048] Embodiment 22: The method, oligonucleotide set, or combination of
5 components of any one of the preceding embodiments, wherein at least one
complementary pair of modified bases includes modified forms of adenine and
thymine.
[0049] Embodiment 23: The method, oligonucleotide set, or combination of
components of embodiment 22, wherein the modified forms of adenine and thymine
10 are 2-aminoadenine and 2-thiothymine, respectively.
15
[0050] Embodiment 24: The method, oligonucleotide set, or combination of
components of any one of the preceding embodiments, wherein at least one
complementary pair of modified bases includes modified forms of guanine and
cytosine.
10051] Embodiment 25: The method, oligonucleotide set, or combination of
components of embodiment 24, wherein the modified forms of guanine includes
deoxyinosine, 7-alkyl-7-deazaguanine, 2' -hy-poxanthine, or 7-nitro-7-
deazahypoxanthine, and the modified form of cytosine includes 3-(2'-deoxy-beta-Driboturanosyl)
pyrro1o-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, or 2-
20 thiocytosine.
25
[0052] Embodiment 26: The method, oligonucleotide set, or combination of
components of any one of the preceding embodiments, wherein the blocker
oligonucleotide and the capture oligonucleotide each comprise at least 2, 3, 4, 5, 6, 7,
8, 9, or 10 modified bases.
10053] Embodiment 27: The method, oligonucleotide set, or combination of
components of any one of the preceding embodiments, wherein the blocker
oligonucleotide is blocked to 3' extension.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0054] Figure 1 A: A schematic drawing showing an amplification-based
assay for the presence of one sequence (e.g., the mutant sequence) in the presence of a
blocking oligonucleotide that destabilizes hybridization of the second sequence (e.g.,
5 the wild-type sequence). The blocker oligonucleotide preferentially blocks
amplification of wild-ty-pe sequences to allow better detection of mutations.
[0055] Figure lB: A schematic drawing showing how excess blocker
oligonucleotide carried over into hybridization-based mutation detection can interfere
with capture ofthe amplified, mutant DNA. rn this case, the capture oligonucleotide
10 is the probe for the mutant sequence. Note: ifthe other strand were to be captured,
the blocker oligonucleotide would interfere by binding to it.
[0056] Figure 2: A schematic drawing showing how the use of modified (e.g.,
pseudo-complementary bases in the blocker and capture oligonucleotides reduce or
prevent the interference shown in Figure 1 B. (Modi tied bases are identified with as
15 A' and T' corresponding to modified (e.g., pseudo-complementary) fom1s of adenine
and thymine.
10057] Figure 3: A schematic drawing showing common next-generation
DNA sequencing protocols for DNA and Rl"'JA.
[0058] Figure 4A: Base-pairing schemes for Watson-Crick doublets between
20 thymine and adenine (Formula 1a), thymine and 2-aminoadenine (Formula lb), 2-
thiothymine and adenine (Formula 2b), and 2-thiothymine and 2-aminoadenine
(Formula 2b). The 2-thiothymine and 2-aminoadenine base pair is destabilizing,
whereas the thymine and 2-aminoadenine and the 2-thiothymine and adenine base
pairs are stabilizing.
25 [0059] Figure 4B: Base-pairing schemes for Watson-Crick doublets between
cytosine and guanine (Formula 3a), cytosine and inosine (Fonnula 3b), dP and
guanine (Formula 4a), and dP and inosine (Formula 4b). The dP and inosine base pair
is destabilizing, whereas the cytosine and inosine and the dP and bruanine base pairs
are stable.
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[0060] Figure 5: Results from a liquid-phase hybridization assay
demonstrating that, in testing desirable hybridization between a 3' labeled fluorescent
capture oligonucleotide and complementary oligonucleotides with either a 3' or 5'
attached quencher, the 5' quencher performed better. The results are from a Melt
5 analysis run on QUANTSTUDIO 7. The analysis employed 300 ru\1 fluorescent,
biotinylated oligonucleotide (the biotin was not required in the present assay, but the
available oligonucleotide happened to be biotinylated) with 3000 nl\tf quencher
oligonucleotide. The absence of salts in the assay led to a lower Tm than would occur
in PCK (See Example L)
10 [0061.] Figure 6A: The same assay as shown in Figure 5 was conducted with
300 nM 3' labeled fluorescent capture oligonucleotide, 300 nM 5' quencher
oligonucleotide and 3000 ru\1, 300 nl\tf, 30 nJVI, or 0 ru"1 of pseudo-complementary
blocker oligonucleotide. The results show that the pseudo-complementary blocker
did not interfere with hybridization of the quencher oligonucleotide to a pseudo-
15 complementary capture oligonucleotide.
20
[00621 Figure 6B: Results from an assay identical in format to that of 6A,
exct.:pt that a blocker oligonucleotide that did not contain any modified bases
("'complementary blocker") interfered with hybridization of the quencher
oligonucleotide to a pseudo-complementary capture oligonucleotide.
DETAILED DESCRIPTION
Definitions
[0063] Tem1s used in the claims and specification are defined as set forth
below unless otherwise specified.
[0064] The term "'nucleic acid" refers to a nucleotide polymer, and unless
25 otherwise limited, includes analogs of natural nucleotides that can function in a
similar manner (e.g., hybridize) to naturally occurring nucleotides.
[0065] The term nucleic acid includes any form of DNA or RNA, including,
for example, genomic DNA; complementary DNA (eDNA), which is a DNA
representation ofmRNA, usually obtained by reverse transcription ofmessenger RNA
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(rnRNA) or by arnplitkation; DNA molecules produced syntheticaHy or by
amplification; mRNA; and non-coding RNA.
[0066] The term nucleic acid encompasses double- or triple-stranded nucleic
acid complexes, as well as single-stranded molecules. In double- or triple-stranded
5 nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e., a
double-stranded nucleic acid need not be double-stranded along the entire length of
both strands).
[0067] The term nucleic acid also encompasses any modifications thereof,
such as by methylation and/or by capping. Nucleic acid modifications can include
10 addition of chemical groups that incorporate additional charge, polarizability,
hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic
acid bases or to the nucleic acid as a whole. Such modit1cations may include base
modifications such as 2' -position sugar modifications, 5-position pyrimidine
modifications, 8-position purine modifications, modifications at cytosine exocydic
15 amines, substitutions of 5-bromo-uracil, sugar-phosphate backbone modifications,
unusual base pairing combinations such as the isobases isocytidine and isoguanidine,
and the like.
[0068] More particularly, in some embodiments, nucleic acids, can include
polydeoxyribonucleotides (containing 2-deoxy-D-ribose ), polyribonucleotides
20 (containing D-ribose), and any other type of nucleic acid that is anN- or C-glycoside
of a purine or pyrimidine base, as weU as other polymers containing nonnucleotidic
backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and
po1ymorpholino polymers (see, e.g., Summerton and \VeUer (1997) "'Morpholino
Antisense Oligomers: Design, Preparation, and Properties," Antisense & Nucleic
25 Acid Drug Dev. 7:1817-195; Okamoto et al. (20020) "Development of
electrochemically gene-analyzing method using DNA-modified electrodes," Nucleic
Acids Res. Supplement No. 2:171-172), and other synthetic sequence-specific nucleic
acid polymers providing that the polymers contain nucleobases in a configuration
which allows for base pairing and base stacking, such as is found in DNA and RNA.
30 The term nucleic acid also encompasses locked nucleic acids (LNAs), which are
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described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which
are incorporated herein by reference in their entirety for their disclosure ofLNAs.
[0069] The nucleic acid(s) can be derived from a completely chemical
synthesis process, such as a solid phase-mediated chemical synthesis, from a
5 biological source, such as through isolation from any species that produces nucleic
acid, or from processes that involve the manipulation of nucleic acids by molecular
biology tools, such as DNA replication, PCR amplification, reverse transcription, or
from a combination ofthose processes.
[0070] As used herein, the term "'complementary" refers to the capacity for
10 precise pairing between two nucleotides; i.e., if a nucleotide at a given position of a
nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid
to fom1 a canonical base pair, then the two nucleic acids are considered to be
complementary to one another at that position. Complementarity between two singlestranded
nucleic acid molecules may be "'partial," in which only some of the
15 nudeotides hind, or it may be complete when total complementarity exists between
the single-stranded molecules. The degree of complementarity between nucleic acid
strands has significant effects on the efficiency and strength of hybridization between
nucleic acid strands.
10071] '"Specific hybridization" refers to the binding of a nucleic acid to a
20 target nucleotide sequence in the absence of substantial binding to other nucleotide
sequences present in the hybridization mixture under defined stringency conditions.
Those of skill in the art recognize that relaxing the stringency of the hybridization
conditions allows sequence mismatches to be tolerated.
10072] In some embodiments, hybridizations are carried out under stringent
25 hybridization conditions. The phrase "stringent hybridization conditions" generally
refers to a temperature in a range from about 5°C to about 20°C or 25°C below than
the melting temperature (T rn) for a specific sequence at a defined ionic strength and
pH. As used herein, the Tm is the temperature at which a population of doublestranded
nucleic acid molecules becomes half-dissociated into single strands.
30 Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g.,
Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL152: GUIDE TO
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MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and
Sambrook et aL (1989) MOLECULAR CLONING: A LABOILI-\TORY MANUAL,
2ND ED., VOLS. l-3, Cold Spring Harbor Laboratory), both incorporated herein by
reference for their descriptions of stringent hybridization conditions). As indicated by
5 standard references, a simple estimate of the Tm value may be calculated by the
equation: Tm :===81.5+0.41((% G+C), when a nucleic acid is in aqueous solution at 1 M
NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC
ACID HYBRiDIZATION (1985)). The melting temperature of a hybrid (and thus the
conditions for stringent hybridization) is affected by various factors such as the length
10 and nature (DNA, RNA, base composition) of the primer or probe and nature of the
target nucleic acid (DNA, Rl'lA, base composition, present in solution or
irrunobilized, and the like), as well as the concentration of salts and other components
(e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol).
The effects ofthese factors are well known and are discussed in standard references in
15 the art. Illustrative stringent conditions suitable for achieving specitk hybridization
of most sequences are: a temperature of at least about 60°C and a salt concentration
of about 0.2 molar at pH7. Tm calculation for oligonucleotide sequences based on
nearest-neighbors thermodynamics can carried out as described in "A unified view of
polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics"
20 John SantaLucia, Jr., PNAS Febmary 17, 1998 voL 95 no. 4 1460-1465 (which is
incorporated by reference herein for this description).
10073] The term "'non-specific hybridization" is used herein to refer to
hybridization between tvvo nucleic acids (e.g., two oligonucleotides) that are less than
fully complementary.
25 [0074] The term "oligonucleotide" is used to refer to a nucleic acid that is
relatively short, generally shorter than 200 nucleotides, more particularly, shorter than
100 nudeotides, most particularly, shorter than 50 nucieotides. Typically,
oligonucleotides are single-stranded DNA molecules.
[0075] The tem1 "target nucleic acid" is used herein to refer to particular
30 nucleic acid to be detected or sequenced in the methods described herein.
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5
[0076] As used herein the tenn "target nucleic acid sequence" refers to a the
nucleotide sequence of a target nucleic acid, such as, for example, the amplit1cation
product obtained by amplifying a target nucleic acid or the eDNA produced upon
reverse transcription of an RNA target nucleic acid.
[0077] A "polymorphic marker" or "polymorphic site" is a locus at which
nucleotide sequence divergence occurs. Illustrative markers have at least two alleles,
each occurring at frequency of greater than l %, and more typically greater than 10%
or 20% of a selected population. A polymorphic site may be as srnaU as one base
pair. Polymorphic markers include restriction fragment length polymorphism
10 (RFLPs), variable number of tandem repeats (VNTR's), hypervariable regions,
minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,
simple sequence repeats, deletions, and insertion elements such as Alu. The first
identified allelic form is arbitrarily designated as the reference form and other allelic
fom1s are designated as alternative or variant alleles. The allelic form occurring most
15 frequently in a selected population is sometimes referred to as the "wild-type" fonn.
Rarely occurring polymorphisms may he designated as "mutant" fom1s of a sequence.
Mutant fom1s of a sequence can confer phenotypic difference on an organism, e.g.,
susceptibility to a disease or dmg resistance in a pathogenic organism. Diploid
organisms may be homozygous or heterozygous for allelic fonns. A diallelic
20 polymorphism has two forms. A triallelic polymorphism has three forms.
[0078] A "'single nucleotide polymorphism" (SNP) occurs at a polymorphic
site occupied by a single nucleotide, which is the site of variation between allelic
sequences. The site is usually preceded by and followed by highly conserved
sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000
25 members of the populations). A SNP usually arises due to substitution of one
nucleotide for another at the polymorphic site. A transition is the replacement of one
purine by another purine or one pyrimidine by another pyrimidine. A transversion is
the replacement of a purine by a pyrimidine or vice versa. SNPs can also arise from a
deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
30 [00791 The term "primer" refers to an oligonucleotide that is capable of
hybridizing (also tem1ed "a1mealing") with a nucleic acid and serving as an initiation
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site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e.,
in the presence of four different nucleoside triphosphates and an agent for
polymerization, such as DNA or RNA polymerase or reverse iranscriptase) in an
appropriate buffer and at a suitable temperature. The appropriate length of a primer
5 depends on the intended use of the primer, but primers are typically at least 7
nucleotides long and, in some embodiments, range from 10 to 30 nucleotides, or, in
some embodiments, from 10 to 60 nucleotides, in length. In some embodiments,
primers can be, e.g., 15 to 50 nudeotides long. Short primer molecules generally
require cooler temperatures to form sufficiently stable hybrid complexes with the
10 template. A primer need not reflect the exact sequence of the template but must be
sufficiently complementary to hybridize with a template.
[0080] A primer is said to anneal to another nucleic acid if the primer, or a
portion thereot~ hybridizes to a nucleotide sequence within the nucleic acid. The
statement that a primer hybridizes to a particular nucleotide sequence is not intended
15 to irnply that the primer hybridizes either cornpletely or exclusively to that nucleotide
sequence. For example, in some embodiments, amplification primers used herein are
said to "'anneal to" or be "'specific for" a nucleotide sequence." This description
encompasses primers that anneal wholly to the nucleotide sequence, as well as
primers that anneal partially to the nucleotide sequence.
20 [0081] The tem1 "primer pair" refers to a set of primers including a 5'
''upstream primer" or "forward primer" that hybridizes with the complement of the 5'
end ofthe DNA sequence to be amplified and a 3' "downstrearn primer" or "reverse
primer" that hybridizes with the 3' end of the sequence to be amplitied. As will be
recognized by those of skill in the art, the terms "upstream" and "downstream" or
25 "forward" and "'reverse" are not intended to be limiting, but rather provide illustrative
orientations in some embodiments.
[0082] A "probe" is a nucleic acid capable of binding to a target nucleic acid
of complementary sequence through one or more types of chemical bonds, generally
through complementary base pairing, usually through hydrogen bond formation, thus
30 forming a duplex structure. The probe can be labeled with a detectable label to permit
facile detection of the probe, particularly once the probe has hybridized to its
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complementary target. Alternatively, however, the probe may be unlabeled, but may
be detectable by specit1c binding with a ligand that is labeled, either directly or
indirectly. Probes can vary significantly in size. Generally, probes are at least 7 to 15
nucleotides in length. Other probes are at least 20, 30, or 40 nudeotides long. Still
5 other probes are somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotides
long. Yet other probes are longer still, and are at least 1 00, 150, 200 or more
nucleotides long. Probes can also be of any length that is within any range bounded
by any of the above values (e.g., 15-20 nucleotides in length).
[0083] The primer or probe can be perfectly complementary to the target
10 nucleotide sequence or can he less than perfectly complementary. In some
embodiments, the primer has at least 65% identity to the complement of the target
nucleotide sequence over a sequence of at least 7 nucleotides, more typically over a
sequence in the range of 10-30 nucleotides, and, in some embodiments, over a
sequence of at least 14-25 nucleotides, and, in some embodiments, has at least 75%
15 identity, at least 85% identity, at least 90(Yo identity, or at least 95%, 96%, 97(Yo, 98%,
or 99%, identity. It will be understood that ce1iain bases (e.g., the 3' base of a primer)
are generally desirably perfectly complernentary to corresponding bases of the target
nucleotide sequence. Primer and probes typically anneal to the target sequence under
stringent hybridization conditions.
20 [0084] As used herein with reference to a portion of a primer or a nucleotide
sequence within the primer, the tem1 '"specific for" a nucleic acid, refers to a primer or
nucleotide sequence that can specifically anneal to the target nucleic acid under
suitable mmealing conditions.
[0085] The tem1 "adaptor" is used herein to refer to a nucleic acid that, in use,
25 becomes appended to one or both ends of a nucleic acid, e.g., a nucleic acid fragment.
30
An adaptor may he single-stranded, double-stranded, or may include single- and
double-stranded portions. Illustrative adaptors include DNA sequencing adaptors that
are added to nucleic acid fragments to facilitate DNA sequencing. Different DNA
sequencing platfom1s typically require di±Ierent adaptors.
[0086] The tem1 "template," as used with reference to DNA sequencing refers
to a sequence that contains the necessary components to he sequenced. Thus, a
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template for DNA sequencing can include adaptors that provide nucleotide sequences
that facilitate DNA sequencing, such as a DNA sequencing primer binding site and a
barcode nucleotide sequence.
[0087] The term "DNA sequencing primer binding site" is used herein to refer
5 to a site to which a DNA sequencing primer anneals in a DNA sequencing template.
At least one DNA sequencing primer binding site is oriented a template such that it
primes synthesis of the portion of the template whose sequence is to be detem1ined.
[0088] The term "barcode nucleotide sequence" is a sequence that encodes an
item of information about a larger nucleotide sequence in which it appears. For
10 example, a barcode nucleotide sequence could encode information about the sample
or individual cell that a target nucleotide sequence was obtained from. Alternatively,
a barcode nucleotide sequence can be a unique molecular identit1er, meaning that
each different target nucleotide sequence in a set of target nucleotide sequences has its
own unique barcode nucleotide sequence.
15 [0089] Amplit1cation according to the present teachings encompasses any
means by which at least a part of at least one target nucleic acid is reproduced,
typically in a template-dependent manner, including without limitation, a broad range
of techniques for arnplifying nucleic acid sequences, either linearly or exponentially.
mustrative means fi.x performing an amplifying step include PCR, nucleic acid
20 strand-based amplification (NASBA), two-step multiplexed amplifications, rolling
circle amplification (RCA), and the like, including multiplex versions and
combinations thereof, for example but not limited to, OLA/PCR, PCRJOLA,
LDRIPCR, PCR/PCRILDR, PCRiLDR, LCR/PCR, PCRiLCR (also known as
combined chain reaction--COt), helicase-dependent amplification (HDA), and the
25 like. Descriptions of such techniques can be found in, among other sources, Ausubel
et al.; PCR Primer: A Laboratory Manual, DitTenbach, Ed., Cold Spring Harbor Press
(1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et aL, J. Clin.
Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed.,
Humana Press, Totowa, N.J. (2002); Abramson et al., Cun- Opin Bioteclmol. 1993
30 Feb.;4(1):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT
Publication No. WO 97/31256; \Venz et al., PCT Publication No. WO 011112579;
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wo 2021/222289 PCT/US2021/029448
Day et at, Genomics, 29(1 ): 152-162 (1995), Ehrlich et aL, Science 252:1643-50
(1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic
Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et
at, Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a
5 Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth Intemational
Symposium on Human Identitication, 1995 (available on the world wide web at:
promega.com/geneticidproc/ussymp6proc/blegrad.html- ); LCR Kit Instruction
Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. NatL Acad.
Sci. USA 88:188-93 (1991); Bi and Sambrook:, NucL Acids Res. 25:2924-2951
10 (1997); Zirvi et al., NucL Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad
Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); \Valk:er et
al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC In£: Dis. 2:18- (2002);
Lage et aL, Genome Res. 2003 Feb.; 13(2):294-307, and Landegren et al., Science
241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov.;2(6):542-8.,
15 Cook: et al., J Microbial Methods. 2003 May;53(2):165-74, Schweitzer et al., Curr
Opin Biotechnol. 2001 Feb.;12(1):21-7, U.S. Pat No. 5,830,711, U.S. Pat. No.
6,027,889, U.S. Pat. No. 5,686,243, PCT Publication No. W00056927A3, and PCT
Publication No. \V09803673AL
[0090] In some embodiments, amplification comprises at least one cycle of
20 the sequential procedures of: annealing at least one primer with complementary or
substantially complementary sequences in at least one target nucleic acid;
synthesizing at least one 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. Arnplitication can comprise
25 them1ocyding or can he performed isothermally.
[0091] As used herein, the term "support" refers to any substrate, typically
one to which oligonucleotides can be attached. If oligonucleotides are attached to the
support, the support is generally non-reactive to other components that will contact
the support in use. The support can be insoluble (e.g., a planar surface or a
30 micro head) or soluble (e.g., a water-soluble polymer that can easily be removed from
a reaction mixture by, e.g., centrifugation and/or precipitation).
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[0092] As used herein, the tem1 "microbead" refers to a bead having a
diameter that is less than 1 mM (i.e., less than 1000 microns). Microbeads may be
microscopic or near-microscopic and may have diameters of about 0.005 to l 00 ~un,
about 0.1 to 50 ~nn, or about 0.5 to 30 jlm.
5 [0093] A "multiplex amplification reaction" is one in which two or more
nucleic acids distinguishable by sequence are amplified simultaneously.
[00941 The term "qPCR" is used herein to refer to quantitative real-time
polymerase chain reaction (PCR), which is also known as "real-time PCR" or "kinetic
polymerase chain reaction;" all terms refer to PCR with real-time signal detection.
l 0 [0095] A "reagent" refers broadly to any agent used in a reaction, other than
the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for a nucleic acid
amplification reaction include, but are not limited to, buffer, metal ions, polymerase,
reverse transcriptase, primers, template nucleic acid, nucleotides, labels, dyes,
nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example,
15 substrates, cofactors, buffer, metal ions, inhibitors, and activators.
[0096] The tem1 "label," as used herein, refers to any atom or molecule that
can be used to provide a detectable and/or quantifiable signaL In particular, the label
can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that
can be attached to probes include, but are not limited to, radioisotopes, tluorophores,
20 chromophores, mass labels, electron dense pa1iicles, magnetic particles, spin labels,
molecules that emit cherni1uminescence, electrochemically active molecules,
enzymes, cofactors, and enzyme substrates.
[0097] The term "dye," as used herein, generally refers to any organic or
inorganic molecule that absorbs electromagnetic radiation.
25 !0098] The naturally occurring bases adenine, thymine, uracil, guanine, and
cytosine, which make up DNA and RNA, are described herein as "unmodified bases"
or '\mmodified forms."
[0099] The term "modified base" is used herein to refer to a base that is not a
canonical, naturally occurring base (e.g., adenine, cytosine, guanine, thymine, or
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5
uracil). Examples of modified bases are the pseudo-cornplementary bases 2-
thiothymine and 2-aminoadenine.
[0100] Nucleotides including modified bases are referred to herein as
"modified nucleotides" (e.g., pseudo-complementary nudeotides ).
10101] Oligonucleotides including one or more '"modified nucleotides" (e.g.,
pseudo-complementary nucleotides) are referred to herein as pseudo-complementary
oligonucleotides (e.g., pseudo-complementary blocker oligonucleotide).
Methods of Determining_ Whether A Nudeotlde Se,quence is Present
10102] The present disclosure provides a method of determining whether a
10 particular a nucleotide sequence is present in a target nucleic acid sequence in a
sample, where the target nucleic acid sequence comprises a polymorphic site, such as
a single nucleotide polymorphism. One way of enhancing the discrimination between
two possible sequences, e.g., a wild-type sequence and a rnutant sequence, is to assay
for the presence of one sequence (e.g., the mutant sequence) in the presence of a
15 blocking oligonucleotide that destabilizes hybridization of the second sequence (e.g.,
the wild-type sequence). Such an assay is shown in Fig. lA.
[0103] This approach has been used, for example, in a method termed "wildtype
blocking-polymerase chain reaction" (\VTB-PCR), which is described, for
example, Dominguez (2005) Oncogene 24:6830-6834 and in U.S. Patent No.
20 10,227,657 (issued March 19, 2019 to Maher et aL). Briefly, WTB-PCR was
developed to facilitate the detection and sequencing of minority mutations from
clinical specimens. In \VTB-PCR, a non-extendable: locked nucleic acid (LNA)
oligonucleotide binds tightly to a region of wild-type DNA known to develop point
mutations. This blocker oligonucleotide inhibits primer extension through the
25 polymorphic region for the wild-type allele, whereas primer extension through the
polymorphic region proceeds nommlly for the mutant allele to produce amplified
mutant DNA (see Fig. 1 A). This technique allows sensitive detection of minority
mutations in a tissue sample containing excess wild-type DNA.
[0104] In some embodiments, the amplification product from WTB-PCR is
30 hybridized to a capture oligonucleotide for detection. In such cases, blocker
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5
oligonucleotide remaining in the amplification product mixture can compete with the
amplified mutant DNA for hybridization to the capture oligonucleotide, as shown in
Fig. 2R The cornpetition can prevent the detection of a mutant allele that is present
in a sample.
[0105] The present method overcomes this difficulty by using a blocker
oligonucleotide and capture oligonucleotide pair that each include one or more
pseudo-complementary bases. The one or more pseudo-complementary bases are
positioned so that, ifthe blocker oligonucleotide were to hybridize to the capture
oligonucleotide, at least one (and preferably more) pseudo-complementary base(s) in
10 the blocker oligonucleotide would be faced with pairing with its complernentary
pseudo-complementary base. Since this pairing is disfavored, relative to normal
Watson-Crick base pairing, the degree to which the blocker oligonucleotide can
compete with the amplified mutant DNA for binding to the capture oligonucleotide is
reduced, improving the sensitivity of the assay for the mutant allele.
15 [0106] The blocker oligonucleotide can, but need not, include LNAs. In some
embodiments, is blocked to 3' extension, e.g., by virtue of lacking a 3' hydroxyl
group or using a chemical blocking moiety. This modification can improve the
specificity ofthe amplification.
10107] The present method is described in terms of a "wild-type" allele and a
20 "mutant" allele for ease of understanding, but those of skill in the are readily
appreciate that this method is applicable to the detection of one of at least two
possible forms of a sequence, such as, e.g., the detection of one of two alleles that do
not have a wild-type-mutant relationship.
10108] The pseudo-complementary bases typically replace residues at
25 sequence positions that are common between two forms of the sequence. The number
of pseudo-complementmy bases used in the blocker and capture oligonucleotides wiU
usually be the same (although this is not a requirement ofthe method). The number
used can vary, depending upon the length of the complementary sequences in the
oligonucleotides. In general, longer oligonucleotides can require the use of more
30 pseudo-complernentary bases than shorter oligonucleotides because it may take more
pseudo-complementa1y bases to adequately destabilize blocker and capture
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oligonucleotide hybridization. The degree of destabilization required is the degree
that sufficiently reduces competition of the blocker oligonucleotide with amplified
DNA for binding to the capture oligonucleotide. These parameters can be determined
empirically based on the guidance provided herein. In particular, Example 1 shows
5 how oligonucleotides can be tested for a reduction in binding competition by the
blocker oligonucleotide. In this Example, three pseudo-complementary bases were
used in a 16-nucleotide blocker oligonucleotide. In illustrative embodiments, at least
5%,10%3,15%,16%3, 17(!1(;, 18,%, 19%3,20%,21%,22%,23%,24(%,25%,30%3,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or all of
10 the bases in a blocker and/or capture oligonucleotide can be pseudo-complementmy
bases. In various embodiments, the number of pseudo-complementary bases in a
blocker and/or capture oligonucleotide falls within a range bounded by any of these
values, e.g., 10%-40%, 15%,-35%, 16% - 30%, 17%-25%, or 18%-20%.
[0109] In certain embodiments, the capture oligonucleotide can be attached to
15 a support In illustrative embodiments, the support can be an insoluble support, such
as a planar surface or a microbead or a soluble support, such as a water-soluble
polymer than can easily be recovered from a reaction mixture by, e.g. centrifugation
and/or precipitation. In general, assay fom1at will dictate whether, and what type of
support should be used.
20 [0110] In some embodiments, the method of detem1ining whether a nucleotide
sequence is present also includes quantifying the amount (relative or absolute) of the
nucleotide sequence. In certain embodiments, a probe can be used for
detection/ quanti ticati on.
[0111] The method can be used to assay a sample comprising a small minority
25 (less than 50%) of cells of one type (e.g., cancerous cells) in a large background of
cells of a ditTerent type. In various embodiments, the minority of cells is less than
approximately 45%, 40(Yo, 35%, 30(%, 25%, 201%, 15%, 10%, 5%, 4%), 3%, 2%1, 1%,
0.5%, 0.1 %, 0.05%, 0.01 %,, 0.005%, 0.001%, 0.0005%, or 0.0001% of the total
number of cells. In various embodiments, the percentage of minority cells falls within
30 a range bounded by any of the values, e.g., 10%-0.0001 !%, 5%-0.001%, 1% to 0.01 %,
or 0.5% to 0.1 %.
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[0112] In some embodiments the methods described herein can be used to
assay single cells.
Nucleic Acid Sequencing Methods
[0113] The present disclosure provides a method for simplifying preparations
5 for nucleic acid sequencing that find particular application in next-generation
sequencing protocols. For reviews of next-generation sequencing and sequencing
library generation, see Goodwin et at (2016) "Coming of Age: Ten Years of nextGeneration
Sequencing Technologies." Nature Reviews Genetics 17(6): 33J---51 and
Head et aL (20 14) "Library Construction for next-Generation Sequencing: Overviews
10 and Challenges." BioTechniques 56(2): 61.
[0114] Briefly, "sequencing by synthesis" is perhaps the most well-established
next-generation sequencing method, and is used by the 454, IHumina, Qiagen, and Ion
Torrent (Thermo Fisher) platfonns, with each platform utilizing their own
technologies. Instrument models within a platfonn may come in varying levels of
15 sequencing capabilities and throughput Sample loading chips and kits for a given
instrument may also be scalable to feature additional higher-throughput options.
Those of skill in the art are hnniHar with these platfom1s and with which to select for
different applications. For example, whole-genome or whole-transcriptome
sequencing may require higher throughputs, and de novo sequencing and
20 metagenomic sequencing may bendit from longer read lengths.
[0115] The typical sample preparation workflow for next-generation
sequencing (NGS), shown schematically in Fig. 3, can include: (1) nucleic acid
fragmentation or amplit1cation to produce nucleic acid fragments suitably sized for
sequencing (e.g., typicaHy used for DNA); (2) eDNA synthesis for RNA, (3) addition
25 of sequencing adaptors (to DNA or RNA), typically by ligation (DNA or RNA ligases
can be used to add adaptors to DNA or RNA, respectively; (4) amplification (e.g.,
PCR), (5) target enrichments, and (6) quantitication.
[0116] The sequencing library fragment size depends mainly on the desired
insert size (between the adaptors) and the limitations ofthe NGS platform, Hlmnina's
30 duster amplification step following adapter ligation can acconmwdate a range of up
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to 1500 bp. For ron Torrent, fragment sizes of 100 to 600 bp should be suitable.
Commercial kits are available for enzymatic fragmentation that specify one
sequencing platform and detail fragmentation size outputs.
[0117] Kits are available for RNA sequencing applications that include
5 reagents for reverse transcription into eDNA, either by PCR or PCR-free. Some also
feature enrichment fi.Jr specific RNA types, either by capturing mRNA or depleting
rRl'lA. These allow for streamlined library construction directly from RNA samples
ranging from inputs of 25 to l 000 ng.
[0118] Commercial enzyme kits for adapter ligation contain reagents tailored
10 to the sequencing platfonn. The general workflow involves end repair of the DNA
fragments followed by ligation ofplatforrn-specific adaptors. The rnajor difference
between Illumina and Ion TmTent is that the latter uses blunt-end ligation. Kits
typically include all the enzymes (such as ligases and polymerases) and buffers
necessary, and some feature additional barcodes for multiplexing.
15 [0119] For either Illumina or Ion Torrent platfonns, many commercial library
preparation kits include PCR polyrnerases for subsequent amplification following
adapter ligation. Some feature high-fidelity and hot-start polymerases for improved
coverage and lower duplication rates.
[0120] The large arnount of data generated by whole genome sequencing, for
20 example, can complicate data processing and analysis. As a workaround, portions of
genomes may he enriched to focus on key genes. With target enrichment, DNA
segments can be emiched either by hybridization-based capture or multiplex PCR.
Several kits are available for the in-solution capture using biotinylated RNA or
oligonucleotide probes that bind to streptavidin beads.
25 [0121] Prior to the sequencing run, sequencing libraries may require accurate
quantification to ensure good data output and quality. NGS quantification kits are
available that utilize qPCR, which are selective for the molecules with the right
adaptor sequences. For convenience and consistency, kits include cornplete sets of
reagents and some feature prediluted DNA standards.
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[0122] The present method simplifies library preparation for those workf1ows
in which amplified DNA templates are hybridized to oligonucleotides, which are
typically attached to a solid support Examples include oligonucleotides to which
"paired-end" sequences flanking a DNA template hybridize in mumina's t1ow cells.
5 These oligonucleotides are termed "capture oligonucleotides" for the purposes of the
present discussion. The design considerations for capture oligonucleotides used in a
sequencing method are similar to those described above for capture oligonucleotides
used in determining whether a particular nucleotide sequence is present, except that
capture oligonucleotides include pseudo-complementary bases to reduce the
10 likelihood that free adaptor sequences will hybridize to the capture oligonucleotides,
instead of the desired hybridization betvveen the capture oligonucleotides and the
amplified DNA templates. This problem arises because, typically, the sequences that
allow the amplified DNA templates to hybridize to the capture oligonucleotides are
present in the adaptor sequences.
15 [0123] In the present method, competition between adaptors and amplified
DNA templates for binding capture oligonucleotides is reduced or eliminated by
including pseudo-complementary bases in the adaptors and in corresponding positions
in the capture oligonucleotides. The considerations for reducing or eliminating
adaptor binding to capture oligonucleotides are essentially the same as those
20 discussed above for reducing blocker oligonucleotide to capture oligonucleotides (see
also below, the section entitled "Primer/Probe/Blocker Oligonucleotide/Adaptor/Capture
Oligonucleotide Design"). The method works because free adaptors contain
pseudo-complementary bases which will not pair efficiently with their counterparts in
the capture oligonucleotide(s), whereas amplified DNA templates include the natural
25 bases provided to the amplification reaction, which ·will pair relatively normally with
the pseudo-complementary bases in the capture oligonucleotides.
SamJlles
[0124] Nucleic acid-containing sarnples can be obtained from biological
sources and prepared using conventional methods known in the art. In particular,
30 nucleic useful in the methods described herein can be obtained from any source,
including unicellular organisms and higher organisms such as plants or non-human
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5
animals, e.g., canines, felines, equines, primates, and other non-human rnammals, as
well as humans. In some embodiments, samples may he obtained from an individual
suspected of being, or known to be, infected with a pathogen, an individual suspected
of having, or known to have, a disease, such as cancer, or a pregnant individual.
[0125] Nucleic acids can he obtained from cells, bodily t1uids (e.g., blood, a
blood fraction, urine, etc.), or tissue samples by any of a variety of standard
techniques. In some embodiments, the method employs samples of plasma, serum,
spinal fluid, lymph fluid, peritoneal tluid, pleural tluid, oral fluid, and external
sections ofthe skin; samples from the respiratory, intestinal genital, or urinary tracts;
10 samples of tears, saliva, blood cells, stem cells, or tumors. Samples can be obtained
from live or dead organisms or from in vitro cultures. mustrative samples can include
single cells, paraffin-embedded tissue samples, and needle biopsies. In some
embodiments, the nucleic acids analyzed are obtained from a single cell.
[0126] Nucleic acids of interest can be isolated using methods well known in
15 the art. The sample nucleic acids need not he in pure form, but are typically
sufficiently pure to allow the steps ofthe methods described herein to he performed.
Targ_d Nucleic Acids
10127] Any target nucleic acid that can detected by nucleic acid amplitication
can be detected or sequenced using the methods described herein. In some
20 embodiments, at least some nucleotide sequence information will be known for the
target nucleic acids. For example, ifthe amplification reaction employed is PCR,
sufficient sequence infom1ation is generally available for each end of a given target
nucleic acid to permit design of suitable amplification primers. In nucleic acid
sequencing embodiments, there may be no sequence information known for the
25 '"target nucleic acids," which in this case are sequencing templates, if the sequencing
ternplates are produced by adding DNA sequencing adaptors to both ends of nucleic
acid fragments, since primers that hind in the adaptor sequences can be used for
amplification.
[0128] The targets can include, for example, nucleic acids associated with
30 pathogens, such as viruses, bacteria, protozoa, or fungi; RNAs, e.g., those tor which
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over- or under-expression is indicative of disease, those that are expressed in a tissueor
developmental-specific manner; or those that are induced by particular stimuli;
genomic DNA, which can be analyzed for specific polymorphisms (such as SNPs),
alleles, or haplotypes, e.g., in genotyping. Of particular interest are genomic DNAs
5 that are altered (e.g., amplified, deleted, and/or mutated) in genetic diseases or other
pathologies; sequences that are associated with desirable or undesirable traits; and/or
sequences that uniquely identif.y an individual (e.g., in forensic or paternity
determinations).
Pr:imer/Probe/Blockcr Oligo:uu.cleotide/Ada.Rtor/fai!hlre Oligonudcotide Design
10 [01291 Those of skill in the art are well-versed in the considerations associated
with designing an oligonucleotide that is intended to anneal or hybridize (or not) to
another nucleotide sequence in an assay. These considerations are discussed briefly
below in terms of primers, and most of these considerations apply to other
armealingihybridizing oligonucleotides.
15 [0130] Primers suitable for nucleic acid amplification are suftkiently long to
prime the synthesis of extension products in the presence of a suitable nucleic acid
polymerase. The exact length and composition of the primer will depend on many
factors, including, for example, temperature of the a1mealing reaction, source and
composition of the primer, and where a probe is ernployed, proximity of the probe
20 annealing site to the primer annealing site and ratio of primer:probe concentration.
For example, depending on the complexity of the target nucleic acid sequence, an
oligonucleotide primer typically contains in the range of about 1 0 to about 60
nucleotides, although it may contain more or fewer nucleotides. The primers should
be sufficiently complementary to selectively mmeal to their respective strands and
25 form stable duplexes.
[0131] In general, one skilled in the art knows how to design suitable primers
capable of amplifying a target nucleic acid of interest. For example, PCR primers can
he designed by using any commercially available software or open source software,
such as Primer3 (see, e.g., Rozen and Skaletsky (2000) Meth. Mol. Biol., 132: 365-
30 386; www .broad.mitedu/node/1 060, and the like) or by accessing the Roche UPL
website. The amplicon sequences are input into the Primer3 program with the UPL
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probe sequences in brackets to ensure that the Primer3 program will design primers
on either side of the bracketed probe sequence.
[0132] The Tm of hybrids formed by primers, or any other oligonucleotides in
an assay can be adjusted by including stabilizing or destabilizing bases in the
5 primer/oligonucleotide.
[0133] "Stabilizing bases" include, e.g., stretches of peptide nucleic acids
(PNAs) that can be incorporated into DNA oligonucleotides to increase duplex
stability. Locked nucleic acids (LNAs) and unlocked nucleic acids (UNAs) are
analogues of RNA that can be easily incorporated into DNA oligonucleotides during
10 solid-phase oligonucleotide synthesis, and respectively increase and decrease duplex
stability. Suitable stabilizing bases also include modified DNA bases that increase the
stability ofbase pairs (and therefore the duplex as a whole). These modified bases
can be incorporated into oligonucleotides during solid-phase synthesis and offer a
more predictable method of increasing DNA duplex stability. Examples include AP-
15 dC (G-clamp) and 2-aminoadenine, as well as 5-methylcytosine and C(5)propynylcytosine
(replacing cytosine), and C(5)-propynyluracil (replacing thymine).
[0134] "Destabilizing bases" are those that destabilize double-stranded DNA
by virtue of f()mling less stable base pairs than the typical A-T and/or G-C base pairs.
Inosine (I) is a destabilizing base because it pairs with cytosine (C), but an I-C base
20 pair is less stable than a G-C base pair. This lower stability results from the fact that
inosine is a purine that can make only two hydrogen bonds, compared to the three
hydrogen bonds of a G-C base pair. Other destabilizing bases are known to, or
readily identified by, those of skill in the art.
10135] As discussed above, the present methods are concemed with reducing
25 unwanted hybridization between oligonucleotides (e.g., a blocker oligonucleotide and
a capture oligonucleotide). Unwanted hybridization between any two
oligonucleotides in an assay can be reduced or prevented by including pseudocomplementary
bases in the oligonucleotides. Pseudo-complementmy bases are
described as "modified bases" in the next section.
30 [0136] Primers and other oligonucleotides may be prepared by any suitable
method, including, for example, direct chemical synthesis by methods such as the
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phosphotriester rnethod ofNarang et aL (1979) Meth. Enzymol. 68: 90-99; the
phosphodiester method ofBrown et aL (1979) Meth. EnzymoL 68: 109-151; the
diethylphosphoramidite method of Beaucage et al. ( 1981) Tetra. Lett, 22: 1859-1862;
the solid support method of U.S. Patent No. 4,458,066 and the like, or can be
5 provided from a commercial source. Primers may be purified by using a Sephadex
column (Amersham Biosciences, Inc., Piscataway, NJ) or other methods known to
those skilled in the art Primer purification may improve the sensitivity of the
methods described herein.
10
Modified Bases
[01371 Modified bases useful in the primers and other oligonucleotides
described herein include those wherein the modified base forms stable hydrogenbonded
base pairs with the natural complementa1y base but does not form stable
hydrogen-bonded base pairs with its modified complementa1y base (e.g., pseudocomplementary
bases). (For ease of discussion, cornplementary bases are also
15 referred to herein as "'partners." Also, for ease of discussion, the following
description relates to prirners and primer pairs, but, as those of skill in the art, readily
appreciate, this description also applies to the other oligonucleotides and
oligonucleotide pairs described herein.) In some embodiments, this is accomplished
when the modified base can form two or more hydrogen bonds with its natural
20 partner, but only one or no hydrogen bonds with its modified partner. This allows the
production of primer and other oligonucleotide pairs that do not form substantially
stable hydrogen-bonded hybrids with one another, as manifested in a melting
temperature (under physiological or substantially physiological conditions) of less
than about 40°C. The primers of the primer pair, however, form substantially stable
25 hybrids with the complementary nucleotide sequence in a template strand (e.g., first
template strand) of a single- or double-stranded target nucleic acid and with a strand
complementary to the template strand (e.g., second template strand). In some
embodiments, due to the increased (in some embodiments, double) number of
hydrogen bonds in such hybrids, the hybrids forrned with the primers of the present
30 invention are more stable than hybrids that would be formed using primers with
unmodified bases.
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[0138] In accordance with well-established convention, the naturally
occurring nucleotides of nucleic acids have the designation A, U, G and C, (RNA)
and dA, dT, dG and dC (DNA). The following description applies to both
ribonucleotides and deoxyribonucleotides, and therefore, unless the context otherwise
5 requires, no distinction needs to be made in this description between A and dA, U and
dT, etc.
[0139] Analogs of A that are modified in the base pmiion to form a stable
hydrogen-bonded pair with T, (or U in the case of RNA) but not with a modified Tare
designated A*. Analogs ofT that are modified in the base portion to form a stable
10 hydrogen-bonded pair with A, but not with A* are designated T*. Analogs of G that
are modified in the base portion to form a stable hydrogen-bonded pair with C, hut not
with a modified C are designated G*. Analogs of C that are modified in the base
portion to form a stable hydrogen-bonded pair with G, but not with G* are designated
C*. In some embodiments, the foregoing conditions are satisfied when each of the
15 A*, T*, G*, and C* nucleotides (collectively, the modified nucleotides) tonn two or
more hydrogen bonds with their natural pa1iner, but only one or no hydrogen bonds
with their rnodified partner. This is iUustrated by Forrnulas 1a, 1b, 2a, 2b, 3a, 3b, 4a
and 4b below (and in Fig. 8A-8B), where the hydrogen bonding between natural A-T
(or A-U in case of RNA) and G-C pairs, and hydrogen bonding between exemplary
20 A *-T, T*-A, G*-C, C*-G, A *-T* and G*-C* pairs are illustrated.
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·:.h
!
H
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[0140] In general, a sufficient number of modified nucleotides are
5 incorporated into the primers described herein to preferentially increase the annealing
of the primers to the template strands of a target nucleic acid, as compared to primerto-
primer annealing. It is not necessary to replace each natural nucleotide ofthe
primer with a modified nucleotide in order to accomplish this. In some embodiments,
the primers include, in addition to one or more modified nucleotides, one or more
10 naturally occurring nucleotides and/or variants of naturally occurring nucleotides,
provided that the variations do not interfere significantly with the complernentary
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binding ability ofthe prirners, as discussed above. For example, primers including
modit1ed nucleotides can include pentofuranose moieties other than ribose or 2-
deoxyribose, as well as derivatives of ribose and 2-deoxyribose, for example 3-amino-
2-deoxyribose, 2-fluoro-2-deoxyribose, and 2-0-CJ-6 alkyl or 2-0-allyl ribose,
5 particularly 2-0-methyl ribose. The glycosidic linkage can be in the a or~
configuration. The phosphate backbone of the primer can, if desired, include
phosphorothioate linkages.

CLAIMS
What is claimed is:
1. A method of determining whether a nucleotide sequence is present in a
5 target nucleic acid sequence in a sample, wherein the target nucleic acid sequence
comprises a polymorphic site, wherein the polymorphic site is characterized by a first
nucleotide sequence and a second nucleotide sequence, wherein the first and second
nucleotide sequences differ by at least one nucleotide or ribonucleotide, the method
compnsmg:
10 contacting nucleic acid of, or derived from, the sample with fonvard and
reverse primers capable of amplifying the target nucleic acid sequence, wherein said
contacting is in the presence of a blocker oligonucleotide that is complementary to the
first nucleotide sequence to form a reaction mixture, wherein:
if the target nucleic acid sequence comprises the first nucleotide
15 sequence, the blocker oligonucleotide anneals to the first nucleotide sequence and
inhibits amplification; or
20
25
30
if the target nucleic acid sequence comprises the second nucleotide
sequence, the blocker oligonucleotide does not anneal to the second nucleotide
sequence and does not inhibit amplification;
conducting an amplification reaction in the reaction mixture;
after the amplification reaction, contacting the reaction mixture, or nucleic
acids frorn the reaction mixture, with a capture oligonucleotide that is complementary
to the second nucleotide sequence under conditions suitable for specit1c hybridization;
and
detecting any specific hybridization to the capture oligonucleotide, wherein
the presence of specific hybridization to the capture oligonucleotide indicates that the
second nucleotide sequence is present in the target nucleic acid sequence,
wherein:
the blocker oligonucleotide comprises one or more first modified bases
and the capture oligonucleotide comprises one or more second modified bases,
at least one of which is cornplementary to one of the first modified bases,
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5
10
15
wherein the modified bases preferentially pair with unmodified forms of their
complementa1y bases, as compared to pairing between modified,
cornplementary bases; and
the presence of the one or more modified bases in the blocker
oligonucleotide and in the capture oligonucleotide destabilizes hybridization
between the blocker oligonucleotide and the capture oligonucleotide.
2. The method of claim 1, wherein at least one of the first modified bases and at
least one of the second, complementary modified bases in the capture oligonucleotide
are bases that do not differ between the first and second nucleotide sequence.
3. The method of claim 1 or claim 2, wherein the method comprises quantifying
any specit1c hybridization to the capture oligonucleotide.
4. The method of any one of claims 1-3, wherein the sample consists of nucleic
acids from a single cell.
5. An oligonucleotide set comprising:
forward and reverse primers capable of amplifying a target nucleic acid
sequence, wherein the target nucleic acid sequence comprises a polymorphic site,
wherein the polymorphic site is characterized by a first nucleotide sequence and a
second nucleotide sequence, wherein the first and second nucleotide sequences differ
by at least one nucleotide or ribonucleotide;
20 a blocker oligonucleotide that is complementary to the first nucleotide
sequence; and
a capture oligonucleotide that is complementary to the second nucleotide
sequence,
wherein the blocker oligonucleotide comprises one or more t1rst modified
25 bases and the capture oligonucleotide comprises one or more second modified bases,
at least one of which is complementary to one of the tlrst modified bases, wherein the
modified bases preferentially pair with unmodified forms of their complementary
bases, as compared to pairing between modified, complementary bases; and
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the presence ofthe one or more rnodified bases in the blocker oligonucleotide
and in the capture oligonucleotide destabilizes hybridization between the blocker
oligonucleotide and the capture oligonucleotide.
6. The method or oligonucleotide set of any one of the preceding claims, wherein
5 the capture oligonucleotide is attached to a support.
7. A method of simplifying preparations for nucleic acid sequencing, the method
compnsmg:
adding DNA sequencing adaptors to nucleic acid fragments to produce
sequencing templates;
10 amplifying sequencing templates to produce amplified DNA templates; and
15
20
25
contacting the amplified DNA templates with capture oligonucleotides
attached to a support under conditions suitable for hybridization, wherein the DNA
sequencing adaptors and the capture oligonucleotides comprise complementary
nucleotide sequences,
8.
wherein:
the DNA sequencing adaptors each comprise one or more first
modified bases in their complementary nucleotide sequence, and the capture
oligonucleotides comprise one or more second modified bases in their
cornplementary nucleotide sequence, wherein at least one of the first and
second modit1ed bases are complementary, wherein the modified bases
preferentially pair with umnodified fom1s of their complementary bases, as
compared to pairing between modified, complementary bases; and
hybridization of amplified DNA templates to the capture
oligonucleotide is favored over hybridization of free adaptors to the capture
oligonucleotides, eliminating a need to separate amplified DNA templates
from free adaptors before further DNA sequencing steps.
A combination of components for simplifying nucleic acid sequencing, the
combination comprising:
DNA sequencing adaptors; and
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5
10
15
capture oligonucleotides attached to, or adapted to he attached to, a support,
wherein the DNA sequencing adaptors and the capture oligonucleotides comprise
complementary nucleotide sequences,
wherein:
the DNA sequencing adaptors each comprise one or more first
modified bases in their complementary nucleotide sequence, and the capture
oligonucleotides comprise one or more second modified bases in their
complementary nucleotide sequence, wherein at least one of the first and
second modified bases are complementary, wherein the modified bases
preferentially pair with unmodified forms of their complementary bases, as
compared to pairing between modified, complementary bases; and
hybridization of amplified DNA templates to the capture
oligonucleotide is favored over hybridization of free adaptors to the capture
oligonucleotides, eliminating a need to separate arnplifled DNA templates
from free adaptors.
9. The method of claim 7 or the combination of components of claim 8, wherein
the DNA sequencing adaptors comprise a nucleotide sequence that is a binding site
for a DNA sequencing primer and a barcode nucleotide sequence.
10. The method of claim 7, wherein the method additionally comprises producing
20 the nucleic acid fragments from genomic DNA, or the combination of components of
claim 8, wherein the combination additionally comprises one or more reagents that
produce the nucleic acid fragments from genomic DNA.
11. The method of claim 7, wherein said adding of DNA sequencing adaptors
comprises ligating the DNA sequencing adaptors to the nucleic acid fragments, or the
25 combination of components of claim 8, wherein the combination additionally
comprises a ligase.
12. The method or combination of components of any one of claims 7-11, wherein
the method employs, or the combination comprises, a DNA polymerase for
amplification.
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13. The rnethod or combination of components of any one of claims 7-12, wherein
the method employs, or the combination comprises, a reverse transcriptase for
reverse-transcribing nucleic acid fragment that are RNA.
14. The method, oligonucleotide set, or combination of components of any one of
5 the preceding claims, wherein modified complementary bases t"i.xm fewer hydrogen
bonds with each other than with unmodified complementary bases.
10
15. The method, oligonucleotide set, or combination of components of claim 14,
wherein the Tm of a base pair formed between modified complementmy bases less
than 40 °C.
16. The method, oligonucleotide set, or combination of components of any one of
the preceding claims, wherein at least one complementary pair of modified bases
comprises modified fom1s of adenine and thymine.
17. The method, oligonucleotide set, or combination of components of any one of
the preceding claims, wherein at least one complementary pair of modified bases
15 comprises modified forms of guanine and cytosine.
18. The method, oligonucleotide set, or combination of components of any one of
the preceding claims, wherein the blocker oligonucleotide is blocked to 3' extension.