U.S. patent application number 13/328844 was filed with the patent office on 2012-06-21 for clonal amplification of nucleic acid on solid surface with template walking.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Pius Brzoska, Kellie Haley, Rachel Kasinskas, Jennifer Kunkel, Kai Lao, Bin Li, Zhaochun Ma, Jennifer O'Neil.
Application Number | 20120156728 13/328844 |
Document ID | / |
Family ID | 45478545 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120156728 |
Kind Code |
A1 |
Li; Bin ; et al. |
June 21, 2012 |
CLONAL AMPLIFICATION OF NUCLEIC ACID ON SOLID SURFACE WITH TEMPLATE
WALKING
Abstract
Novel methods of generating a localized population of
immobilized clonal amplicons on a support are provided.
Inventors: |
Li; Bin; (Palo Alto, CA)
; Lao; Kai; (Pleasanton, CA) ; O'Neil;
Jennifer; (Peabody, MA) ; Kunkel; Jennifer;
(Amesbury, MA) ; Haley; Kellie; (Peabody, MA)
; Kasinskas; Rachel; (Amesbury, MA) ; Ma;
Zhaochun; (Sunnyvale, CA) ; Brzoska; Pius;
(Woodside, CA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
45478545 |
Appl. No.: |
13/328844 |
Filed: |
December 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61424599 |
Dec 17, 2010 |
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61445324 |
Feb 22, 2011 |
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61451919 |
Mar 11, 2011 |
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61526478 |
Aug 23, 2011 |
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61552660 |
Oct 28, 2011 |
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Current U.S.
Class: |
435/91.1 |
Current CPC
Class: |
C12Q 1/6874 20130101;
C12P 19/34 20130101; C12Q 1/46 20130101; C12Q 1/6806 20130101; C12Q
2565/537 20130101; C12Q 1/6846 20130101; C12Q 1/6834 20130101; C12Q
2531/119 20130101; C12Q 1/6869 20130101; C12Q 1/6853 20130101; C12Q
1/6846 20130101; C12Q 2531/119 20130101; C12Q 2565/537 20130101;
C12Q 1/46 20130101; C12Q 1/6806 20130101; C12Q 2521/507 20130101;
C12Q 2531/119 20130101; C12Q 2565/537 20130101; C12Q 2527/101
20130101; C12Q 2537/1376 20130101; C12Q 2565/537 20130101; C12Q
1/6806 20130101; C12Q 2521/507 20130101; C12Q 2527/101 20130101;
C12Q 2537/1376 20130101; C12Q 2565/537 20130101 |
Class at
Publication: |
435/91.1 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Claims
1. A method of primer extension, comprising: a) hybridizing a first
primer molecule ("first forward primer") to a complementary
primer-binding sequence ("reverse-strand PBS") on a nucleic acid
strand ("reverse strand"); wherein at least 60% of nucleotide bases
of the first forward primer are adenine, thymine or uracil or are
complementary to adenine, thymine or uracil; b) generating an
extended first forward strand that is a full-length complement of
the reverse strand and is hybridized thereto, by extending the
first forward primer molecule using the reverse strand as template;
and c) hybridizing a second primer molecule ("second forward
primer") to the reverse-strand PBS where the reverse strand is also
hybridized to the first forward strand.
2. The method of claim 1, comprising amplifying the forward strand
by one or more amplification cycles comprising steps (b) and (c),
wherein the second forward primer of step (c) of a first
amplification cycle is the first forward primer of step (b) of a
subsequent amplification cycle; and wherein a substantial
proportion of reverse strands are hybridized to forward strands at
all times during or between said one or more repetitions.
3. The method of claim 1, wherein the substantial proportion of
reverse strands is at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 99% of reverse strands.
4. The method of claim 1, comprising amplifying the reverse strand
by: a) hybridizing a first reverse primer molecule to a
complementary reverse-primer-binding sequence ("forward-strand
PBS") on an extended forward strand; b) generating an extended
first reverse strand that is a full-length complement of the
forward strand and hybridized thereto, by extending the first
reverse primer molecule in template-dependent fashion using the
forward strand as template; and c) hybridizing a second primer
("second reverse primer") to the forward-strand PBS where the
forward strand is also hybridized to the first reverse strand.
5. The method of claim 4, comprising amplifying the reverse strand
by one or more repetitions of steps (b)-(c), wherein the second
reverse primer of step (c) is the first reverse primer of repeated
step (b); and wherein a substantial proportion of forward strands
are hybridized to reverse strands at all times during or between
said one or more repetitions.
6. The method of claim 1, wherein the substantial proportion of
reverse strands is at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 99% of reverse strands.
7. The method of claim 1, wherein the first and/or second forward
primers are immobilized to a single support, or the first and/or
second reverse primers are immobilized to a single support
8. The method of claim 1, further comprising completely separating
the extended forward strands from reverse strands after performing
the desired number of amplification cycles, and optionally removing
separated forward strands from the presence of separated reverse
strands, or vice versa.
9. The method of claim 1, wherein during one or more amplification
cycles all nucleic acid reagents are not in contact with a
recombinase or reverse transcriptase or helicase or nicking enzyme
or any other enzyme that is not a polymerase at any time.
10. The method of claim 1, wherein template-dependent extension of
a forward primer using a reverse strand as template results in
displacement of another forward strand that was already hybridized
to the reverse strand.
11. The method of claim 1, wherein the Tm of all forward primers is
not more than 50.degree. C., 55.degree. C., 60.degree. C. or
65.degree. C., and wherein the Tm of the reverse strands is not
less than 95.degree. C., 90.degree. C., 85.degree. C., 80.degree.
C. or 75.degree. C.
12. The method of claim 1, wherein amplification is performed under
isothermal conditions.
13. The method of claim 1, wherein the isothermal conditions are
adjusted to a temperature that is higher than the Tm of all forward
primers, but lower than the Tm of the reverse strands, wherein the
Tm of a reverse strand is the temperature at which half of the
reverse strands in a clonal population of identical reverse strands
are fully denatured from a perfectly complementary strand that is
fully hybridized to the reverse strand across its entire
length.
14. The method of claim 1, wherein said first and second forward
primers are adjacently immobilized to the same support, whereby
amplification generates an immobilized clonal populations of
extended forward strands.
15. The method of claim 1, wherein said first and second forward
primers are adjacently immobilized to the same support, whereby
amplification generates an immobilized clonal population of
extended forward strands.
16. The method of claim 1, wherein a plurality of template nucleic
acids are individually hybridized to spatially-separated
immobilization sites, whereby amplification generates
spatially-separated clonal populations corresponding to individual
template nucleic acids.
17-25. (canceled)
26. A method of generating separated and immobilized clonal
populations of a first template sequence ("template 1") and a
second template sequence ("template 2"), comprising amplifying the
first and second template sequence, wherein: a) both templates are
in single-stranded form and are both contained within the same
continuous liquid phase, where a first and second immobilization
site (respectively, "IS1" and "IS2") are in contact with said
continuous liquid phase, and where IS1 and IS2 are spatially
separated, b) template 1 comprises a first subsequence ("T1-FOR")
at its 3' end, and a second subsequence ("T1-REV") that is
non-overlapping with T1-FOR and at its 5' end, c) template 2
comprises a first subsequence ("T2-FOR") at its 3' end, and a
second subsequence ("T2-REV") that is non-overlapping with T2-FOR
and at its 5' end, d) IS1 comprises an immobilized primer ("IS1
primer") that can hybridize to both T1-FOR and T2-FOR, e) IS2
comprises an immobilized primer ("IS2 primer") that can hybridize
to both T1-FOR and T2-FOR, f) the reverse complement of T1-REV
cannot hybridize substantially to primers on IS1, and/or the
reverse complement of T2-REV cannot hybridize substantially to
primers on IS2, but can each hybridize substantially to a
non-immobilized primer in the continuous liquid phase; whereby
amplification results in a population of clonal amplicons of
template 1 substantially attached to IS1 and not to IS2, and/or a
population of clonal amplicons of template 2 substantially attached
to IS2 and not to IS1.
27. The method of claim 26, wherein intermixing of non-immobilized
nucleic acid molecules is substantially unretarded in the
continuous liquid phase at some timepoint during amplification.
28. The method of claim 27, wherein intermixing is substantially
unretarded for a period of time during amplification, and
optionally is substantially unretarded during the entire duration
of amplification.
29. The method of claim 27, wherein any nucleic acid that has
dissociated from one immobilization site is capable of
substantially hybridizing to both immobilization sites and any
movement (e.g., movement by diffusion, convection) of said
dissociated nucleic acid to another immobilization site is not
substantially retarded in the continuous liquid phase.
30. The method of claim 26, wherein the continuous liquid phase is
in simultaneous contact with IS1 and IS2.
31-103. (canceled)
Description
[0001] This application claims priority to U.S. Provisional App.
No. 61/552,660 filed Oct. 28, 2011; U.S. Provisional App. No.
61/526,478 filed Aug. 23, 2011; U.S. Provisional App. No.
61/451,919 filed Mar. 11, 2011; U.S. Provisional App. No.
61/445,324 filed Feb. 22, 2010; and U.S. Provisional App. No.
61/424,599 filed Dec. 17, 2010; each of which is incorporated by
reference in its entirety.
BACKGROUND
[0002] Nucleic acid amplification is very useful in molecular
biology and has wide applicability in practically every aspect of
biology, therapeutics, diagnostics, forensics and research.
Generally, multiple amplicons are generated from a starting
template using one or more primers, where the amplicons are
homologous or complementary to the template from which they were
generated. Multiplexed amplification can also streamline processes
and reduce overheads. A single set of primers can be mixed with
different templates, or a single template can be contacted with
multiple different primers, or multiple different templates can be
contacted with multiple different primers. This application relates
to methods and reagents for nucleic acid amplification and/or
analysis.
SUMMARY
[0003] Methods, reagents and products of nucleic acid amplification
and/or analysis are provided herein. Amplification can make use of
immobilized and/or soluble primers. Amplicons generated from
methods provided herein are suitable substrates for further
analysis, e.g., sequence determination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1: Schematic showing an embodiment of template walking
In an alternative embodiment, the immobilized primer comprises an
adenosine-rich sequence designated as (A).sub.n, e.g., (A).sub.30,
and the primer binding site for the immobilized primer on the
template comprises a complementary T-rich sequence, e.g.,
(T).sub.30.
[0005] FIG. 2: Overview of amplification on beads by template
walking and deposition of beads onto a planar array for
sequencing
[0006] FIG. 3: Alternative embodiments using semiconductor-based
detection of sequencing by synthesis. Template walking can be used
to generate a population of clonal amplicons on a bead or on the
base or bottom of a reaction chamber. In an alternative embodiment,
the immobilized primer comprises an adenosine-rich sequence
designated as (A).sub.n, e.g., (A).sub.30, and the primer binding
site for the immobilized primer on the template comprises a
complementary T-rich sequence, e.g., (T).sub.30.
[0007] FIG. 4: Alternative embodiments of immobilization sites in
the form of primer lawns onplanar substrates. Arrays of separated
immobilization sites can be used or else a single continuous lawn
of primers can be considered to be a random array of immobilization
sites. Optionally, the location of one or more immobilization sites
in the continuous lawn of primers can be undetermined as yet, where
the location is determined at the time of attachment of the initial
template before walking or is determined by the space occupied by
the amplified cluster. In an alternative embodiment, the
immobilized primer comprises an adenosine-rich sequence designated
as (A).sub.n, e.g., (A).sub.30, and the primer binding site for the
immobilized primer on the template comprises a complementary T-rich
sequence, e.g., (T).sub.30.
[0008] FIG. 5: Influence of temperature on the template walking
reaction. A graphical plot of the delta Ct before and after the
template walking amplification was calculated and plotted against
reaction temperature.
[0009] FIG. 6: Table of the Ct values of the 96 duplex TaqMan qPCR
reactions.
[0010] FIG. 7: 100,000-fold amplification by template walking on
beads. Delta Ct before and after the template walking reaction and
fold of amplification before and after the template walking
reaction was calculated and plotted against reaction time.
[0011] FIG. 8: Schematic depiction of an exemplary strand-flipping
and walking strategy. (A) Template walking, (B) Strand flipping to
generate flipped strands, (C) addition of new primer-binding
sequence Pg' on final flipped strands.
DEFINITIONS
[0012] Any active verb (or its gerund) is intended to indicate that
the corresponding action occurs at a specific, significant or
substantial level (e.g., more than random, or more than an
appropriate control). For example the act of "hybridizing"
indicates that a significant or substantial level of specific
hybridization takes place. For example in the case of hybridization
of a primer to a template during an amplification process,
hybridizing is optionally sufficient to achieve a desired fold of
amplification--e.g., at least 10.sup.3 or 10.sup.4 or 10.sup.5 or
10.sup.6 amplicons from a single template. In another example,
specific hybridization is more than would happen between two
nucleic acids that share no significant amount of sequence
homology. In yet another example, specific hybridization comprises
binding of a nucleic acid to a target nucleotide sequence in the
absence of substantial binding to other nucleotide sequences
present in the hybridization mixture under defined stringency
conditions. Optionally, the sample is derived from tissues or
fluids taken from living or dead organisms (e.g., humans) for the
purposes of diagnostics or forensics). Optionally, the same
comprises a genomic library or an exome library.
[0013] Two sequences can be considered to be complementary if one
sequence hybridizes substantially and specifically under the
conditions of choice to the other sequence. Two sequences can be
considered to be homologous if the reverse complement of one
sequence can hybridize substantially and specifically under the
conditions of choice to the other sequence. Substantial
hybridization for example is where more than 5%, optionally 10%,
30%, 50% or 80% of one of the nucleic acids is hybridized to the
other nucleic acid. Hybridization between two single-stranded
nucleic acids often but not necessarily involves the formation of a
double stranded structure that is stable under conditions of
choice. The conditions of choice are for example conditions in
which hybridization is intended, e.g., during an annealing step of
an amplification cycle. Two single-stranded polynucleotides are
optionally considered to be hybridized if they are bonded to each
other by two or more sequentially adjacent base pairings.
Optionally, a substantial proportion of nucleotides in one strand
of the double stranded structure undergo Watson-Crick base-pairing
with a nucleoside on the other strand. Hybridization also includes
the pairing of nucleoside analogs, such as deoxyinosine,
nucleosides with 2-aminopurine bases, and the like, that may be
employed to reduce the degeneracy of the probes, whether or not
such pairing involves formation of hydrogen bonds.
[0014] A nucleic acid can be considered immobilized if it is
attached to a support in a manner that is substantially stable, at
least during conditions of choice (e.g., during the amplification
reaction). The attachment can be by any mechanism, including but
not limited to non-covalent bonding, ionic interactions, covalent
linkage. If a first nucleic acid is hybridized to a second nucleic
acid immobilized on a support, then the first nucleic acid can also
be considered to be immobilized to the support during
amplification, if amplification conditions are such that
substantial amounts of the first and second nucleic acids are
associated or connected with each other at any or all times during
amplification. For example the first and second nucleic acids can
be associated together by hybridization involving Watson-Crick base
pairing or hydrogen bonding. In an example, the amplification
conditions of choice allow at least 50%, 80%, 90%, 95% or 99% of
the first nucleic acid to remain hybridized with the second nucleic
acid, or vice versa. A nucleic acid can be considered unimmobilized
or non-immobilized if it is not directly or indirectly attached to
or associated with a support.
[0015] A medium can be considered flowable under conditions of
choice if the medium is under those conditions at least temporarily
a fluid medium that does not substantially or completely restrain
or impede transfer or movement of an unimmobilized molecule. The
unimmobilized molecule is not itself immobilized to a solid support
or surface or associated with another immobilized molecule. In an
embodiment, the unimmobilized molecule is a solute (e.g., a nucleic
acid) through the flowable medium. Exemplary transfer or movement
in the medium can be by means of diffusion, convection, turbulence,
agitation, Brownian motion, advection, current flows, or other
molecular movements within the liquid) from any first point in the
continuous phase to any other point in fluid communication or in
the same continuous phase. For example, in a flowable medium a
significant amount of an unimmobilized nucleic acid is transferred
from one immobilization site to another immobilization site that is
within the same continuous phase of the flowable medium, or in
fluid communication with the first immobilization site. Optionally,
the rate of transfer or movement of the nucleic acid in the medium
is comparable to the rate of transfer or movement of the nucleic
acid in water. In some instances, the conditions of choice are
conditions that the medium is subjected to during amplification.
The conditions of choice may or may not allow the flowable medium
to remain substantially motionless. The conditions may or may not
subject the flowable medium to active mixing, agitation or shaking.
The medium is optionally flowable at least temporarily during
amplification. For example the medium is flowable under at least
one preamplification and/or amplification condition of choice.
Optionally, a flowable medium does not substantially prevent
intermingling of different unimmobilized nucleic acids or transfer
of an unimmobilized nucleic acid between different zones of a
continuous phase of the flowable medium. The movement or transfer
of nucleic acids for example can be caused by means of diffusion or
convection. A medium is optionally considered nonflowable if
unimmobilized nucleic acids upon amplification fail to spread or
move between different immobilization sites or over the entire
continuous phase. Generally, a flowable medium does not
substantially confine unimmobilized nucleic acids (e.g., the
templates or amplicons) within limited zones of the reaction volume
or at fixed locations during the period of amplification.
Optionally, a flowable medium can be rendered non-flowable by
various means or by varying its conditions. Optionally, a medium is
flowable if it is liquid or is not semisolid. A medium can be
considered flowable if its fluidity is comparable to pure water. In
other embodiments, a medium can be considered flowable if it a
fluid that is substantially free of polymers, or if its viscosity
coefficient is similar to that of pure water.
[0016] In an embodiment, a support comprises one or more
immobilization sites. A nucleic acid (e.g., a template or an
amplicon) optionally associates with the support at its
corresponding immobilization site. An immobilization site
optionally comprises a specific portion or area of a support, or a
position or location on a support. An immobilization site can have
or optionally can lack any specific or predetermined location,
position, dimension, size, area or structure on the support, or any
other distinguishing parameter or characteristic. Optionally, one
or more of such parameters are determined for an immobilization
site at the time of association of the template with the support,
and/or during or at the completion of amplification. In an
embodiment, one or template-attachment moieties (e.g.,
amplification primers) are attached to the support, which
optionally are or are not arranged on the support at pre-determined
or defined positions or angles or distances from the center of a
defined point. Optionally, an arrangement of template molecules
over one or more immobilization sites on a support is or is not
achieved through an intentional design or placement of individual
attachment moieties. Such a "randomly-patterned" or "random" array
of attachment moieties (e.g., primers) can be achieved by dropping,
spraying, plating or spreading a solution, emulsion, aerosol, vapor
or dry preparation comprising a pool of nucleic acid molecules onto
a support and allowing the nucleic acid molecules to settle onto
the support, with or without any intervention that would direct
them to specific or predetermined immobilization sites thereon.
[0017] A support or immobilization site optionally comprises one or
more oligonucleotides (e.g., amplification primers) attached to a
support. Preferably but not necessarily, the attachment of the
oligonucleotide to the support or immobilization site is stable
during subsequent amplification or other assays (e.g., more than
10%, 30%, 50%, 70% or 90% of oligonucleotides remain attached after
being subjected to the amplification conditions of choice).
[0018] Optionally, in any method described herein, all primers on
at least one support or immobilization site comprise the same
sequence. The support or immobilization site can optionally
comprise other nucleic acids which do not hybridize to one strand
of the template of interest or its complement. The support or
immobilization site optionally does not comprise any other nucleic
acid which hybridizes to one strand of the template of interest or
its complement (i.e., the immobilization site optionally lacks any
other primers). Optionally, a support or immobilization site
comprises a plurality of primers having at least two different
sequences. Optionally, the support or immobilization site comprises
a species of immobilized primers that is complementary to a first
portion of a single-stranded template, and does not comprise an
immobilized primer that is homologous to a second non-overlapping
portion of the template (or can hybridize to the
template-complement). The two portions are non-overlapping if they
do not contain any subportions that hybridize to each other or to a
complement of the other portion.
[0019] Two or more different types of primer (e.g., different in
sequence) can be present in substantially the same concentrations
as one another, or alternatively in different concentrations.
Optionally, the primers are substantially homogeneously dispersed
over the support or immobilization site. Optionally, in any method
described herein, two different immobilization sites are spatially
separated subcomponents (e.g., portions or areas) of a single
support and/or are on different (e.g., structurally disconnected)
supports. Optionally, in any method described herein, at least one
immobilization site includes the entire surface of the support or
the entire volume of a support. The immobilized primers are
optionally uniformly distributed over the one or more supports or
immobilization sites.
[0020] Optionally, in any method described herein, the two
different immobilization sites are located in a predetermined
arrangement in or on a shared support (e.g. in a grid pattern). In
other embodiments, the metes, bounds or positioning of one or more
immobilization sites is not known or predetermined before
immobilization of the template to the support. In an example, the
support comprises multiple immobilized primers, and the primer to
which a starting template hybridizes (e.g., before any
amplification occurs) can be considered to be included within
(e.g., a central point of) the immobilization site for that
template. In such an embodiment, the positioning of an
immobilization site is determined during or after hybridization of
the primer to the template. The metes and bounds of an
immobilization site can also be determined during or after
extension and/or amplification.
[0021] A population of nucleic acids is considered clonal if a
substantial portion of its members have substantially identical (or
substantially complementary) sequence. It will be understood that
members of a population need not be 100% identical or
complementary, e.g., a certain number of "errors" may occur during
the course of synthesis. In an embodiment, at least 50% of the
members of a population are substantially identical (or
complementary) to each other or to a reference nucleic acid
molecule (i.e., a molecule of defined sequence used as a basis for
a sequence comparison). More preferably at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 99%, or more of
the members of a population are substantially identical (or
complementary) to the reference nucleic acid molecule. Two
molecules can be considered substantially identical if the percent
identity between the two molecules is at least 75%, 80%, 85%, 90%,
95%, 98%, 99%, 99.9% or greater, when optimally aligned. Two
molecules can be considered substantially complementary if the
percent complementarity between the two molecules is at least 75%,
80%, 85%, 90%, 95%, 98%, 99%, 99.9% or greater, when optimally
aligned. In addition, a low or insubstantial level of mixing of
non-homologous nucleic acids may occur during methods described
herein, and thus a clonal population may contain a minority of
diverse nucleic acids (e.g., less than 30%, e.g., less than
10%).
[0022] As will be appreciated by one of ordinary skill in the art,
references to templates, initializing oligonucleotides, extension
probes, primers, etc., can refer to populations or pools of nucleic
acid molecules that are substantially identical within a relevant
portion, rather than single molecules. For example, a "template"
can refer to a plurality of substantially identical template
molecules; a "probe" can refer to a plurality of substantially
identical probe molecules, etc. In the case of probes that are
degenerate at one or more positions, it will be appreciated that
the sequence of the probe molecules that comprise a particular
probe will differ at the degenerate positions, i.e., the sequences
of the probe molecules that constitute a particular probe may be
substantially identical only at the nondegenerate position(s).
These terms within this application are intended to provide support
for either a population or a molecule. Where it is intended to
refer to a single nucleic acid molecule (i.e., one molecule), the
terms "template molecule", "probe molecule", "primer molecule",
etc., may be used instead. In certain instances the plural nature
of a population of substantially identical nucleic acid molecules
will be explicitly indicated.
[0023] "Template", "oligonucleotide", "probe", "primer",
"template", "nucleic acid" and the like are intended to be
interchangeable terms herein. These terms refer to polynucleotides,
not necessarily limited to any length or function. The same nucleic
acid can be regarded as a "template", "probe" or "primer" depending
on the context, and can switch between these roles with time. A
"polynucleotide," also called a "nucleic acid," is a linear polymer
of two or more nucleotides joined by covalent internucleosidic
linkages, or variant or functional fragments thereof. In naturally
occurring examples of these, the internucleoside linkage is
typically a phosphodiester bond. However, other examples optionally
comprise other internucleoside linkages, such as phosphorothiolate
linkages and may or may not comprise a phosphate group.
Polynucleotides include double- and single-stranded DNA, as well as
double- and single-stranded RNA, DNA:RNA hybrids, peptide-nucleic
acids (PNAs) and hybrids between PNAs and DNA or RNA, and also
include known types of modifications. Polynucleotides can
optionally be attached to one or more non-nucleotide moieties such
as labels and other small molecules, large molecules such proteins,
lipids, sugars, and solid or semi-solid supports, for example
through either the 5' or 3' end. Labels include any moiety that is
detectable using a detection method of choice, and thus renders the
attached nucleotide or polynucleotide similarly detectable using a
detection method of choice. Optionally, the label emits
electromagnetic radiation that is optically detectable or visible.
In some cases, the nucleotide or polynucleotide is not attached to
a label, and the presence of the nucleotide or polynucleotide is
directly detected. A "nucleotide" refers to a nucleotide,
nucleoside or analog thereof. Optionally, the nucleotide is an N-
or C-glycoside of a purine or pyrimidine base. (e.g.,
deoxyribonucleoside containing 2-deoxy-D-ribose or ribonucleoside
containing D-ribose). Examples of other analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides. Referring to a nucleic acid by any one of these
terms should not be taken as implying that the nucleic acid has any
particular activity, function or properties. For example, the word
"template" does not indicate that the "template" is being copied by
a polymerase or that the template is not capable of acting as a
"primer" or a "probe".
[0024] It will be appreciated that in certain instances nucleic
acid reagents involved in amplification such as a template, probe,
primer, etc., may be a portion of a larger nucleic acid molecule
that also contains another portion that does not serve the same
function. Optionally, this other portion does not serve any
template, probe, or primer function. In some instances, a nucleic
acid that substantially hybridizes to an optionally-immobilized
primer (e.g., on an immobilization site) is considered to be the
"template". Any one or more nucleic acid reagents that are involved
in template walking (template, immobilized strands, immobilized or
unimmobilized primer, etc.) may be generated before or during
amplification from other nucleic acids. The nucleic acid reagent is
optionally generated from (and need not be identical to) an input
nucleic acid by making one or more modifications to the nucleic
acid that was initially introduced into the template walking
medium. An input nucleic acid can for example be subjected to
restriction digestion, ligation, one or more amplification cycles,
denaturation, mutation, etc, to generate a nucleic acid that serves
as the template, primer, etc, during amplification or further
amplification. For example, a double-stranded input nucleic acid
can be denatured to generate a first single-stranded nucleic acid
which optionally is used to generate a second complementary strand.
If so desired, the first single-stranded nucleic acid can be
considered the "template" for our purposes herein. Alternatively,
the second complementary strand generated from the first
single-stranded nucleic acid can be considered the "template" for
our purposes herein. In another example, a template is derived from
an input nucleic acid and is not necessarily identical to the input
nucleic acid. For example, the template can comprise additional
sequence not present an input nucleic acid. In an embodiment the
template can be an amplicon generated from an input nucleic acid
using one or more primers with a 5' overhang that is not
complementary to the input nucleic acid.
[0025] The term "amplifying" refers to production of copies of a
nucleic acid molecule, for example via repeated rounds of primed
enzymatic synthesis. Optionally, such amplifying takes place with
an immobilized template nucleic acid molecule and/or one or more
primers that are immobilized. An amplicon is for example a
single-stranded or double-stranded nucleic acid that is generated
by an amplification procedure from a starting template nucleic
acid. The amplicon comprises a nucleic acid strand, of which at
least a portion is substantially identical or substantially
complementary to at least a portion of the starting template. Where
the starting template is double-stranded, an amplicon comprises a
nucleic acid strand that is substantially identical to at least a
portion of one strand and is substantially complementary to at
least a portion of either strand. The amplicon can be
single-stranded or double-stranded irrespective of whether the
initial template is single-stranded or double-stranded.
[0026] The term "support" includes any solid or semisolid article
on which reagents such as nucleic acids can be immobilized. Nucleic
acids may be immobilized on the solid support by any method
including but not limited to physical adsorption, by ionic or
covalent bond formation, or combinations thereof. A solid support
may include a polymeric, a glass, or a metallic material. Examples
of solid supports include a membrane, a planar surface, a
microtiter plate, a bead, a filter, a test strip, a slide, a cover
slip, and a test tube, means any solid phase material upon which a
oligomer is synthesized, attached, ligated or otherwise
immobilized. A support can optionally comprise a "resin", "phase",
"surface" and "support". A support may be composed of organic
polymers such as polystyrene, polyethylene, polypropylene,
polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as
co-polymers and grafts thereof. A support may also be inorganic,
such as glass, silica, controlled-pore-glass (CPG), or
reverse-phase silica. The configuration of a support may be in the
form of beads, spheres, particles, granules, a gel, or a surface.
Surfaces may be planar, substantially planar, or non-planar.
Supports may be porous or non-porous, and may have swelling or
non-swelling characteristics. A support can be shaped to comprise
one or more wells, depressions or other containers, vessels,
features or locations. A plurality of supports may be configured in
an array at various locations. A support is optionally addressable
(e.g., for robotic delivery of reagents), or by detection means
including scanning by laser illumination and confocal or deflective
light gathering. An amplification support (e.g., a bead) can be
placed within or on another support (e.g., within a well of a
second support).
[0027] In an embodiment the solid support is a "microparticle,"
"bead" "microbead", etc., (optionally but not necessarily spherical
in shape) having a smallest cross-sectional length (e.g., diameter)
of 50 microns or less, preferably 10 microns or less, 3 microns or
less, approximately 1 micron or less, approximately 0.5 microns or
less, e.g., approximately 0.1, 0.2, 0.3, or 0.4 microns, or smaller
(e.g., under 1 nanometer, about 1-10 nanometer, about 10-100
nanometers, or about 100-500 nanometers). Microparticles (e.g.,
Dynabeads from Dynal, Oslo, Norway) may be made of a variety of
inorganic or organic materials including, but not limited to, glass
(e.g., controlled pore glass), silica, zirconia, cross-linked
polystyrene, polyacrylate, polymehtymethacrylate, titanium dioxide,
latex, polystyrene, etc. Magnetization can facilitate collection
and concentration of the microparticle-attached reagents (e.g.,
polynucleotides or ligases) after amplification, and can also
facilitate additional steps (e.g., washes, reagent removal, etc.).
In certain embodiments of the invention a population of
microparticles having different shapes sizes and/or colors can be
used. The microparticles can optionally be encoded, e.g., with
quantum dots such that each microparticle can be individually or
uniquely identified.
DETAILED DESCRIPTION
[0028] In some embodiments, the disclosure relates generally to
methods, compositions, systems, apparatuses and kits for clonally
amplifying one or more nucleic acid templates to form clonally
amplified populations of nucleic acid templates. Any amplification
method described herein optionally comprises repeated cycles of
nucleic acid amplification. A cycle of amplification optionally
comprises (a) hybridization of primer to a template strand, (b)
primer extension to form a first extended strand, (c) partial or
incomplete denaturation of the extended strand from the template
strand. The primer that hybridizes to the template strand
(designated "forward" primer for convenience) is optionally
immobilized on or to a support. The support is for example solid or
semi-solid. Optionally, the denatured portion of the template
strand from step (c) is free to hybridize with a different forward
primer in the next amplification cycle. In an embodiment, primer
extension in a subsequent amplification cycle involves displacement
of the first extended strand from the template strand. A second
"reverse" primer can for example be included which hybridizes to
the 3' end of the first extended strand. The reverse primer is
optionally not immobilized.
[0029] In an embodiment, the templates are amplified using primers
immobilized on/to one or more solid or semi-solid supports.
Optionally the support comprises immobilized primers that are
complementary to a first portion of a template strand. Optionally,
the support does not significantly comprise an immobilized primer
that is homologous to a second non-overlapping portion of the same
template strand. The two portions are non-overlapping if they do
not contain any subportions that hybridize to each other or to a
complement thereof. In another example, the support optionally does
not significantly comprise an immobilized primer that can hybridize
to the complement of the template strand).
[0030] Optionally, a plurality of nucleic acid templates are
amplified simultaneously in a single continuous liquid phase in the
presence of one or more supports, where each support comprises one
or more immobilization sites. In an embodiment, each template is
amplified to generate a clonal population of amplicons, where
individual clonal populations are immobilized within or on a
different support or immobilization site from other amplified
populations. Optionally, the amplified populations remain
substantially clonal after amplification.
[0031] A template is for example amplified to generate clonal
populations which comprise template-homologous strands (called
"template strands" or "reverse strands" herein) and/or
template-complementary strands (called "primer strands" or "forward
strands" herein). In an embodiment clonality is maintained in the
resulting amplified nucleic acid populations by maintaining
association between template strands and its primer strands,
thereby effectively associating or "tethering" associated clonal
progeny together and reducing the probability of
cross-contamination between different clonal populations.
Optionally, one or more amplified nucleic acids in the clonal
population is attached to a support. A clonal population of
substantially identical nucleic acids can optionally have a
spatially localized or discrete macroscopic appearance. In an
embodiment a clonal population can resemble a distinct spot or
colony (e.g., when distributed in a support, optionally on the
outer surface of the support).
[0032] In some embodiments, the disclosure relates generally to
novel methods of generating a localized clonal population of clonal
amplicons, optionally immobilized in/to/on one or more supports.
The support can for example be solid or semisolid (such as a gel or
hydrogel). The amplified clonal population is optionally attached
to the support's external surface or can also be within the
internal surfaces of a support (e.g., where the support has a
porous or matrix structure).
[0033] In some embodiments, amplification is achieved by multiple
cycles of primer extension along a template strand of interest
(also called a "reverse" strand). For convenience, a primer that
hybridizes to the template strand of interest is termed a "forward"
primer, and is optionally extended in template-dependent fashion to
form a "forward" strand that is complementary to the template
strand of interest. In some methods, the forward strand is itself
hybridized by a second primer termed the "reverse" primer, which is
extended to form a new template strand (also called a reverse
strand). Optionally, at least a portion of the new template strand
is homologous to the original template ("reverse") strand of
interest.
[0034] As mentioned, one or more primers can be immobilized
in/on/to one or more supports. Optionally, one primer is
immobilized by attachment to a support. A second primer can be
present and is optionally not immobilized or attached to a support.
Different templates can for example be amplified onto different
supports or immobilization sites simultaneously in a single
continuous liquid phase to form clonal nucleic acid populations. A
liquid phase can be considered continuous if any portion of the
liquid phase is in fluid contact or communication with any other
portion of the liquid body. In another example, a liquid phase can
be considered continuous if no portion is entirely subdivided or
compartmentalized or otherwise entirely physically separated from
the rest of the liquid body. Optionally, the liquid phase is
flowable. Optionally, the continuous liquid phase is not within a
gel or matrix. In other embodiments, the continuous liquid phase is
within a gel or matrix. For example the continuous liquid phase
occupies pores, spaces or other interstices of a solid or semisolid
support.
[0035] Where the liquid phase is within a gel or matrix, one or
more primers are optionally immobilized on a support. Optionally
the support is the gel or matrix itself. Alternatively the support
is not the gel or matrix itself. In an example one primer is
immobilized on a solid support contained within a gel and is not
immobilized to gel molecules. The support is for example in the
form of a planar surface or one or more microparticles. Optionally
the planar surface or plurality of microparticles comprises forward
primers having substantially identical sequence. In an embodiment,
the support does not contain significant amounts of a second
different primer. Optionally, a second non-immobilized primer is in
solution within the gel. The second non-immobilized primer for
example binds to a template strand (i.e., reverse strand), whereas
the immobilized primer binds to a forward strand.
[0036] For convenience, the portion of a nucleic acid template
strand that is hybridized by a primer will be referred to as the
"primer-binding sequence" or PBS. Thus, a forward primer binds to a
forward-primer binding sequence ("forward PBS") on a reverse
strand, while a reverse primer binds to a reverse PBS on the
forward strand.
[0037] An embodiment includes a method of primer extension,
comprising: (a) a primer-hybridization step, (b) an extension step,
and (c) a walking step. Optionally, the primer-hybridization step
comprises hybridizing a first primer molecule ("first forward
primer") to a complementary forward-primer-binding sequence
("forward PBS") on a nucleic acid strand ("reverse strand").
Optionally the extension step comprises generating an extended
first forward strand that is a full-length complement of the
reverse strand and is hybridized thereto. The extended first
forward strand is for example generated by extending the first
forward primer molecule in template-dependent fashion using the
reverse strand as template. Optionally the walking step comprises
hybridizing a second primer ("second forward primer") to the
forward PBS where the reverse strand is also hybridized to the
first forward strand. For example, the walking step comprises
denaturing at least a portion of the forward PBS from the forward
strand ("free portion"), where another portion of the reverse
strand remains hybridized to the forward strand.
[0038] In an embodiment, the primer extension method is an
amplification method, in which any one or more steps of
primer-hybridization, extension and/or walking are repeated at
least once. For example, the method can comprise amplifying the
forward strand by one or more amplification cycles. An
amplification cycle optionally comprises extension and walking. An
exemplary amplification cycle comprises or consists essentially of
extension followed by walking. Optionally, the second forward
primer of a first amplification cycle acts as the first forward
primer of a subsequent amplification cycle. For example, the second
forward primer of a walking step in a first amplification cycle
acts as the first forward primer of an extension step of a
subsequent amplification cycle.
[0039] Optionally, the method of primer extension or amplification
further comprises extending or amplifying the reverse strand by (a)
hybridizing a first reverse primer molecule to a complementary
reverse-primer-binding sequence ("reverse PBS") on an extended
forward strand; (b) generating an extended first reverse strand
that is a full-length complement of the forward strand and
hybridized thereto, by extending the first reverse primer molecule
in template-dependent fashion using the forward strand as template;
and (c) hybridizing a second primer ("second reverse primer") to
the reverse PBS where the forward strand is also hybridized to the
first reverse strand. One or more repetitions of steps (b)-(c) are
optionally performed, wherein the second reverse primer of step (c)
is the first reverse primer of repeated step (b); and wherein a
substantial proportion of forward strands are hybridized to reverse
strands at all times during or between said one or more
repetitions. In embodiments, the substantial proportion is
optionally at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or 99%.
[0040] Optionally, during amplification the reverse strand and/or
forward strand is not exposed to totally-denaturing conditions that
would result in complete separation of a significant fraction
(e.g., more than 10%, 20%, 30%, 40% or 50%) of a large plurality of
strands from their extended and/or full-length complements.
[0041] In an embodiment a substantial proportion of forward and/or
reverse strands are optionally hybridized to extended and/or
full-length complements at all times during or between one or more
amplification cycles (e.g., 1, 5, 10, 20, or all amplification
cycles performed). In embodiments, the substantial proportion of
strands is optionally at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or 99% of strands. In an embodiment this is achieved by
maintaining the amplification reaction at a temperature higher than
the T.sub.m of unextended primers, but lower than the T.sub.m of
the primer-complementary strands. For example, amplification
conditions are kept within a temperature that is higher than the
T.sub.m of unextended forward primers, but lower than the T.sub.m
of extended or full-length reverse strands. Also for example,
amplification conditions are kept within a temperature that is
higher than the T.sub.m of unextended reverse primers, but lower
than the T.sub.m of extended or full-length forward strands.
[0042] Optionally, one or more forward primers, and/or one or more
reverse primers are breathable, e.g., have a low T.sub.m. In an
example at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of
nucleotide bases of a breathable primer are adenine, thymine or
uracil or are complementary to adenine, thymine or uracil.
[0043] The T.sub.m of a nucleic acid strand (e.g., a primer or
template strand) is for example the temperature at which at least a
desired fraction of a clonal population of duplexes are rendered
completely single-stranded under the chosen reagent conditions,
where an individual duplex comprises the nucleic acid strand in
question hybridized to its full-length complement. By default, the
desired fraction is 50%. In embodiments, the desired fraction is
optionally at least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or 99%. In an embodiment the T.sub.m(N) is
theoretically predicted using known methods, e.g., as discussed
herein. In another embodiment the T.sub.m is empirically measured
by known methods. (e.g., Spink, Methods Cell Biol. 2008; 84:115-41;
Meunier-Prest et al., Nucleic Acids Res. 2003 Dec. 1; 31(23): e150;
Brewood et al., Nucleic Acids Res. 2008 September; 36(15):
e98.)
[0044] The T.sub.m for a desired fraction can be depicted as
T.sub.m(N) where N denotes the desired fraction in percentage
terms. In an embodiment the T.sub.m(50) of the forward and/or
reverse primer (at which 50% of all primer molecules are completely
dissociated from their complementary PBSs) is not more than
50.degree. C., 55.degree. C., 60.degree. C., 65.degree. C.,
70.degree. C., 75.degree. C. or 80.degree. C. Optionally, the
T.sub.m(50) of the extended or full-length forward and/or reverse
strands (at which 50% of all strand molecules are completely
dissociated from their complementary sequences) is not less than
80.degree. C., 75.degree. C., 70.degree. C., 65.degree. C.,
60.degree. C. or 55.degree. C. In another embodiment the
T.sub.m(80) of the forward and/or reverse primer is not more than
50.degree. C., 55.degree. C., 60.degree. C., 65.degree. C.,
70.degree. C., 75.degree. C. or 80.degree. C. Optionally, the
T.sub.m(80) of the extended or full-length forward and/or reverse
strands is not less than 80.degree. C., 75.degree. C., 70.degree.
C., 65.degree. C., 60.degree. C. or 55.degree. C. In another
embodiment the T.sub.m(90) of the forward and/or reverse primer is
not more than 50.degree. C., 55.degree. C., 60.degree. C.,
65.degree. C., 70.degree. C., 75.degree. C. or 80.degree. C.
Optionally, the T.sub.m(90) of the extended or full-length forward
and/or reverse strands is not less than 80.degree. C., 75.degree.
C., 70.degree. C., 65.degree. C., 60.degree. C. or 55.degree. C. In
another embodiment the T.sub.m(95) of the forward and/or reverse
primer is not more than 50.degree. C., 55.degree. C., 60.degree.
C., 65.degree. C., 70.degree. C., 75.degree. C. or 80.degree. C.
Optionally, the T.sub.m(95) of the extended or full-length forward
and/or reverse strands is not less than 80.degree. C., 75.degree.
C., 70.degree. C., 65.degree. C., 60.degree. C. or 55.degree. C.
Optionally, the T.sub.m(99) of the extended or full-length forward
and/or reverse strands is not less than 80.degree. C., 75.degree.
C., 70.degree. C., 65.degree. C., 60.degree. C. or 55.degree. C. In
another embodiment the T.sub.m(95) of the forward and/or reverse
primer is not more than 50.degree. C., 55.degree. C., 60.degree.
C., 65.degree. C., 70.degree. C., 75.degree. C. or 80.degree. C.
Optionally, the T.sub.m(99) of the extended or full-length forward
and/or reverse strands is not less than 80.degree. C., 75.degree.
C., 70.degree. C., 65.degree. C., 60.degree. C. or 55.degree.
C.
[0045] Optionally, one or more amplification cycles (e.g., 1, 5,
10, 20, or substantially all amplification cycles) are performed at
temperature that is higher than the T.sub.m(70) of an unextended
primer and lower than the T.sub.m(20) of the complementary
full-length strand. For example, the temperature is higher than the
T.sub.m(80) of an unextended primer and lower than the T.sub.m(20)
or T.sub.m(15) or T.sub.m(10) or T.sub.m(5) or T.sub.m(1) of the
complementary full-length strand. Also for example the temperature
is higher than the T.sub.m(90) of an unextended primer and lower
than the T.sub.m(20) or T.sub.m(15) or T.sub.m(10) or T.sub.m(5) or
T.sub.m(1) of the complementary full-length strand. Also for
example the temperature is higher than the T.sub.m(95) of an
unextended primer and lower than the T.sub.m(20) or T.sub.m(15) or
T.sub.m(10) or T.sub.m(5) or T.sub.m(1) of the complementary
full-length strand. Also for example the temperature is higher than
the T.sub.m(98) of an unextended primer and lower than the
T.sub.m(20) or T.sub.m(15) or T.sub.m(10) or T.sub.m(5) or
T.sub.m(1) of the complementary full-length strand. Also for
example the temperature is higher than the T.sub.m(99) of an
unextended primer and lower than the T.sub.m(20) or T.sub.m(15) or
T.sub.m(10) or T.sub.m(5) or T.sub.m(1) of the complementary
full-length strand. Optionally, the one or more amplification
cycles are performed at temperature that is at least 5, 10, 15, 20,
25, 30, 35, or 45.degree. C. higher than the T.sub.m(50) of an
unextended primer. Optionally, the temperature is at least 5, 10,
15, 20, 25, 30, 35, or 45.degree. C. lower than the T.sub.m(50) of
a full-length primer-complementary strand. In an embodiment the
unextended primer is a forward primer and the complementary
full-length strand is a reverse strand, or vice versa.
[0046] Optionally, template-dependent extension of a forward primer
using a reverse strand as template in the extension step results in
displacement of another forward strand that was already hybridized
to the reverse strand. Optionally, template-dependent extension of
a reverse primer using a forward strand as template in the
extension step results in displacement of another reverse strand
that was already hybridized to the forward strand.
[0047] In an embodiment the method further comprises completely
separating the extended forward strands from reverse strands after
performing primer extension or a desired number of amplification
cycles, and optionally removing separated forward strands from the
presence of separated immobilized reverse strands, or vice
versa.
[0048] Optionally, one or more nucleic acid reagents are not in
contact with a recombinase and/or reverse transcriptase and/or
helicase and/or nicking enzyme and/or any other enzyme that is not
a polymerase during any one or more steps. For example the primers
and/or template are not in contact with one or more such enzymes at
any time.
[0049] Denaturation is optionally achieved non-enzymatically, e.g.,
by raising the temperature. In an embodiment amplification is
performed under substantially isothermal conditions, as described
herein.
[0050] Optionally, any one or more nucleic acids, e.g., primers are
attached (e.g., covalently attached) to a support. In an embodiment
the first and/or second forward primers are immobilized to a single
(same) support.
[0051] In an embodiment, first and second forward primers are
closely immobilized to the same support, whereby amplification
generates an immobilized clonal population of extended forward
strands. Optionally, the distance between the first and second
forward primers is not more than twice the length of the primer or
the PBS.
[0052] Optionally, a plurality of template nucleic acids are
individually hybridized to spatially-separated immobilization
sites, whereby amplification generates spatially-separated clonal
populations corresponding to individual template nucleic acids.
[0053] In an embodiment, first forward primer is hybridized to a
forward PBS on a "reverse" template strand. Optionally, the first
forward primer is immobilized on a support. The first forward
primer can be extended along the reverse strand to form (an
extended) first forward strand. The extension is optionally
template-dependent, using the reverse strand as a template. After
extension, the first forward strand and the reverse strand are
optionally hybridized to each other in a duplex. Optionally, at
least part of the PBS of the first reverse strand and the
forward-primer portion of the first forward strand are separated
from one another (e.g., via denaturation or melting), but the first
reverse strand and the first forward strand remain associated with
(e.g., hybridized to) each other over another portion. The
separated portion of the reverse strand including at least part of
the forward-PBS can then be annealed (e.g., by hybridization) to a
second, different forward primer. Optionally, the second forward
primer is immobilized on a support. The second forward primer can
for example be immobilized on the same support as the first forward
primer, and is optionally situated sufficiently close to the first
primer so that the a portion of the reverse strand can hybridize to
the first forward strand while another portion of the reverse
strand is hybridized to the second forward primer simultaneously.
The second forward primer can then be extended in turn to form an
extended second forward strand. Optionally, the second forward
primer is extended along the reverse strand in a template-dependent
fashion. Extension of the second forward primer optionally
displaces the first forward strand from the reverse strand. As
before, the primer binding sequence of the reverse strand can be
separated from the primer portion of the second forward strand,
where another portion of the reverse strand remains associated
(e.g., by hybridization) with the second forward strand. These
steps can be repeated to with further forward primers to form
further extended forward strands (for example, extended third,
fourth, fifth, sixth, seventh, eighth, ninth, tenth or higher order
extended strands). The process can optionally be repeated for a
desired number of amplification cycles to provide a population of
amplified, immobilized nucleic acid molecules. The population can
be substantially clonal in nature. For example, the amplified
nucleic acid molecules of the amplified clonal population can
include a plurality of nucleic acids that are substantially
identical and/or substantially complementary to each other.
[0054] Optionally, the extended forward strands comprise a
reverse-primer-binding sequence ("reverse PBS"), to which a reverse
primer hybridizes. The reverse PBS on the forward strand optionally
comprises or is near the 3' end of the forward strands. In some
embodiments, amplification involves dissociating least part of the
reverse PBS from any hybridized or associated sequence, such as
sequence on a reverse strand. The reverse PBS on the forward strand
is optionally contacted with a reverse primer which hybridizes to
it. The reverse primer is then extended using the forward strand as
template to form an extended reverse strand. Optionally, the
newly-generated reverse strand acts as template for forward primer
extension. The reverse strand can also participate as template,
primer, or probe in another reaction, including any method
described herein.
[0055] The amplified nucleic acid populations can be used for many
different purposes, including sequencing, screening, diagnosis, in
situ nucleic acid synthesis, monitoring gene expression, nucleic
acid fingerprinting, etc.
[0056] Optionally, any one or more primer extension and/or
amplification methods herein generate one or more immobilized
nucleic-acid extension products. In a variation, a solid support
comprises primers that all identical or substantially identical.
The solid support can comprise other nucleic acids. Optionally,
these other nucleic acids do not hybridize to a template strand of
interest or its complement. The solid support optionally does not
comprise any other nucleic acid which hybridizes to the template
strand of interest or its complement.
[0057] In some embodiments, the disclosure relates generally to
methods, compositions, systems, apparatuses and kits for clonally
amplifying a nucleic acid template onto a support in an
amplification reaction solution. Optionally, the nucleic acid
template is contacted with a support in a solution comprising a
continuous liquid phase. The support can include a population of
primers, including at least a first primer and a second primer. The
population of primers can be immobilized on the support, for
example by covalent attachment to the support. In some embodiments,
the nucleic acid template includes a primer binding sequence
adjacent to a target sequence. The primer binding sequence can by
complementary to a sequence of the first primer and optionally a
sequence of the second primer. The target sequence can be
noncomplementary to the primers in the population. In some
embodiments, the primer binding sequence of the nucleic acid
template is hybridized to the first primer. The first primer can be
extended along the template using a polymerase, thereby forming an
extended first primer. At least a portion of the primer binding
sequence of the template can be separated (e.g., denatured or
melted) from the extended first primer. The separating is
optionally performed while maintaining hybridization between a
portion of the template and the extended first primer. The
separated portion of the primer binding sequence can be
subsequently hybridized to the second primer. Optionally, such
hybridization is performed while maintaining hybridization between
the other portion of the template and the extended first primer.
The second primer can be extended along the template using a
polymerase, thereby forming a support including an extended first
primer and an extended second primer. The extended portion of the
extended first primer and/or the extended second primer can include
sequence complementary to the target sequence.
[0058] In some embodiments, the disclosure relates generally to
methods for clonally amplifying a nucleic acid template onto a
support in an amplification reaction solution, comprising:
contacting a nucleic acid template with a support in a liquid
solution, wherein the support includes a population of immobilized
primers including at least a first primer and a second primer, and
wherein the nucleic acid template includes a primer binding
sequence adjacent to a target sequence, where the primer binding
sequence is complementary to a sequence of the first primer and a
sequence of the second primer, and the target sequence is
noncomplementary to the primers in the population; hybridizing the
primer binding sequence of the nucleic acid template to the first
primer; extending the first primer along the template using a
polymerase, thereby forming an extended first primer; denaturing at
least a portion of the primer binding sequence of the template from
the extended first primer while maintaining hybridization between
another portion of the template and the extended first primer;
hybridizing the denatured portion of the primer binding sequence to
the second primer while maintaining hybridization between the other
portion of the template and the extended first primer; and
extending the second primer along the template using a polymerase,
thereby forming a support including an extended first primer and an
extended second primer, where the extended first primer and the
extended second primer each include sequence complementary to the
target sequence. The population of primers can be comprised of
substantially identical primers that differ in sequence by no more
than one, two, three, four or five nucleotides. In some
embodiments, the primer population is comprised of different
primers, at least some of which include a sequence that is
complementary to the primer binding sequence of the template. In
some embodiments, the primers of population are noncomplementary to
the sequence of the 5' terminal half of the template. In some
embodiments, the primers of the population are noncomplementary to
the sequence of the 3' terminal half of any of the extended primers
of the support. In some embodiments, the primers of the population
are noncomplementary to any sequence of the template other than the
primer binding sequence.
[0059] In some embodiments, the disclosure relates generally to
methods for clonally amplifying a population of nucleic acid
templates onto a population of supports in an amplification
reaction solution, comprising: clonally amplifying a first template
onto a first nucleic acid template onto a first support according
to any of the methods disclosed herein, and clonally amplifying a
second nucleic acid template onto a second support according to the
same method, wherein both supports are included within a single
continuous liquid phase during the amplifying.
[0060] As will be appreciated by one of ordinary skill in the art,
references to templates, initializing oligonucleotides, extension
probes, primers, etc., can in some embodiments refer to populations
or pools of nucleic acid molecules that are substantially identical
within a relevant region rather than single molecules. Thus, for
example, a "template" can in some embodiments refer to a plurality
of substantially identical templates; a "probe" can in some
embodiments refer to a plurality of substantially identical probe
molecules, etc. In the case of probes that are degenerate at one or
more positions, it will be appreciated that the sequence of the
probe molecules that comprise a particular probe will differ at the
degenerate positions, i.e., the sequences of the probe molecules
that constitute a particular probe may be substantially identical
only at the nondegenerate position(s). For purposes of description
the singular form can be used to refer not only to single molecules
but also to populations of substantially identical molecules. In
certain instances the singular nature of a nucleic acid molecule,
or the plural nature of a population of substantially identical
nucleic acid molecules, will be explicitly indicated.
[0061] It will be understood that members of a population need not
be 100% identical. For example, all members of a clonally amplified
population of nucleic acid sequence need not be identical since a
certain number of "errors" may occur during the course of
synthesis; similarly, not all primers within a population of
primers may be identical to each other. In some embodiments, at
least 50% of the members of a population are identical to a
reference nucleic acid molecule (i.e., a molecule of defined
sequence used as a basis for a sequence comparison). In some
embodiments, at least 50% of the members of a population are at
least 70%, 75%, 85%, 90%, or more preferably at least 95% identical
to a reference nucleic acid molecule. More preferably at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, at least
99%, or more of the members of a population are at least 85%, 90%,
or more preferably at least 95% identical, or yet more preferably
at least 99% identical to the reference nucleic acid molecule.
Preferably the percent identity of at least 95% or more preferably
at least 99% of the members of the population to a reference
nucleic acid molecule is at least 98%, 99%, 99.9% or greater.
Percent identity may be computed by comparing two optimally aligned
sequences, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in
both sequences to yield the number of matched positions, dividing
the number of matched positions by the total number of positions,
and multiplying the result by 100 to yield the percentage of
sequence identity. It will be appreciated that in certain instances
a nucleic acid molecule such as a template, probe, primer, etc.,
may be a portion of a larger nucleic acid molecule that also
contains a portion that does not serve a template, probe, or primer
function. In that case individual members of a population need not
be substantially identical with respect to that portion.
[0062] A nucleic acid optionally comprises one or more nucleotides.
In an embodiment a nucleotide comprises any one or more of a
nucleobase (nitrogenous base), a five-carbon sugar (either ribose
or 2'-deoxyribose), and a phosphate group. Optionally, a nucleotide
comprises all three components or derivatives thereof. Optionally,
the nucleobases is a purine or a pyrimidine base. Exemplary purine
bases include adenine and guanine, while exemplary pyrimidines are
thymine, uracil and cytosine
[0063] As used herein, the term "complementary" and its variants,
when used in reference to individual nucleotides, include
nucleotides that are efficiently incorporated by DNA polymerases
opposite each other during DNA replication under physiological
conditions. In an typical embodiment, complementary nucleotides can
form base pairs with each other, such as the A-T/U and G-C base
pairs formed through specific Watson-Crick type hydrogen bonding
between the nucleobases of nucleotides and/or polynucleotides
positions antiparallel to each other; other types of base pairing
can also occur. For example, the complementarity of other
artificial base pairs can be based on other types of hydrogen
bonding and/or hydrophobicity of bases and/or shape complementarity
between bases.
[0064] As used herein, the term "complementary" and its variants,
when used in reference to nucleic acid sequences, refers to nucleic
acid sequences that can undergo cumulative base pairing with each
other at two or more individual corresponding positions in
antiparallel orientation, as in a hybridized duplex. Optionally
there can be "complete" or "total" complementarity between a first
and second nucleic acid sequence where each nucleotide in the first
nucleic acid molecule or sequence can undergo a stabilizing base
pairing interaction with a nucleotide in the corresponding
antiparallel position on the second nucleic acid sequence;
alternatively, two nucleic acid sequences can be complementary when
at least 50% of the nucleotide residues of one nucleic acid
sequence are complementary to nucleotide residues in the other
nucleic acid sequence. The complementary residues within a
particular complementary nucleic acid sequence need not always be
contiguous with each other, and can be interrupted by one or more
noncomplementary residues within the complementary nucleic acid
sequence. In some embodiments, at least 50%, but less than 100%, of
the residues of one of the two complementary nucleic acid sequences
are complementary to residues in the other nucleic acid sequence.
In some embodiments, at least 70%, 80%, 90%, 95% or 99% of the
residues of one nucleic acid sequence are complementary to residues
in the other nucleic acid sequence. Sequences are said to be
"substantially complementary" when at least 85% of the residues of
one nucleic acid sequence are complementary to residues in the
other nucleic acid sequence.
[0065] As used herein, "complementary" when used in reference to
two or more nucleic acid molecules, can include any nucleic acid
molecules where each molecule comprises a sequence that is
complementary to a sequence in the other nucleic acid molecules.
The complementary nucleic acid molecules need not be complementary
across their entire length, and each molecule can include one or
more regions that is noncomplementary to the other molecules. For
example, a template and a primer molecule can be referred to as
"complementary" even when they are of different lengths; in some
embodiments, the template can be longer than the primer and include
sequence that is noncomplementary to any sequence of the primer, or
vice versa.
[0066] The term "noncomplementary" and its variants, as used herein
with reference to two nucleic acid molecules or sequences,
typically refers to nucleic acid molecules or sequences in which
less than 50% of the residues of one nucleic acid molecule or
sequence are complementary to residues in the other nucleic acid
molecule or sequence. A "mismatch" is present at any position in
the nucleic acid molecule molecules or sequences where the two
opposed nucleotides are not complementary. Similarly, two
nucleotide sequences or portions thereof are considered to match
each other if the sequences or portions are identical or
complementary to each other.
[0067] As used herein, the term "hybridization" refers to the
process of base pairing between any two nucleic acid molecules
including complementary nucleotides at one or more positions.
Typically, such base pairing can occur according to established
paradigms, for example the Watson-Crick paradigm wherein A-T/U and
G-C base pairs are formed through specific Watson-Crick type
hydrogen bonding between the nucleobases of nucleotides and/or
polynucleotides positions antiparallel to each other. In some
embodiments, hybridization can occur according to non-Watson Crick
paradigms as well; for example, artificial base pairs can be formed
through other types of hydrogen bonding and/or hydrophobicity of
bases and/or shape complementarity between bases. The hybridized
nucleic acid molecules need not be hybridized across their entire
length, and each molecule can include one or more regions not
hybridized to the other molecule. For example, a template and a
primer molecule can be described as hybridized to each other even
when a substantial region of the template, primer or both remain
non-hybridized to each other. Furthermore, a region of
hybridization can include one or more contiguous nucleotides that
are not base paired with each other. Nucleic acid molecules (or
sequences within nucleic acid molecules) that are base paired with
each other in this manner are referred to as "hybridized". In some
embodiments, a single nucleic acid molecule may undergo
self-hybridization (e.g., hairpin formation) with itself.
[0068] Typically, hybridizing nucleic acid molecules (for example,
hybridizing a primer with a template) includes contacting nucleic
acid molecules with each other under conditions where one or more
nucleotide residues within each nucleic acid molecule base pairs
with one or more nucleotides of another nucleic acid molecule. The
contacting can be performed using any suitable conditions,
depending on the desired application. In one exemplary assay, two
nucleic acid molecules are contacted in a buffered solution
comprising salts and/or detergents, e.g., SDS, for a desired length
of time and for a desired period. For example, the hybridization
can be performed using low stringency, medium stringency or high
stringency hybridization conditions. The stringency of
hybridization can be adjusted by varying various hybridization
parameters, including for example temperature, salt concentration,
SDS concentration, at the like. Methods of hybridization and of
controlling the stringency of hybridization are well known in the
field.
[0069] Among other things, a method is provided of generating a
localized clonal population of immobilized clonal amplicons of a
single-stranded template sequence, comprising: (a) attaching the
single-stranded template sequence ("template 1") to an
immobilization site ("IS1"), wherein IS1 comprises multiple copies
of an immobilized primer ("IS1 primer") which can hybridize
substantially to template 1, and template 1 is attached to IS1 by
hybridization to an IS1 primer, and (b) amplifying template 1 using
IS1 primer and a non-immobilized primer ("SP1 primer") in solution,
wherein amplified strands that are complementary to the
single-stranded template 1 cannot hybridize substantially when
single-stranded to primers on IS1, wherein amplification generates
a localized clonal population of immobilized clonal amplicons
around the point of initial hybridization of template 1 to IS1.
[0070] Also provided is a method of generating separated and
immobilized clonal populations of a first template sequence
("template 1") and a second template sequence ("template 2"),
comprising amplifying the first and second template sequence to
generate a population of clonal amplicons of template 1
substantially attached to first immobilization site ("IS1") and not
to a second immobilization site ("IS2"), or a population of clonal
amplicons of template 2 substantially attached to IS2 and not to
IS1, wherein: (a) both templates and all amplicons are contained
within the same continuous liquid phase, where the continuous
liquid phase is in contact with a first and second immobilization
site (respectively, "IS1" and "IS2"), and where IS1 and IS2 are
spatially separated, (b) template 1 when in single-stranded form
comprises a first subsequence ("T1-FOR") at one end, and a second
subsequence ("T1-REV") at its opposite end, (c) template 2 when in
single-stranded form comprises a first subsequence ("T2-FOR") at
one end, and a second subsequence ("T2-REV") at its opposite end,
(d) IS1 comprises multiple copies of an immobilized nucleic acid
primer ("IS1 primer") that can hybridize substantially to T1-FOR
and T2-FOR when T1 and T2 are single-stranded, (e) IS2 comprises
multiple copies of an immobilized primer ("IS2 primer") that can
hybridize substantially to both T1-FOR and T2-FOR when T1 and T2
are single-stranded, (f) the reverse complement of T1-REV when
single-stranded cannot hybridize substantially to primers on IS1,
but can hybridize substantially to a non-immobilized primer ("SP1")
in the continuous liquid phase; and (g) the reverse complement of
T2-REV when single-stranded cannot hybridize substantially to
primers on IS2, but can hybridize substantially to a
non-immobilized primer ("SP2") in the continuous liquid phase.
[0071] In addition, a method is provided of generating separated
and immobilized clonal populations of a first template sequence
("template 1") and a second template sequence ("template 2"),
comprising amplifying the first and second template sequence,
wherein: (a) both templates are in single-stranded form and are
both contained within the same continuous liquid phase, where a
first and second immobilization site (respectively, "IS1" and
"IS2") are in contact with said continuous liquid phase, and where
IS1 and IS2 are spatially separated, (b) template 1 comprises a
first subsequence ("T1-FOR") at its 3' end, and a second
subsequence ("T1-REV") that is non-overlapping with T1-FOR and at
its 5' end, (c) template 2 comprises a first subsequence ("T2-FOR")
at its 3' end, and a second subsequence ("T2-REV") that is
non-overlapping with T2-FOR and at its 5' end, (d) IS1 comprises an
immobilized primer ("IS1 primer") that can hybridize to both T1-FOR
and T2-FOR, (e) IS2 comprises an immobilized primer ("IS2 primer")
that can hybridize to both T1-FOR and T2-FOR, and (f) the reverse
complement of T1-REV cannot hybridize substantially to primers on
IS1, and/or the reverse complement of T2-REV cannot hybridize
substantially to primers on IS2, but can each hybridize
substantially to a non-immobilized primer in the continuous liquid
phase; whereby amplification results in a population of clonal
amplicons of template 1 substantially attached to IS1 and not to
IS2, and/or a population of clonal amplicons of template 2
substantially attached to IS2 and not to IS1.
[0072] Optionally, in any method described herein, the continuous
medium is flowable. Optionally, intermixing of non-immobilized
nucleic acid molecules is substantially unretarded in the
continuous liquid phase during at least a portion of the
amplification process, e.g., during any one or more steps or cycles
described herein.
[0073] Optionally, in any method described herein, intermixing is
substantially unretarded for a period of time during amplification.
For example, intermixing is substantially unretarded during the
entire duration of amplification.
[0074] Optionally, in any method described herein, any nucleic acid
that has dissociated from one immobilization site is capable of
substantially hybridizing to both immobilization sites and any
movement (e.g., movement by diffusion, convection) of said
dissociated nucleic acid to another immobilization site is not
substantially retarded in the continuous liquid phase.
[0075] Optionally, in any method described herein, the continuous
liquid phase is in simultaneous contact with IS1 and IS2.
[0076] Optionally, in any method described herein, a first portion
of a template that is bound by an immobilized primer does not
overlap with a second portion of the template whose complement is
bound by a non-immobilized primer.
[0077] Optionally, in any method described herein, at least one
template to be amplified is generated from an input nucleic acid
after the nucleic acid is placed in contact with at least one
immobilization site.
[0078] Optionally, any method described herein comprising the steps
of: (a) contacting a support comprising immobilized primers with a
single-stranded nucleic acid template, wherein: hybridizing a first
immobilized primer to a primer-binding sequence (PBS) on the
template (b) extending the hybridized first primer in
template-dependent extension to form an extended strand that is
complementary to the template and at least partially hybridized to
the template; (c) partially denaturing the template from the
extended complementary strand such that at least a portion of the
PBS is in single-stranded form ("free portion"); (d) hybridizing
the free portion to a non-extended, immobilized second primer (e)
extending the second primer in template-dependent extension to form
an extended strand that is complementary to the template (f)
optionally, separating the annealed extended immobilized nucleic
acid strands from one another.
[0079] Optionally, in any method described herein (a) during
amplification, nucleic acid duplexes are formed comprising a
starting template and/or amplified strands; which duplexes are not
subjected during amplification to conditions that would cause
complete denaturation of a substantial number of duplexes.
[0080] Optionally, in any method described herein, the
single-stranded templates are produced by taking a plurality of
input double-stranded or single-stranded nucleic acid sequences to
be amplified (which sequence may be known or unknown) and appending
or creating a first universal adaptor sequence and a second
universal adaptor sequence onto the ends of at least one input
nucleic acid; wherein said first universal adaptor sequence
hybridizes to IS1 primer and/or IS2 primer, and the reverse
complement of said second universal adaptor sequence hybridizes to
at least one non-immobilized primer. The adaptors can be
double-stranded or single-stranded.
[0081] Optionally, in any method described herein, first and second
nucleic acid adaptor sequences are provided at first and second
ends of said single-stranded template sequence.
[0082] Optionally, in any method described herein, a tag is also
added to one or more nucleic acid sequences (e.g., a template or a
primer or an amplicons), said tag enabling identification of a
nucleic acid containing the tag.
[0083] Optionally, in any method described herein, all primers on
at least one immobilization site or support have the same sequence.
Optionally, an immobilization site or support comprises a plurality
of primers having at least two different sequences. Optionally, two
or more different types of primer (e.g., different in sequence) are
present in substantially the same concentrations as one another, or
alternatively in different concentrations. Optionally, the primers
of at least one immobilization site or support are substantially
homogeneously dispersed over the immobilization site or support.
Optionally, in any method described herein, two different
immobilization sites are spatially separated subcomponents of a
single support and/or are on different unconnected supports.
[0084] Optionally, in any method described herein, the support
forms a three-dimensional matrix and the two different
immobilization sites are two different three-dimensional portions
of the support that are not completely overlapping. Optionally, in
any method described herein, the two different immobilization sites
are two different areas on the surface of a support that are not
completely overlapping. Optionally, in any method described herein,
the two different immobilization sites are on different
supports.
[0085] At least one support can be a bead, e.g., a microbead or
nanobead. In an embodiment, the bead is a "scaffolded nucleic acid
polymer particle" or SNAPP, described in U.S. Publ. App. No.
2010-0304982, incorporated by reference.
[0086] Optionally, in any method described herein, at least one
immobilization site includes the entire surface of the support or
the entire volume of the support. Optionally, in any method
described herein, the two different immobilization sites are
located in a predetermined arrangement (e.g. in a grid pattern). In
other embodiments, one or more immobilization sites are not
predetermined (e.g., the support comprises immobilized primers at
non-predetermined locations), and the primer to which a starting
template hybridizes (e.g., before any amplification occurs) can be
considered to be the immobilization site for that template.
[0087] Optionally, in any method described herein, heating is used
to partially separate annealed nucleic acid strands. Optionally,
primer extension is achieved by hybridizing the primer to a
template, and contacting with a polymerase and nucleotides. The
contacting and hybridizing can be achieved simultaneously or
sequentially. Optionally, one or more nucleotides are detectably
labeled.
[0088] Optionally, in any method described herein, the method
further includes the step of treating one or more extended
immobilized nucleic acid strands so as to release a nucleic acid
molecule or a part thereof. The treating for example can optionally
consist of nucleic acid cleavage, e.g., with a restriction
endonuclease or with a ribozyme. For example, one or more of said
primers has a restriction endonuclease recognition site or a
ribozyme recognition site or has part of such a site, which part
becomes complete when primer extension occurs.
[0089] Optionally, in any method described herein, the method is
used to amplify a plurality of different nucleic acid sequences,
e.g., sequentially or simultaneously. The plurality is for example
more than 10.sup.3, 10.sup.5, 10.sup.7, 10.sup.9, 10.sup.11,
10.sup.14, or 10.sup.20 target nucleic acids.
[0090] Any method herein can be used to provide amplified nucleic
acid molecules for diagnosis or for screening or for genotyping, or
to provide amplified nucleic acid molecules to be used as a support
for other components, or to generate additional nucleic acid
molecules in free (e.g., non-immobilized rather than immobilized)
form. For example any method can be used to monitor gene
expression, or to identify nucleic acid molecules with gene
products that are rarely expressed, identifying heterozygous
individuals, nucleic acid fingerprinting.
[0091] Optionally, in any method described herein, said different
nucleic acid sequences are each provided with a first and second
nucleic acid "adaptor" sequence as described anywhere herein, said
first and second "adaptor" sequences being the same for the each of
the different nucleic acid sequences.
[0092] Optionally, in any method described herein, said different
nucleic acid sequences are each provided with a different tag so
that the different sequences can be distinguished from one
another.
[0093] In an embodiment, amplification is achieved using RPA, i.e.,
recombinase-polymerase amplification (see, e.g., WO2003072805,
incorporated by reference herein). RPA optionally is carried out
without substantial variations in temperature or reagent
conditions. In an embodiment herein, partial denaturation and/or
amplification, including any one or more steps or methods described
herein, can be achieved using a recombinase and/or single-stranded
binding protein. Suitable recombinases include RecA and its
prokaryotic or eukaryotic homologues, or functional fragments or
variants thereof, optionally in combination with single-strand
binding proteins (SSBs). In an embodiment, the recombinase agent
optionally coats single-stranded DNA (ssDNA) such as an
amplification primer to form a nucleoprotein filament strand which
invades a double-stranded region of homology on a template. This
optionally creates a short hybrid and a displaced strand bubble
known as a D-loop. In an embodiment, the free 3'-end of the
filament strand in the D-loop is extended by DNA polymerases to
synthesize a new complementary strand. The complementary strand
displaces the originally-paired partner strand of the template as
it elongates. In an embodiment, one or more of a pair of
amplification primers are contacted with one or more recombinase
agents before be contacted with a template which is optionally
double-stranded.
[0094] In any method described herein, amplification of a template
(target sequence) comprises contacting a recombinase agent with one
or more of at least one pair of amplification primers, thereby
forming one or more "forward" and/or "reverse" RPA primers. Any
recombinase agent that has not associated with the one or more
primers is optionally removed. Optionally, one or more forward RPA
primers are then contacted with a template strand, which optionally
has a region of complementarity to at least one RPA primer. The
template strand can be hybridized to a Contacting of a RPA primer
with a complementary template optionally results hybridization
between said primer and the template. Optionally, the 3' end of the
primer is extended along the template with one or more polymerases
(e.g., in the presence of dNTPs) to generate a double stranded
nucleic acid and a displaced template strand. The amplification
reaction can comprise repeated cycles of such contacting and
extending until a desired degree of amplification is achievable.
Optionally the displaced strand of nucleic acid is amplified by a
concurrent RPA reaction. Optionally, the displaced strand of
nucleic acid is amplified by contacting it in turn with one or more
complementary primers; and (b) extending the complementary primer
by any strategy described herein.
[0095] In an embodiment the one or more primers comprise a
"forward" primer and a "reverse" primer. Placing both primers and
the template in contact optionally results in a first double
stranded structure at a first portion of said first strand and a
double stranded structure at a second portion of said second
strand. Optionally, the 3' end of the forward and/or reverse primer
is extended with one or more polymerases to generate a first and
second double stranded nucleic acid and a first and second
displaced strand of nucleic acid. Optionally the second displaced
strand is at least partially complementary to each other and can
hybridize to form a daughter double stranded nucleic acid which can
serve as double stranded template nucleic acid in a subsequent
amplification cycles.
[0096] Optionally said first and said second displaced strands is
at least partially complementary to said first or said second
primer and can hybridize to said first or said second primer.
[0097] In an alternative embodiment of any method or step or
composition or array described herein, the support optionally
comprises immobilized primers of more than one sequence. After a
template nucleic acid strand hybridizes to first complementary
immobilized primer, the first primer can then be extended and the
template and the primer can be separated partially or completely
from one another. The extended primer can then be annealed to a
second immobilised primer that has different sequence from the
first, and the second primer can be extended. Both extended primers
can then be separated (e.g., fully or partially denatured from one
another) and can be used in turn as templates for extension of
additional immobilized primers. The process can be repeated to
provide amplified, immobilised nucleic acid molecules. In an
embodiment, this amplification results in immobilized primer
extension products of two different sequences that are
complementary to each other, where all primer extension products
are immobilized at the 5' end to the support.
[0098] The amplified nucleic acids generated from any method herein
can be used for many different purposes, including sequencing,
screening, diagnosis, in situ nucleic acid synthesis, monitoring
gene expression, nucleic acid fingerprinting, etc.
[0099] Optionally, the template concentration is adjusted such that
immobilized templates are generally spaced as a sufficient distance
from each other that the clonal clusters generated from individual
templates have little or substantially no overlap with each other,
or do not contaminate one another during or after amplification or
replication. Optionally, any of the amplification methods herein
involve a step of adjusting the concentration of template nucleic
acid before it is contacted with a solid support so that individual
template molecules hybridize to primers immobilized on the solid
support at a density of at least 10000, 100000, 400000, 500000,
1,000,000 or 10.sup.7 molecules per mm.sup.2. Optionally,
individual template molecules are amplified in-situ on the support,
giving rise to clonal populations that are spatially-centered
around the point of hybridization of the initial template.
Strand Flipping
[0100] In a "flipping" embodiment described below, two or more
primers are extended to form two or more corresponding extended
strands. Optionally, the two or more primers that are extended
comprise or consist essentially of substantially identical
sequence, and the extended portions of corresponding extended
strand are at least partly non-identical and/or complementary to
each other.
[0101] One exemplary embodiment of flipping is as follows. A
starting template is amplified, e.g., by template walking, to
generate a plurality of primer-extended strands (which for
convenience will be designated as "forward" strands). Optionally,
the forward strands are complementary to the starting template.
Optionally, the forward strands are immobilized on the support.
Optionally, the forward strands comprise substantially identical
sequence, e.g., the forward strands are substantially identical to
each other. In an embodiment the forward strands are formed by
extension of one or more primers immobilized on a support
("forward" primers). The forward primers and/or the forward strands
are optionally attached to the support at or near their 5' ends.
Optionally, one or more of the primer-extended forward strands
comprises a 3' sequence (called a self-hybridizing sequence) that
is absent in the unextended primer and can hybridize under the
conditions of choice to a 5' sequence (this process will be termed
"self-hybridization"). The 5' sequence is optionally part of the
unextended forward primer. In an example, the forward extension
product forms a "stem-loop" structure upon such hybridization.
Optionally, the unextended forward primer comprises a "cleavable"
nucleotide at or near its 3' terminus that is susceptible to
cleavage. In an embodiment, the cleavable nucleotide is linked to
at least one other nucleotide by a "scissile" internucleoside
linkage that can be cleaved under conditions that will not
substantially cleave phosphodiester bonds.
[0102] After extension, the forward-primer extension product (i.e.,
the forward strand) is optionally allowed to self-hybridize. In a
further embodiment, after allowing for self-hybridization the
forward strand is cleaved at a scissile linkage of a cleavable
nucleotide (for example a nucleotide which forms a scissile linkage
with a neighboring nucleotide). The cleavage results in two
fragments of the primer-extension product (i.e., the extended
forward strand). In an embodiment, a first fragment comprises at
least a portion of the original unextended forward primer.
Optionally, the first fragment does not comprise any extended
sequence. Optionally, the first fragment is immobilized (e.g.,
because the unextended forward primer was already immobilized). In
an embodiment, a second fragment comprises extended sequence.
Optionally the second fragment comprises any 3' portion of the
unextended primer beyond the cleavable nucleotide or does not
comprise any portion of the unextended primer. Optionally, the
second fragment is hybridized to the first portion through its
self-hybridizing sequence.
[0103] In an example, the cleavable nucleotide is one that is
removed by one or more enzymes. The enzyme can for instance be a
glycosylase. The glycosylase optionally has N-glycosylase activity
which releases the cleavable nucleotide from double stranded DNA.
Optionally, the removal of the cleavable nucleotide generates an
abasic, apurinic or apyrimidinic site. The abasic site can
optionally be further modified, for example by another enzymatic
activity. Optionally, the abasic site is modified by a lyase to
generate a base gap. The lyase for example cleaves 3' and/or 5' to
the abasic site. Cleavage optionally occurs at both the 5' and 3'
end by the lyase, resulting in removing the abasic site and leaving
a base gap. Exemplary cleavable nucleotides such as
5-hydroxy-uracil, 7,8-dihydro-8-oxoguanine (8-oxoguanine),
8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine,
aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine can be recognized and
removed by various glycosylases to form an apurinic site. One
suitable enzyme is formamidopyrimidine [fapy]-DNA glycosylase, also
known as 8-oxoguanine DNA glycosylase or FPG. FPG acts both as a
N-glycosylase and an AP-lyase. The N-glycosylase activity
optionally releases damaged purines from double stranded DNA,
generating an apurinic (AP site), where the phosphodiester backbone
is optionally intact. The AP-lyase activity cleaves both 3' and 5'
to the AP site thereby removing the AP site and leaving a one-base
gap. In an example the cleavable nucleotide is 8-oxoadenine, which
is coverted to a one-base gap by FPG with both glycosylase and
lyase activities.
[0104] In another embodiment the cleavable nucleotide is uridine.
Optionally, the uridine is cleaved by "USER" reagent, which
includes Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase
Endonuclease VIII, where UDG catalyses the excision of a uracil
base, forming an abasic (apyrimidinic) site while leaving the
phosphodiester backbone intact, and where the lyase activity of
Endonuclease VIII breaks the phosphodiester backbone at the 3' and
5' sides of the abasic site so that base-free deoxyribose is
released; after which kinase is optionally used to convert the
phosphate group on the 3' end of cleaved product to an --OH
group).
[0105] At least one cleaved fragment is optionally contacted with a
polymerase. Optionally the first immobilized fragment can be
extended by the polymerase. If so desired the second hybridized
fragment can act as template for extension of the first fragment.
In an embodiment a "flipped" double-stranded extension product is
formed. This flipped product can optionally be subjected to
template walking in any manner described herein. When both flipped
and unflipped are subjected to template walking, a cluster of two
different extension products is formed, where both extension
products have an identical portion (corresponding to the unextended
primers) and portion complementary to each other, corresponding to
the extended portions of the extension products.
[0106] In an embodiment, a sequence of interest, such as a
self-hybridizing sequence or a new primer-binding site, can be
optionally be added at the 3' ends of extended forward strands by
contacting the extended forward strands with a single-stranded
"splice" adaptor sequence in the presence of extension reagents
(e.g., a polymerase and dNTPs). This splice sequence optionally
comprises a 3' portion that is substantially complementary to a 3'
end portion of the extended forward strand, and a 5' portion that
is substantially complementary to the sequence of interest to be
added. After hybridizing the splice adaptor to the 3' end of the
extended forward strand, the forward strand is subjected to
template-dependent polymerase extension using the splice adaptor as
template. Such extension results in the addition of the sequence of
interest to the 3' end of the extended forward strand.
[0107] Thus, any method of primer extension and/or amplification
described herein can include any one or more of the following
steps: [0108] a) extension of immobilized forward primers by
template walking to generate a plurality of extended forward
strands which are optionally identical; [0109] b) optionally
hybridizing a splice adaptor to a 3' end of the extended forward
strands and subjecting the forward strands to template-dependent
extension using the splice adaptor as a template, thereby adding a
further 3' sequence to the further-extended forward strands,
wherein a portion of the added 3' sequence is complementary to a
portion of the unextendedforward primer and hybridizes thereto to
form a stem-loop structure [0110] c) cleaving the forward strands
at a scissile linkage of a cleavable nucleotidelocated at or near
the junction of unextended forward primer sequence and extended
forward strand sequence; and optionally removing the cleavable
nucleotide, thereby generating two cleaved fragments, the first
fragment comprising a portion of an unextended forward primer
hybridized to a 3' primer-complementary sequence on the second
fragment; [0111] d) optionally subjecting the first fragment to
polymerase extension using the second fragment as template to
generate a flipped forward strand; [0112] e) optionally hybridizing
a second splice adaptor to a 3' end of the flipped forward strand,
and subjecting the forward strands to template-dependent extension
using the splice adaptor as a template, thereby adding a further 3'
sequence to the flipped forward strands, wherein a portion of the
added 3' sequence is a new primer-binding sequence that is absent
in the flipped strands; [0113] f) selectively extending or
amplifying the flipped strands which comprise the new
primer-binding sequence by contacting with the new primer and
extending or amplifying by any method, e.g., as described herein.
The new primer will not bind to unflipped strands or to flipped
strands that were not further extended in step (e).
[0114] FIG. 8 shows a schematic depiction of an exemplary
strand-flipping and walking strategy. (A) Template walking, (B)
Strand flipping to generate flipped strands, (C) addition of new
primer-binding sequence Pg' on final flipped strands.
[0115] I. Methods of Clonal Amplification
[0116] Overview
[0117] A nucleic acid is associated with (e.g., hybridized to) an
appropriate primer, which is optionally immobilized. The hybridized
nucleic acid may for convenience be designated as the "template"
strand or "reverse strand." This word "template" is not intended to
imply any particular functional, structural or sequence
relationship with an input nucleic acid that was originally
introduced into solution or with a final nucleic acid product that
is generated by the amplification process. In an embodiment, a
forward primer can hybridize to a first portion of the reverse
strand. Another "reverse" primer is optionally present, which is
substantially identical to a second non-overlapping portion of the
reverse strand. The two portions are for example non-overlapping if
they do not contain any subportions that are identical or
complementary to each other.
[0118] In an embodiment the template strand is optionally
single-stranded over at least a portion that is complementary to
the forward primer, which portion is designated the forward
"primer-binding sequence" or forward PBS. The forward primer is
optionally extended using the reverse strand as template to form an
extended forward strand, resulting in a duplex between the
hybridized template (reverse strand) and the forward strand. At
least part of the primer portion of the forward strand can be
separated from the hybridized reverse strand. The forward strand
comprises a reverse PBS portion that can hybridize to a "reverse"
primer and this reverse primer can in turn be extended to form an
extended reverse strand. Both the forward and reverse strand can
then be separated from each other and the process can be repeated
to provide a clonal population of amplified, immobilized nucleic
acid molecules.
[0119] Optionally, any one or more steps of separating forward and
reverse strands from each other involves partial separation, e.g.,
separation that dissociates a portion of the forward strand from a
portion of the reverse strand, but does not abolish all association
between the two strands. Optionally, a portion of a forward strand
is dissociated from a reverse strand, while another portion of the
same forward strand remains associated (e.g., by hybridization)
with a reverse strand.
[0120] During partial separation, at least a portion of the forward
PBS on the reverse strand is dissociated from the first forward
strand. However, separation is "partial" because the forward strand
and reverse strand remain associated with eachother overall. For
example, another portion of the reverse strand remains hybridized
to the forward strand. Optionally, the denatured portion of the
reverse strand re-hybridizes with a second forward primer. Thus a
portion of the reverse strand is hybridized to the first forward
strand, while another portion of the same reverse strand is
hybridized to the second forward primer. The second forward primer
is then extended along the reverse strand (using the reverse strand
as template) to generate a second forward strand. Repeated cycles
of amplification, where an amplification cycle optionally comprises
hybridization, =extension and (partial) separation, generate a
clonal population of nucleic acids.
[0121] Optionally, one or more forward primers are immobilized on a
support which lacks any immobilized reverse primers, or vice versa.
In an embodiment, the first and second forward primers are
immobilized close together or adjacent to each other. The resulting
clonal population of nucleic acids comprises forward strands that
are immobilized close to or adjacent to each other. In an
embodiment, at least 10.sup.6, 10.sup.8, 10.sup.10, 10.sup.12, or
10.sup.14 primers are immobilized on a cm.sup.2 or a cm.sup.3 of an
individual support or immobilization site. Optionally, all forward
primers are identical in sequence, or have an identical 3'
portion.
[0122] In alternative embodiments, both the forward and/or reverse
primer are optionally immobilized on a support. Alternatively both
primers are non-immobilized.
[0123] Generally, a nucleic acid is clonally amplified onto a
support on which multiple copies of a primer are immobilized. An
exemplary nucleic acid is one of a collection of nucleic acids.
Individual nucleic acids of the collection for example can have one
or more adaptor sequences at their 5' and/or 3' ends and variable
sequences in between, such as gDNA or cDNA. In an embodiment, the
3' adaptor has a low T.sub.m region (where T.sub.m is the
temperature at which half of the DNA molecules are in non-denatured
or double-stranded state and half are in denatured, e.g., random
coil, state), and the 5' adaptor optionally has a higher Tm region,
or vice versa. The low T.sub.m region is for example an A-rich,
T-rich or pyrimidine-rich region, such as an AT (or U)-rich
sequence, such as polyT, polyA, polyU and any combinations of A, T
and U bases. Exemplary methods are described herein.
[0124] The methods described herein can be used for many different
purposes, including sequencing, screening, diagnosis, in situ
nucleic acid synthesis, monitoring gene expression, nucleic acid
fingerprinting, etc.
[0125] Non-limiting exemplary methods of clonal nucleic acid
amplification on a support are as follows.
A) Amplification on a Support
[0126] In some embodiments, the disclosed methods, compositions,
systems, apparatuses and kits include nucleic acids (e.g., primers,
templates etc.,) that are attached to a support. The nucleic acids
can be attached using any suitable method. In some embodiments, the
attachment between the nucleic acid molecule and the support is
mediated by covalent bonding, by hydrogen bonding (for example
attachment of a template nucleic acid to a support mediated by
hybridization of the template to another nucleic acid, e.g.,
primer, which is covalently attached to the support), Van Der
Waal's forces, affinity interactions, and the like. Any suitable
method for attachment of the nucleic acid sequence to the support
can be used, including the use of binding pairs (e.g.,
avidin/biotin; antigen/antibody). In some embodiments, one member
of the binding pair is attached to the support, the other member of
a binding pair is attached to the nucleic acid, and the nucleic
acid is attached to the support via interaction of the two members
of the binding pair.
[0127] The support can be comprised of any material and have any
dimensions or shape. The support can be selected to have properties
or reactivities that interfere only minimally with the
amplification process. In some embodiments, the support is
comprised of solid material; alternatively, it can be comprised at
least partially of semi-solid, fluid or liquid material. In some
embodiments, the support is spherical, spheroidal, tubular,
pellet-shaped, rod-shaped, octahedral, hexagonal, square or
trapezoidal in shape. In some embodiments, the support is porous.
In some embodiments, the support can be comprised of a hydrophilic
porous matrix such as a hydrogel. See, e.g., U.S. Patent
Publication No. 2010-0304982, Hinz et al.; and US Patent
Publication No. 2010-0136544, Agresti et al.; all of which
foregoing applications are incorporated by reference herein.
[0128] Among other things, novel methods of generating a localized
clonal population of immobilized clonal amplicons in or on a
support are provided. The support can for example be solid or
semisolid. The amplified clonal population is optionally
immobilized to the support's external surface or can also be within
the internal surfaces of a support (e.g., where the support is
semisolid, e.g., with a gel or matrix structure). Exemplary
supports can be solid or semi-solid. Optionally, the semi-solid
support comprises polyacrylamide, cellulose, polyamide (nylon) and
cross-linked agarose, dextran and -polyethylene glycol.
[0129] Optionally, the disclosed methods and compositions include
the attachment of one or more individual members of a collection
(e.g., a collection) of nucleic acids to one or more supports of a
population of supports. For example, different nucleic acids of the
collection can be attached to different supports. The resulting
population of supports includes a plurality of supports each
comprising a single nucleic acid. In some embodiments, the nucleic
acids of the collection are double stranded, and the collection is
denatured to form a population of single-stranded nucleic acids. In
some embodiments, the support includes primers, and one or more of
the single stranded nucleic acids can be attached to the support
through hybridization to primers on the surface.
[0130] Optionally before amplification the collection of nucleic
acids can be appropriately diluted and contacted with the
population of supports in solution, such that at least 40%, 50%,
60%, 70%, 80%, 90% or 95% of the supports (or immobilization sites
where the population of supports consists of one or a few supports)
become attached to no more than one nucleic acid. In some
embodiments, the ratio of the number of nucleic acids to the total
number of supports can be set to facilitate mono-clone formation
by, e.g., maximizing the number of resulting supports (or number of
immobilization sites on a single support) that include only a
single nucleic acid, or choosing a ratio that is statistically
predicted to give more clonal supports (e.g., beads) than lower or
higher ratios.
[0131] Optionally, a single support is used in any of the
amplification methods herein, where the single support has a
plurality of primers that can hybridize to the templates. In such
an embodiment, the concentration of the template collection is
adjusted before it is contacted with a solid support so that
individual template molecules in the collection get attached or
associated (e.g., by hybridization to primers immobilized on the
solid support) at a density of at least 10.sup.2, 10.sup.3,
10.sup.4, 10.sup.5, 4.times.10.sup.5, 5.times.10.sup.5,
6.times.10.sup.5, 8.times.10.sup.5, 10.sup.6, 5.times.10.sup.6 or
10.sup.7 molecules per mm.sup.2.
[0132] Optionally, individual template molecules are amplified
in-situ on the support, giving rise to clonal populations that are
spatially-centered anound the point of hybridization of the initial
template. Optionally, the amplification generates no more than
about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.15, or
10.sup.20 amplicons from a single amplified template. Optionally,
the colonies of clonal amplicons are situated on the solid support
at a density of at least 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5,
4.times.10.sup.5, 5.times.10.sup.5, 6.times.10.sup.5,
8.times.10.sup.5, 10.sup.6, 5.times.10.sup.6 or 10.sup.7 molecules
per mm.sup.2.
[0133] In some embodiments, the nucleic acid collection can be
contacted with one or more supports under conditions where multiple
nucleic acids bind to the same support. Such contacting can be
particularly useful in methods that involve parallel clonal
amplification of nucleic acids in different regions of the same
support. The ratio of the number of nucleic acids to the surface
area of the support can be adjusted to facilitate mono-clone
formation by, e.g., ensuring that the nucleic acids are
appropriately spaced in the support to favor formation of
monoclonal populations of amplified nucleic acids without
substantial cross-contamination between different clonal
populations. For example where a single support us used, the
collection of nucleic acids to be amplified is adjusted to such a
dilution that the resulting amplified clonal populations generated
from individual nucleic acids are generally discrete or distinct,
e.g., without overlap. For example, individual nucleic acids within
50%, 70%, 80% or 90% or more of the amplified clonal populations
are not interspersed with substantially non-identical nucleic
acids. Optionally, different amplified populations are not in
contact or completely overlapping with other amplified populations,
or are distinguishable from each other using a detection method of
choice.
[0134] In some embodiments, the nucleic acids are attached to the
surface of a support. In some embodiments, the nucleic acids can be
attached within the support. For example, for supports comprised of
hydrogel or other porous matrices, the nucleic acids can be
attached throughout the volume of the support including on the
surface and within the support.
[0135] In some embodiments, the support (or at least one support in
a population of supports) can be attached to at least one primer,
optionally to a population of primers. For example, the support (or
at least one support) can include a population of primers. The
primers of the population can be substantially identical each
other, or may include a substantially identical sequence. One, some
or all of the primers can include a sequence that is complementary
to a sequence within one or more nucleic acid templates. In some
embodiments, the population of primers can include at least two
noncomplementary primers.
[0136] The primers can be attached to the support through their 5'
end, and have free 3' ends. The support can be the surface of a
slide or the surface of a bead. The primers have low melting
temperature, such as oligo (dT).sub.20, and can hybridize to the
low T.sub.m region of the collection adaptor. The distances between
the primers need to be shorter than the adapter length to allow
templates waking, or alternatively, a long primer with 5' end long
linker will increase the chance of walking
[0137] In some embodiments, the support is attached to and/or
contacted with a primer and a template (or reverse strand) under
conditions where the primer and template hybridize to each other to
form a nucleic acid duplex. The duplex can include a double
stranded portion that comprises complementary sequences of the
template and primer, where at least one nucleotide residue of the
complementary sequences are base paired with each other. In some
embodiments, the duplex can also include a single stranded portion.
The duplex can also include a single stranded portion. The single
stranded portion can include any sequence within the template (or
primer) that is not complementary to any other sequence in the
primer (or template).
[0138] A non-limiting exemplary method of clonal nucleic acid
amplification on a support is as follows. A nucleic acid (which
shall be designated for convenience as the reverse strand) is
clonally amplified onto a support on which multiple copies of a
complementary forward primer are attached. An exemplary nucleic
acid is one of a plurality of DNA collection molecules, that for
example the plurality of nucleic acid members have one or more
common ("adaptor") sequences at or near their 5' and/or 3' ends and
variable sequences in between, such as gDNA or cDNA. In an
embodiment, the 3' common portion, e.g., adaptor, has a breathable
(e.g., low T.sub.m) region, and the 5' common sequence (e.g.,
adaptor) optionally has a less breathable (e.g., higher T.sub.m)
region, or vice versa. In another embodiment, both the 5' and 3'
common sequences are breathable. The breathable (e.g., low T.sub.m)
region is for example a region that is rich in A, T and/or U, such
as an AT (or U)-rich sequence, such as polyT, polyA, polyU and any
combinations of A, T and U bases, or bases complementary to such
bases. Exemplary methods are described herein.
[0139] One non-limiting exemplary method of clonal nucleic acid
amplification on a support is shown in FIG. 1. A non-limiting
description of an exemplary method is as follows.
[0140] A double stranded DNA library molecule is denatured and the
single stranded DNA is attached to the support through
hybridization to the primers on the surface. The ratio of number of
DNA molecules to support area or number of beads is set to
facilitate mono-clone formation.
[0141] Primers are attached on a support through their 5' and have
free 3'. The support can be the surface of a slide or the surface
of a bead. The primers have low melting temperature, such as oligo
(dT).sub.20 or oligo (dA).sub.30 and can hybridize to the low Tm
region of the library adaptor. The distances between the primers
can be shorter than the adapter length to allow templates waking,
or alternatively, a long primer with 5' end long linker will
increase the chance of walking
[0142] A nucleic acid is clonally amplified onto a support on which
multiple copies of a primer are attached. An exemplary nucleic acid
is one of a plurality of DNA library molecules, which for example
have one or more common (e.g., "adaptor") sequences at their 5'
and/or 3' ends and variable sequences in between, such as gDNA or
cDNA. In an embodiment, the 3' adaptor has a low Tm region, and the
5' adaptor optionally has a higher Tm region, or vice versa. The
low Tm region is for example a pyrimidine-rich region, such as an
AT (or U)-rich sequence, such as polyT, polyA, polyU and any
combinations of A, T and U bases or bases complementary to such
bases. Exemplary methods are described herein.
B) Primer Extension
[0143] One or more primers, whether in soluble form or attached to
a support, is incubated with a DNA polymerization or extension
reaction mix, which optionally comprises any one or more of
reagents such as enzyme, dNTPs and buffers. The primer (e.g., a
forward primer) is extended. Optionally, the extension is a
template-dependent extension of a primer along a template
comprising the successive incorporation of nucleotides that are
individually complementary to successive nucleotides on the
template, such that the extended or nonextended forward primer is
complementary to the reverse strand (also termed antiparallel or
complementary). Optionally, the extension is achieved by an enzyme
with polymerase activity or other extension activity, such as a
polymerase. The enzyme can optionally have other activities
including 3'-5' exonuclease activity (proofreading activity) and/or
5'-3' exonuclease activity. Alternatively, in some embodiments the
enzyme can lack one or more of these activities. In an embodiment
the polymerase has strand-displacing activity. Examples of useful
strand-displacing polymerases include Bacteriophage .PHI.29 DNA
polymerase and Bst DNA polymerase. Optionally, the enzyme is active
at elevated temperatures, e.g., at or above 45.degree. C., above
50.degree. C., 60.degree. C., 65.degree. C., 70.degree. C.,
75.degree. C., or 85.degree. C.
[0144] An exemplary polymerase is Bst DNA Polymerase (Exonuclease
Minus), is a 67 kDa Bacillus stearothermophilus DNA Polymerase
protein (large fragment), exemplified in accession number 2BDP_A,
which has 5'-3' polymerase activity and strand displacement
activity but lacks 3'-5' exonuclease activity. Other polymerases
include Taq DNA polymerase I from Thermus aquaticus (exemplified by
accession number 1TAQ), Eco DNA polymerase I from Echerichia coli
(accession number P00582), Aea DNA polymerase I from Aquifex
aeolicus (accession number 067779), or functional fragments or
variants thereof, e.g., with at least 80%, 85%, 90%, 95% or 99%
sequence identity at the nucleotide level.
[0145] Generally, the extension step produces a nucleic acid, which
comprises a double-stranded duplex portion in which two
complementary strands are hybridized to each other. In one
embodiment, walking involves subjecting the nucleic acid to
partially-denaturing conditions that denature a portion of the
nucleic acid strand but are insufficient to fully denature the
nucleic acid across its entire length. In an embodiment, the
nucleic acid is not subjected to fully-denaturing conditions during
a portion or the entire duration of the walking procedure. As
intended herein, a nucleic acid molecule can be considered
partially-denatured when a portion of at least one strand of the
nucleic acid remains hybridized to a complementary strand, while
another portion is in an unhybridized state (even if it is in the
presence of a complementary sequence). The unhybridized portion is
optionally at least 5, 7, 8, 10, 12, 15, 17, 20, or 50 nucleotides
long. The hybridized portion is optionally at least 5, 7, 8, 10,
12, 15, 17, 20, or 50 nucleotides long.
[0146] Optionally, a nucleic acid can be considered to be partially
denatured when a substantial fraction of individual molecules of
the nucleic acid (e.g., above 20%, 30%, 50%, or 70%) are in a
partially denatured state. Optionally less than a substantial
amount of individual molecule are fully denatured, e.g., not more
than 5%, 10%, 20%, 30% or 50% of the nucleic acid molecules in the
sample. Similarly a nucleic acid is optionally considered fully
denatured when it lacks any double-strandedness (or lacks any
hybridization to a complementary strand) in more than 80% or 90% of
individual molecules of the nucleic acid. Under exemplary
conditions at least 50% of the nucleic acid is partly denatured,
but less than 20% or 10% is fully denatured. In other situations,
at least 30% of the nucleic acid is partly denatured, but less than
10% or 5% is fully denatured. Similarly, a nucleic acid can be
considered to be non-denatured when a minority of "breathable"
portions of interest in the nucleic acid, e.g., a PBS, are
denatured. In exemplary annealing conditions at least 10%, 30%,
50%, 60%, 70%, 80% or 90% of PBSs of nucleic acid molecules in the
sample are hybridized to corresponding primers.
[0147] In an embodiment, partially denaturing conditions are
achieved by maintaining the duplexes as a suitable temperature
range. For example, the nucleic acid is maintained at temperature
sufficiently elevated to achieve some heat-denaturation (e.g.,
above 45.degree. C., 50.degree. C., 55.degree. C., 60.degree. C.,
65.degree. C., or 70.degree. C.) but not high enough to achieve
complete heat-denaturation (e.g., below 95.degree. C. or 90.degree.
C. or 85.degree. C. or 80.degree. C. or 75.degree. C.). Complete
heat-denaturation conditions are for example conditions that would
result in complete separation of a significant fraction (e.g., more
than 10%, 20%, 30%, 40% or 50%) of a large plurality of strands
from their extended and/or full-length complements. In an
embodiment the nucleic acid is subjected to isothermal conditions,
where temperature variation is constrained within a limited range
during at least some portion of the amplification (e.g., the
temperature variation is within 20.degree. C., optionally within
10.degree. C., for example within 5.degree. C., or 2.degree. C.).
Optionally, the temperature is maintained at or around 50.degree.
C., 55.degree. C., 60.degree. C., 65.degree. C., or 70.degree. C.
for at least about 10, 15, 20, 30, 45, 60 or 120 minutes.
Optionally, any temperature variation is not more than 20.degree.
C., optionally within 10.degree. C., for example within 5.degree.
C., or 2.degree. C. during one or more amplification cycles (e.g.,
e.g., 1, 5, 10, 20, or all amplification cycles performed).
Optionally, thermocycling can be performed (where temperature
variance is within isothermal or non-isothermal ranges). In an
example, the temperature variation is constrained between the
denaturation step and another step such as annealing and/or
extension. In an example, the difference between the denaturation
temperature and the annealing or extension temperature is not more
than 20.degree. C., optionally within 10.degree. C., for example
within 5.degree. C., or 2.degree. C., for one or more cycles of
amplification. The temperature is for example constrained for at
least 5, 10, 15, 20, 30, 35 or substantially all cycles of
amplification.
[0148] Partial denaturation can also be achieved by other means,
e.g., chemical means using chemical denaturants such as urea or
formamide, with concentrations suitably adjusted, or using high or
low pH (e.g., pH between 4-6 or 8-9). In an embodiment, partial
denaturation and amplification is achieved using
recombinase-polymerase amplification (RPA). Exemplary RPA methods
are described herein.
[0149] In an embodiment, the sequence of the negative and/or
positive strand designed such that a primer-binding sequence or a
portion thereof is breathable, i.e., is susceptible to denaturation
under the conditions of choice (e.g., amplification conditions).
The breathable portion is optionally more susceptible than a
majority of nucleic acids of similar length with randomized
sequence, or more susceptible than at least another portion of the
strand comprising the breathable sequence. Optionally, the
breathable sequence shows a significant amount of denaturation
(e.g., at least 10%, 20%, 30%, 50%, 70%, 80%, 90% or 95% of
molecules are are completely denatured across the breathable
sequence) at the amplification conditions of choice. For example
the breathable sequence is designed to be fully-denatured in 50% of
strand molecules at 30, 35, 40, 42, 45, 50, 55, 60, 65 or
70.degree. C. under the conditions of choice (e.g., amplification
conditions).
[0150] Optionally, the T.sub.m of a nucleic acid strand (e.g., a
primer or template strand) is the temperature at which at least a
desired fraction of a clonal population of duplexes are rendered
completely single-stranded under the chosen reagent conditions,
where an individual duplex comprises the nucleic acid strand in
question hybridized to its full-length complement. By default, the
desired fraction is 50% if the fraction is not specified. In
alternative embodiments, the desired fraction is optionally at
least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or 99%. Also for example a sequence can be considered
breathable if the theoretically-predicted melting temperature of
the breathable sequence is not more than 20, 30, 35, 40, 45, 50,
55, 60 or 65.degree. C. under the amplification conditions of
choice, using known theoretical calculations for predicted melting
temperature (T.sub.m). In an example, the thermal stability,
melting behavior and/or T.sub.m is a theoretically-predicted
temperature according to the teachings of Breslauer et al., Proc.
Nat. Acad. Sci. 83, 3746-50 (1986). In an exemplary calculation,
T.sub.m is predicted as follows:
T m = .DELTA. H kcal C * Mol .DELTA. S + R ln ( [ primer ] / 2 ) -
273.15 .smallcircle. C . ##EQU00001##
[0151] where .DELTA.H is the enthalpy of base stacking interactions
adjusted for helix initiation factors; .DELTA.S is the entropy of
base stacking adjusted for helix initiation factors, and for the
contributions of salts to the entropy of the system, and R is the
universal gas constant (1.987 Cal/.degree. C.*Mol). Further details
and assumptions are set forth in SantaLucia, J. (1998) Proc. Nat.
Acad. Sci. USA 95, 1460); Rychlik, W. and Rhoads, R. E. (1989)
Nucl. Acids Res. 17, 8543; and Borer P. N. et al. (1974) J. Mol.
Biol. 86, 843.
[0152] In another embodiment the T.sub.m is empirically measured by
known methods. (e.g., Spink, Methods Cell Biol. 2008; 84:115-41;
Meunier-Prest et al., Nucleic Acids Res. 2003 Dec. 1; 31(23): e150;
Brewood et al., Nucleic Acids Res. 2008 September; 36(15):
e98.)
[0153] In an embodiment, at least one PBS on each strand is
breathable--e.g., the forward PBS and the reverse PBS are both
breathable. Optionally, a nucleic acid such as a forward or reverse
strand comprises two breathable sequences. For example, a 5'
portion and a 3' portion can be breathable.
[0154] Where partial denaturation is achieved by heating or
elevated temperatures, an exemplary breathable PBS may be
pyrimidine-rich (e.g., with a high content of As and/or Ts and/or
Us). The PBS comprises for example a poly-A, poly-T or puly-U
sequence, or a polypyrimidine tract. One or more amplification or
other primers (e.g., an immobilized primer) are optionally designed
to be correspondingly complementary to these primer-binding
sequences. An exemplary PBS of a nucleic acid strand comprises a
poly-T sequence, e.g., a stretch of at least 10, 15, 20, 25 or 30
thymidine nucleotides, while the corresponding primer has a
complementary sequence to the PBS, e.g., a stretch of at least 10,
15, 20, 25 or 30 adenosine nucleotides. Exemplary low-melt primers
optionally have a high proportion (e.g., at least 50%, 60%, 65%,
70%, 75%, 80%, 85% 90% 95% or 100%) of nucleobases that generally
(e.g., under amplification conditions of choice) form no more than
two hydrogen bonds with a complementary base when the primer is
hybridized to a complementary template. Examples of such
nucleobases include A (adenine), T (thymine) and U (uracil).
Exemplary low-melt primers optionally have a high proportion of any
one or more of A (adenine), T (thymine) and/or U (uracil)
nucleotides or derivatives thereof. In an embodiment, the
derivatives comprise nucleobases that are complementary to A
(adenine), T (thymine) and/or U (uracil). The portion of the primer
that hybridizes to the PBS optionally has at least 50%, 60%, 65%,
70%, 75%, 80%, 85%, 90% 95% or 100% of A (adenine), T (thymine) or
U (uracil) nucleotides, or any combination thereof. In another
example, the portion of the primer that hybridizes to the PBS
comprises a polyA sequence (e.g., at least 5, 10, 15, 20, 25 or 30
nucleotides long). Other exemplary primers comprise
(NA.sub.x).sub.n repeats. Optionally, n (in lower case) is from 2
to 30, e.g., from 3 to 10, for example 4 to 8. "N" (in upper case)
is any nucleotide--and optionally, N is C or G. "A" is the
shorthand convention for adenine, and "x" denotes the number of
adenine residues in the repeat, for example 2, 3, 4, 5, 6, 10 or
more. Exemplary primers comprise multiple repeats of (CAA).sub.n,
CA).sub.n, (CAAA).sub.n or even (GAA).sub.n.
[0155] Optionally only one strand (e.g., the forward or the reverse
strand) has a breathable PBS. In another embodiment, both the
forward and reverse strands have a breathable PBS. The breathable
PBS is optionally complementary to a primer that is either
immobilized to a support or is not immobilized (e.g., in soluble
form). Optionally the strand comprising the breathable PBS is
either immobilized to a support or is not immobilized (e.g., in
soluble form). Optionally both primers are immobilized, or or both
strands are immobilized. Optionally neither primer is immobilized,
or neither strand is immobilized.
[0156] An amplification cycle optionally comprises breathing,
annealing and extension. The nucleic acid to be amplified is
optionally subjected to conditions which are suitable for or
optimized for at least one of these steps. In an embodiment, the
nucleic acid is subjected to conditions which are suitable for more
than one of these steps, (e.g., annealing and extension, or
breathing and extension). In some instances, all three of these
steps can take place simultaneously under the same conditions.
[0157] In an exemplary method the nucleic acid can be subjected to
conditions which permit or facilitate breathing. In an embodiment,
"breathing" is said to occur when the two strands of a
double-stranded duplex are substantially hybridized to each other,
but are denatured across a local portion of interest (e.g., the
terminal ends or primer-binding sites). One or more breathable
sequences (e.g., a forward and/or reverse PBS having a low Tm
portion) of the nucleic acid gets locally denatured ("breathes")
from a first complementary strand (e.g., a forward or reverse
strand) which it is hybridized to, and is thus made available to
hybridize to another second strand. An exemplary first strand is a
primer extension product from a first primer. An exemplary second
strand is for example a second unextended primer (e.g., a
PBS-complementary oligonucleotide comprising, e.g., a dT or dA
sequence). Optionally, the first and second strands are immobilized
on a support, and can be closely situated (e.g., in close enough
proximity to allow walking). The conditions for breathing are
optionally partially-denaturing conditions under which the PBS is
generally denatured but another portion of nucleic acid remains in
a hybridized or double-stranded state. Optionally, DNA helicase can
be included in the reaction mix to facilitate the partial
denaturing.
[0158] Optionally, the nucleic acid is then subjected to conditions
which facilitate annealing, e.g., the temperature is decreased, to
enable hybridization between the breathable PBS and the second
strand. In an embodiment, the same conditions are used to
facilitate both breathing and extension. In another embodiment,
annealing conditions are different from breathing conditions--for
example, the annealing conditions are nondenaturing conditions or
conditions that favor denaturation less than the breathing
conditions. In an example, annealing conditions involve a lower
temperature (such as 37.degree. C.) than breathing conditions, in
which a higher temperature (e.g., 60-65.degree. C.) is used.
Optionally, fully denaturing conditions are avoided during one or
more cycles of amplification (e.g., the majority of amplification
cycles or substantially all amplification cycles).
[0159] Extension conditions are generally permissive or highly
suitable for primer extension. In an embodiment, the extension
conditions of choice are the same as or different from annealing
and/or breathing conditions. In an embodiment, the same set of
conditions is used for all three steps (e.g., isothermal
amplification), such that subjecting the sample to a single set of
conditions for a sustained period enables multiple amplification
cycles of breathing, annealing and extension to take place.
[0160] In an embodiment, strand extension is performed for example
by a strand displacing DNA polymerase, such as Bst DNA polymerase
large fragment, Klenow DNA polymerase, phi29 DNA polymerase, Vent
DNA polymerase, any functional fragments and/or variants, or any
combination of such enzymes. The strand-displacement capability
optionally facilitates extension through duplex portions of
partially-denatured nucleic acids.
[0161] Optionally, one or more of the PBS-breathing and primer
extension steps are repeated multiple times to amplify an initial
nucleic acid. Where one or more nucleic acid reagents (e.g.,
primers) are immobilized to a support, the primer-extension
products remain substantially attached to the support, e.g., by
virtue of attachment of an unextended extended primer to the
support prior to amplification, or by hybridization to such a
primer). Optionally, a localized clonal population of clonal
amplicons is formed around a discrete site on the support. An
exemplary discrete site is a point of attachment of an initial
nucleic acid strand to the support, and from which other nucleic
acids within the clonal population are directly or indirectly
generated by primer extension, using the initial nucleic acid or
its copies as a template.
[0162] Optionally, a sample is prepared of a population of one or
more nucleic acids to be amplified. The population of nucleic acids
can be in single-stranded or double-stranded form; optionally one
or more nucleic acids individually comprises a nucleic acid strand
with a known 3' end sequence and a known 5' end sequence which are
substantially identical or complementary to the one or more primers
used in the amplification. A 3' portion of the nucleic acid strand
can for example be complementary to an immobilized primer, whereas
a 5' portion can be identical to a soluble primer. The 5' and/or 3'
portions can be common ("universal") or invariant between
individual nucleic acids within the population. Optionally, the
nucleic acids within the population individually comprise variant
(e.g., unknown) sequence between the common portions, such as
genomic DNA, cDNAs, mRNAs, mate-pair fragments, exomes, etc. The
collection can for example have enough members to ensure over 50%,
70%, or 90% coverage of the corresponding genetice source (e.g.,
the genome or the exome).
[0163] II. Compositions, Arrays and Kits
[0164] Also provided herein is a composition comprising any one or
any subset or all of the following: at least one reverse nucleic
acid strand, a plurality of forward primers immobilized on at least
one support, a plurality of reverse primers in solution, and a
polymerase. The forward and/or reverse primers are optionally
low-melt or rich in adenine, thymine or uracil as described herein.
An exemplary composition comprises clonal populations of nucleic
acid strands ("reverse strands"), where individual reverse strands
of each clonal population comprise a low-melt (e.g., breathable)
primer-binding sequence at the 3' end and/or a low-melt primer
sequence on the 5' end. The composition optionally includes a
plurality of reverse primers that are substantially identical to
the low-melt primer sequence on a 5' portion or end of the reverse
strand. The composition optionally includes a plurality of forward
primers that are substantially complementary to the low-melt
primer-binding sequence on a 3' portion or end of the reverse
strand. In an embodiment the forward primer and/or the reverse
primer is immobilized by attachment to a support. For example the
forward primer is immobilized and the reverse primer is not
immobilized, or vice versa. An exemplary composition comprises any
one or more of: (1) a reverse nucleic acid strand, (2) a plurality
of low-melt forward primers immobilized on a support, (3) a
plurality of low-melt reverse primers in solution, and (4) a
polymerase.
[0165] Optionally, the composition further comprises one or more
extended forward strands that are londer than unextended forward
primers and are optionally full-length complements of one or more
reverse strands. In an embodiment one or more extended forward
strands are hybridized to a complementary reverse strand, where the
reverse strand is optionally also hybridized to another different
forward primer or to a different forward strand. The different
forward strand is optionally a less-than-full-length complement of
the reverse strand. The composition can contain any one or more
reagents described herein, and/or be subjected to any one or more
procedures or conditions (e.g., temperatures) described herein.
[0166] Optionally, the composition comprises a plurality of
spatially-separated clonal populations are attached to one or more
solid supports. For example, a plurality of spatially-separated
clonal populations are attached to the same support. The
composition is optionally free of another enzyme that is not a
polymerase, e.g., a recombinase or reverse transcriptase or
helicase or nicking enzyme.
[0167] Optionally the composition comprises a collection of nucleic
acids producible by any one or more methods described herein. For
example, the collection can comprise immobilized nucleic acids
which occupy one or more distinct areas on a surface, each area
comprising a plurality of identical nucleic acid strands and
optionally, a plurality of identical complementary strands
hybridized thereto, where the complementary strands have no
attachment or linkage or association with the solid support except
by virtue of hybridization to the immobilized nucleic acid.
Optionally, an individual nucleic acid strand within such an area
is located so that another nucleic acid strand is located on the
surface within a distance of the length of that strand. Optionally
there is at least one distinct area present per mm.sup.2 of surface
on which the nucleic acids are immobilized. For example the number
of distinct areas/mm.sup.2 of surface on which the nucleic acids
are immobilized is greater than 10.sup.2, greater than 10.sup.3,
greater than 10.sup.4, greater than 10.sup.5, greater than
10.sup.6, greater than 10.sup.7, or greater than 10.sup.8.
[0168] The collections of amplified clonal populations can form
arrays, which can be one-dimensional (e.g., a queue of generally
monoclonal microbeads) or two-dimensional (e.g., the amplified
clonal populations are situated on a planar support), or
three-dimensional. The individual clonal populations of an array
are optionally but not necessarily situated or arranged such that
they are addressed or addressable. Optionally, different clonal
populations are spaced at an appropriate distance from one another,
which distance is generally sufficient to permit different clonal
populations to be distinguished from each other. In an embodiment,
localized clonal populations are scattered in an ordered or
disordered, e.g., random, pattern over a planar substrate.
[0169] The features of an exemplary array are individual
distinguishable clonal populations of nucleic acids, where
optionally the features are distributed over one or more supports.
In an exemplary microbead embodiment, an array comprises a
plurality of microbeads, where an individual microbead generally
comprises a monoclonal population of nucleic acids, and different
microbeads generally comprise different clonal populations (e.g.,
which differ in sequence). Optionally, the microbeads are
distributed or packed in a monolayer over a planar substrate. In
other embodiments, the array comprises a single (e.g., planar)
support, the single support comprising a plurality of spatially
discrete clonal populations of nucleic acids, where different
clonal populations optionally differ in sequence.
[0170] Optionally, one or more nucleic acids within individual
clonal popluations can be attached to the planar substrate
directly. In another example, the nucleic acids of individual
clonal populations are attached to microbeads, for example as
discussed herein. The clonal microbeads are optionally packed
closely together over a planar substrate, in random or ordered
fashion. Optionally, more than 20%, 30%, 50%, 70%, 80%, 90%, 95% or
99% of the microbeads are in contact with at least one, two, four
or six other microbeads. Optionally, less than 10%, 20%, 30%, 50%,
70%, 80%, 90%, 95% or 99% of the microbeads are in contact with
one, two, four or six other microbeads.
[0171] Optionally the features of an array (such as immobilization
sites or amplified DNA clonal populations) are generally discrete
or distinct from each other. For example, 50%, 70%, 80% or 90% or
more of the features of an array are not in contact or not
completely overlapping with other features on the same array, or
are distinguishable from each other using a detection method of
choice. Optionally, the features can partially overlap with each
other as long as they remain distinguishable from each other.
[0172] Also provided herein are kits comprising any one or more
reagents (e.g., nucleic acids or enzymes or supports) described
herein. For example a kit can contain one or more primers,
optionally immobilized. An exemplary kit comprises two primers,
where the denaturation temperature of one primer is optionally at
least 10, 20, 30 or 40.degree. C. from the other. Optionally the
lower-melting primer comprises a adenine/thymine/uracil-rich
portion such as a polyA tract such as those described herein, e.g.,
a polyA, T or U sequence that is at least 10, 15, 20, 25 or 30
nucleotides long. The lower-melting primer is optionally
immobilized, where the higher-melting primer is optionally
non-immobilized.
[0173] Optionally, the kit further contains one or more supports
described herein, comprising the immobilized primers. An exemplary
support comprises a planar surface. Where multiple supports are
used, the kit can comprise microbeads bearing identical
primers.
[0174] The kit optionally contains one or more polymerases, e.g., a
strand-displacing polymerase, and/or any combination of
amplification reagents described herein.
[0175] The kit can also comprise instructions for diluting an
initial population of templates to be amplified, and/or a dilution
medium.
[0176] III. Uses
[0177] The amplified and/or immobilized nucleic acid molecules
generated from methods described herein can be subjected to many
different applications, including sequencing, screening, diagnosis,
in situ nucleic acid synthesis, monitoring gene expression, nucleic
acid fingerprinting, forensics, diagnostics, etc.
[0178] Any method or plurality of nucleic acids described herein
can be used in providing nucleic acid molecules for sequence
analysis. For example, one or more nucleic acid molecules (e.g.,
amplicons) produced by any method described herein can be contacted
with at least one sequencing analysis primer and/or at least one
probe. Optionally, at least one sequencing analysis primer is or
comprises an oligonucleotide. In an embodiment, at least one probe
comprises one or more nucleotides or one or more oligonucleotides.
Oligonucleotides used as sequence analysis primers or probes for
example can hybridize to at least one of the same sequences as at
least one immobilization primer, e.g., the oligonucleotides
optionally have the same sequences as the immobilization
primers.
[0179] In an embodiment, sequence analysis is performed by
contacting one or more target nucleic acid molecules to be analyzed
with one or more sequence analysis primers and/or probes, removing
any unhybridized probes and determining the label of the ligated
probe.
[0180] In another embodiment, sequence analysis is performed by
contacting one or more target nucleic acid molecules to be analyzed
with one or more sequence analysis primers and/or probes, and
detecting any resulting hybridization between at least one target
nucleic acid and at least one labeled sequence analysis primer
and/or probe. In an embodiment the sequence analysis method
comprises extending one or more sequence analysis primers
hybridized to target nucleic acid molecules by ligating any
adjacently-hybridized oligonucleotides probes to the sequence
analysis primer, removing any unligated probes and determining the
label of the ligated probe.
[0181] In another embodiment, sequence analysis is performed by
contacting one or more target nucleic acid molecules to be analyzed
with one or more sequence analysis primers and labeled nucleotide
probes and a template-dependent polymerase, allowing the polymerase
to incorporate a labeled nucleotide into a polymerase extension
product of the sequence analysis primer, removing any
unincorporated nucleotides, and determining the identity of the
incorporated nucleotide. The nucleotide probes are for example
fluorescently-labeled, and/or the identity of the incorporated
nucleotide is determined from its label. In another embodiment the
nucleotide probe is not labeled and the presence or incorporation
of the nucleotide into a polymerase extension product is measured
by virtue of a by-product generated by incorporation. For example,
incorporation of a nucleotide into an extension product is
optionally detected by measuring changes in pH or ions or an
electric current, for example by using a field-effect
transistor.
[0182] In any method described herein, the probe (e.g., a
nucleotide or an oligonucleotide) is optionally further extendable
by polymerase or ligase. Alternatively, the probe is optionally not
extendable by a polymerase or ligase. Such non-extendable probes
are optionally rendered extendable during the sequence analysis
process (e.g., after their identity is determined). Optionally,
nucleotide probes include A, G, C, T bases, where nucleotide probes
with a different base at the interrogation position of the probe
have a different fluorescent label.
[0183] Optionally, all or less than all (e.g., at least one)
sequencing analysis primer or sequencing analysis probe comprises a
labeled nucleotides/oligonucleotides. Optionally, the labeled
nucleotides/oligonucleotides all have the same label or are
differentially labeled. In an embodiment the labeled
nucleotides/oligonucleotides are fluorescence labeled. For example
a mixture of labeled and non-labeled nucleotides is used.
[0184] Optionally a plurality of different sequences are determined
in parallel. For example, the sequence analysis is parallel
sequence analysis of nucleic acid molecules present in at least 2,
e.g., at least 10, 1000, 1000, 106, 109 or 1012 different distinct
areas. The sequence analysis technology can be polymerase-based
sequence analysis, for example Sanger sequence analysis. Examples
of sequence analysis technology include sequence analysis by
synthesis, or sequence analysis using reversible terminators, or
ligation based sequence analysis, e.g., two-base encoding using
SOLiD.
[0185] Also provided is an apparatus for performing any method
described herein, comprising any one or more reagents or devices
mentioned herein. For example the apparatus can comprise one or
more supports, each support comprising a plurality of immobilized
primers (which are optionally identical). For example the apparatus
can comprise a nucleic acid polymerase or ligase, and/or a
plurality of nucleotides (optionally labeled) and means for
partially separating annealed nucleic acid strands. Exemplary means
for separating annealed nucleic acid strands comprises a controlled
heating means, e.g., a heating means that can maintain the reagents
at a constant temperature. The apparatus optionally comprises a
source of one or more reactants described herein and detector means
for detecting one or more signals produced after one or more of
said reactants have been applied to said nucleic acid molecules.
The means for detecting optionally has sufficient resolution to
distinguish between the distinct immobilization sites of the
support, or between multiple supports (if in the form of
microbeads). Optionally, the apparatus comprises a support which
has primers immobilized on a planar surface.
[0186] In an embodiment the apparatus comprises a charge coupled
device (CCD), which optionally is operatively connected with an
imaging device.
[0187] In an embodiment each immobilization site is a separate
bead, and wherein each bead comprises a clonal population of
amplicons after amplification. Optionally, each bead can be
distributed into or on an array before, during or after
amplification. The array for example is any array of wells, where
beads bearing amplicons are distributed individually into separate
wells. Optionally the array is a large-scale FET array.
[0188] Optionally, at least some of the clonal populations
generated by any method herein appear discrete from each other when
detected or analyzed (e.g., by optical or electrochemical
detection) and/or spatially separated. In an embodiment where
nucleotide incorporation is detected by the generation of H+ ions,
such ions can only diffuse a short distance, for example <100 nm
in a buffer solution. This limited capacity for diffusion ensures
H+ generated in one clonal population will generally not interfere
with nearby clonal populations and will only be significantly
detectable by the sensor directly under the clonal population.
[0189] The following examples are provided purely for illustrative
purposes and are not intended to limit the scope of the present
disclosure or claims.
EXAMPLES
Example 1
[0190] 5'-dual-biotin labeled oligo(dT)35 were bound to DynaBeads
MyOne streptavidin C1 magnetic beads. 80 million oligo(dT)35-bound
beads and 800 ul DNA template at 0.01 pg/ul were mixed in
hybridization buffer. The DNA template sequence was as follows:
[0191] TTTTTTTTTTTTTTTTTTTT CCACTACGCCTCCGCTTTC CTC TCT ATG GGC AGT
CGG TGA TTC GTG GAA GAC GGG GGC AGT CTA TAC CCC TGT GGC GAC CAC TGC
GCG GTG GTT TGC TAG GAG AGA ATG AGG AAC CCG GGG CAG. The following
amplification protocol was performed to hybridize the DNA templates
to the beads: 95.degree. C. 2 min, 50.degree. C. 1 min, 40.degree.
C. 1 min, 30.degree. C. 1 min, 25.degree. C. 2 min. Beads were
washed with washing buffer and the primer was extended with
Exo-Klenow by adding to the washed beads 10.times.NEB buffer2 (40
ul), 25 mM dNTP, 4 ul, Exo-Klenow 40 u/ul, 4 ul, and water, 352 ul,
and incubating at room temperature for 10 minutes. The beads were
washed again and a template-walking reaction was performed as
follows. First, the following reaction mixture was prepared:
[0192] 5 million beads
[0193] 10.times. ThermoPol buffer 10 ul
[0194] 25 mM dNTP 1 ul
[0195] P2 primer (CTG CCC CGG GTT OCT CAT TOT) 50 uM 2 ul
[0196] 100.times.BSA 10 ul
[0197] Bst DNA polymerase large fragment 8 u/ul 10 ul
[0198] H2O 67 ul
[0199] This reaction mix was incubated at 60.degree. C. for 45
minutes and 90 minutes with shaking. Beads were washed and the
amplified DNA on the beads quantified with TaqMan qPCR, using the
following reactants:
TABLE-US-00001 TaqMan forward primer: AGTCGGTGATTCGTGGAAGAC TaqMan
reverse primer: CTCATTCTCTCCTAGCAAACCAC TaqMan probe:
Fam-CCCCTGTGGCGACCAC-NFQ
[0200] The fold of amplification before and after the template
walking reaction was calculated and plotted against reaction time.
Sufficient amplification for detection purposes was seen by 30
minutes or less.
Example 2
[0201] Binding of template to bead was done as described in Example
1, using the following template: TTTTTTTTTTTTTTTTTTTT
CCACTACGCCTCCGCTTTC CTC TCT ATG GGC AGT CGG TGA TTC GTG GAA GAC GGG
GGC AGT CTA TAC CCC TGT GGC GAC CAC TGC GCG GTG GTT TGC TAG GAG AGA
ATG AGG AAC CCG GGG CAG
[0202] A template-walking reaction was performed as described in
Example 1, except that 5 ul of 1 uM P2 primer (CTG CCC CGG GTT CCT
CAT TOT) and 5 ul of 8 u/ul Bst DNA polymerase large fragment was
added to the reaction, and the beads were incubated at a range of
temperatures--specifically, 46.degree. C., 51.degree. C.,
58.degree. C., 60.degree. C., 63.degree. C., 65.degree. C. for 45
minutes with shaking
[0203] The DNA templates on the beads with TaqMan qPCR as described
in Example 1, using the following reagents:
TABLE-US-00002 TaqMan forward primer: AGTCGGTGATTCGTGGAAGAC TaqMan
reverse primer: CTCATTCTCTCCTAGCAAACCAC TaqMan probe:
Fam-CCCCTGTGGCGACCAC-NFQ
[0204] The delta Ct before and after the template walking reaction
was calculated and plotted against reaction temperature, as shown
in FIG. 5. This figure shows the influence of temperature on the
template walking reaction.
Example 3
[0205] Oligo(dT)35 beads were prepared as in Example 1, and 80
million oligo(dT)35-bound beads were incubated with 800 ul of a
mixture of two DNA templates (at equal molar ratio) at 0.04 pg/ul
in hybridization buffer. The DNA template sequence is as
following.
TABLE-US-00003 DNA template 1: TTTTTTTTTTTTTTTTTTTT
CCACTACGCCTCCGCTTTC CTC TCT ATG GGC AGT CGG TGA TTC GTG GAA GAC GGG
GGC AGT CTA TAC CCC TGT GGC GAC CAC TGC GCG GTG GTT TGC TAG GAG AGA
ATG AGG AAC CCG GGG CAG DNA template 2:
TTTTTTTTTTTTTTTTTTTTCCACTACGCCTCCGCTTTCCTCTCTATGGGCAG
TCGGTGATGAAACAGTTGATCATGGACAACCATATTCTGCTGTACGGCCAAGGCGG
ATGTACGGTACAGCAGATACTAAGATGATGAAGAGAATGAGGAACCCGGGGCAG
[0206] Binding of template to beads and template-walking was
performed as described in Example 1, except that 20 ul of 8 u/ul
Bst DNA polymerase large fragment was added, and beads were
incubated at 60.degree. C. for 45 minutes with shaking. The beads
were washed and diluted in a 96-well qPCR plate to approximately 5
beads per well. 96 duplex TaqMan qPCR reactions were performed with
the following primers and probes.
TABLE-US-00004 TaqMan forward primer1: AGTCGGTGATTCGTGGAAGAC TaqMan
reverse primer1: CTCATTCTCTCCTAGCAAACCAC TaqMan probe1:
Vic-CCCCTGTGGCGACCAC-NFQ TaqMan forward primer2:
GAAACAGTTGATCATGGACAACCAT TaqMan reverse primer2:
TCATCTTAGTATCTGCTGTACCGTACAT TaqMan probe2:
Fam-CCGCCTTGGCCGTACAG-NFQ
[0207] FIG. 6 shows the Ct values of the 96 duplex TaqMan qPCR
reactions.
[0208] The following Table 1 summarizes the Ct values. The
experimental percentages of beads populations are consistent with
calculated probabilities based on monoclonal amplification model
and Poison distribution.
TABLE-US-00005 TABLE 1 Positive percentages Probability 96 wells
100 copies cut off counts (%) (%) FAM Ct < 37 7.00 Vic Ct <
34 8.00 FAM Ct < 37 and Vic Ct < 34 1.00 empty wells or beads
81.00 84.38 84.98 single template beads 14.00 14.58 14.41 FAM and
Vic 1.00 1.04 0.61
Example 4
[0209] Beads were prepared as in Example 1, except 5'-dual-biotin
labeled oligo(dA)35 was used instead of Oligo(dt)35 as the
bead-immobilized primer.
[0210] The following DNA template was bound to the prepared beads
as described in Example 1:
TABLE-US-00006 AAAAAAAAAAAAAAAAAAAA CCACTACGCCTCCGCTTTC CTC TCT ATG
GGC AGT CGG TGA TTC GTG GAA GAC GGG GGC AGT CTA TAC CCC TGT GGC GAC
CAC TGC GCG GTG GTT TGC TAG GAG AGA ATG AGG AAC CCG GGG CAG
[0211] The primer was not extended with Exo-Klenow. Template
walking was performed as in Example 1 except that 20 ul of 120 u/ul
Bst DNA polymerase large fragment was added and the beads incubated
at 37 C for 3 minutes and then at 60 C for 45 minutes and 90
minutes with shaking. The amplified DNA on the beads were
quantified using TaqMan qPCR and the following reagents:
TABLE-US-00007 TaqMan forward primer: AGTCGGTGATTCGTGGAAGAC TaqMan
reverse primer: CTCATTCTCTCCTAGCAAACCAC TaqMan probe:
Fam-CCCCTGTGGCGACCAC-NFQ
[0212] The fold of amplification before and after the template
walking reaction was calculated and plotted against reaction time,
as shown in FIG. 7. After 90 minutes of template walking reaction,
the DNA templates on beads were amplified about 100,000 fold.
Example 5
[0213] 5'-dual-biotin labeled oligo(dA)35 were bound to DynaBeads
MyOne streptavidin C1 magnetic beads. 80 million oligo(dA)35-bound
beads were mixed with 800 ul E. coli DH10B genomic DNA fragment
library at 0.1 pg/ul in hybridization buffer. Hybridization buffer
was used as a negative control. The DH10B fragment library had the
following structure.
[0214] AAAAAAAAAAAAAAAAAAAA--P1 adaptor--150 bp to 200 bp DH10B
genomic DNA fragments--P2 adaptor
[0215] The following program was run on a thermo cycler to
hybridize the DNA templates to the beads: 95 C 2 min, 50 C 1 min,
40 C 1 min, 30 C 1 min, 25 C 2 min. The beads were washed and a
template walking reaction was done as follows:
[0216] 5 million beads with DH10B library hybridized, or negative
control beads
[0217] 10.times. ThermoPol buffer 10 ul
[0218] 25 mM dNTP 1 ul
[0219] P2 primer (CTG CCC CGG GTT CCT CAT TCT) 50 uM 2 ul
[0220] 100.times.BSA 10 ul
[0221] Bst DNA polymerase large fragment 120 u/ul 10 ul
[0222] H2O 67 ul
[0223] The beads were incubated at 37 C for 3 minutes and then at
60 C for 45 minutes with shaking. The beads were then washed and
stained with fluorescent label by hybridizing a cy5 labeled
oligonucleotide targeting the P1 adaptor sequence in the DH10B
fragment library. The stained beads were laid on a microscope slide
and imaged on a fluorescent microscope.
[0224] A shifted overlay of the white light image of the beads and
the cy5 fluorescent image of the beads revealed the white light
images of the beads as black dots and the cy5-labeled beads as red
dots. About 15% of the beads were seen to be positively stained
with cy5 and the rest were negative.
[0225] Another exemplary protocol for template walking was perfomed
as follows:
[0226] Hybridization and primer extension: template was diluted in
1.times.NEB Buffer 2 to a final concentration of 100 pM. Template
was heated at 95.degree. C. for 3 min and then keep on ice.
Hybridization/reaction mixes were prepared as follows:
TABLE-US-00008 100 pM denatured template: 48.5 .mu.l 100 mM dNTP:
0.5 .mu.l 5 U/.mu.l Klenow: 1 .mu.l Total volume: 50 .mu.l
[0227] 10 .mu.l of reaction mixture was added to chosen lanes of
StarLight flow cell and the flow cell at 37.degree. C. for 20
minutes. Each lane was washed twice with 1 ml 1.times.TMAX
buffer
[0228] Template walking: A walking reaction mixture was prepared
was follows:
TABLE-US-00009 10x NEB ThermoPol Buffer: 5 .mu.l 100 mM dNTP: 1
.mu.l 50 .mu.M Primer Pe: 0.5 .mu.l 120 U/.mu.l Bst (from NEB): 20
.mu.l H.sub.2O: 23.5 .mu.l Total volume: 50 .mu.l
[0229] 10 .mu.l of reaction mixture was added to each lane, and the
flow cell incubated at 60.degree. C. for 20 minutes. Each lane was
washed twice with 1 ml 1.times.TMAX buffer, and the nucleic acid
subjected to analysis as desired.
Example 6
[0230] This example describes clonal amplification of nucleic acids
onto supports in the form of matrices, followed by sequencing of
the resulting amplified population using the Ion Torrent PGM.TM.
sequencer, which is an ion-based sequencing system that detects
byproducts of nucleotide incorporation using a chemically sensitive
field effect transistor (chemFET). Exemplary embodiments are shown
in FIG. 3. In an embodiment, at least one primer for template
walking is immobilized on a surface (e.g., the floor) of individual
wells, where an individual well is operatively connected to a
corresponding transistor.
[0231] Template Preparation: Libraries were prepared as described
in the Life Technologies SOLiD 4.0 User Manual. Briefly, 2 ug of
genomic DNA isolated from the E. coli strain DH10B was sheared
using a Covaris S2 system. The sheared DNA was end-repaired and was
purified with the SOLiD Library Column Purification Kit.
[0232] A double-stranded adapter ("ION Adaptor 1") was prepared by
hybridizing the single-stranded complementary oligonucleotides ION
Adaptor Oligo 1 and ION Adaptor Oligo 1' to each other according to
the procedure in Appendix C of the User Manual for the SOLiD.TM. 4
System (Applied Biosystems, Life Technologies). ION Adaptor Oligo 1
included a priming sequence that was 30 mer long (i.e., was 30
nucleotides in length) and ION Adaptor Oligo 1' included the
complement of this 30mer priming sequence at the 5' end, plus an
additional two thymidine residues ("TT") at the 3' end.
Hybridization of ION Adaptor Oligo 1 with ION Adaptor Oligo 1'
forms the double-stranded adaptor "Ion Adaptor 1", which includes a
30-mer double-stranded priming sequence and has one blunt end and
one "sticky" end including a 3' overhang of two additional T
residues. A third primer, Ion Adapter A PCR primer, which contained
only the first 20 nucleotides of ION Adaptor Oligo 1, was used in
the PCR step as described below.
[0233] Similarly, a second double-stranded adaptor ("ION Adaptor
2") was prepared by hybridizing the complementary single-stranded
oligonucleotides Ion T-Tailed Adapter Oligo 2 and Ion A-Tailed
Adapter Oligo 2' using the procedure in Appendix C of the SOLiD 4.0
User Manual. ION T-tailed Adaptor Oligo 2 was 52mer in length and
included a priming sequence of 30 nucleotides to the 5' end,
followed by a stretch of 22 thymidine ("T") residues at the 3' end.
ION A-tailed Adaptor Oligo 2' was 50mer in length and included a
stretch of 20 adenine ("A") residues at the 5' end, followed by a
30-nucleotide stretch complementary to the priming sequence of ION
T-tailed Adaptor Oligo 2. Hybridization of the ION T-tailed Adaptor
Oligo 2 with the ION A-tailed Adaptor Oligo 2' forms the
double-stranded adaptor "Ion Adaptor 2", which includes a 30mer
double stranded priming sequence flanking a 20mer poly-A/poly-T
duplex, and has one blunt end and one "sticky" end including a 3'
overhang of two additional T residues.
[0234] The double-stranded adaptors Ion Adaptor 1 and Ion Adaptor 2
were ligated to the end-repaired DNA. The ligation product was
size-selected and subjected to 8 cycles of amplification using the
following primers: Ion Adapter A PCR primer (which contained only
the first 20 nucleotides of ION Adaptor Oligo 1) and ION T-tailed
Adapter Oligo 2. The amplified reaction product was subjected to
size selection, yielding a final adapted double-stranded DNA
library with a median size of 209 bp.
[0235] Isothermal amplification of nucleic acids: The adapted
double-stranded library obtained as described above was mixed with
polymer matrices including 30mer polyA primers (these matrices are
referred to herein as "SNAPPs" for Scaffolded Nucleic Acid Polymer
Particles, and were prepared essentially as described in U.S.
Patent Publication No. 20100304982, Hinz et al.).
[0236] Annealing templates to particles: The double-stranded
library was annealed to the SNAPP particles. Briefly, 10 million
SNAPPs were washed 2.times. in buffer (each wash carried out by
pipetting up and down to resuspend the SNAPPs, then pelleting the
SNAPPs by centrifugation)
[0237] The dsDNA adapter-ligated and size-selected library was
diluted in buffer, such that the library was added at an average
ratio of 1 double-stranded template molecule per SNAPP, in a total
volume of 100 ul.
[0238] The template library was hybridized to the SNAPPs by running
the following thermocycling program: [0239] 95.degree. C. for 3
minutes [0240] 50.degree. C. for 1 minute [0241] 40.degree. C. for
1 minute [0242] 30.degree. C. for 1 minute [0243] 25.degree. C. for
2 minutes
[0244] The template-annealed SNAPPs were washed twice in
buffer.
[0245] Isothermal templating reaction and processing: The annealed
and washed SNAPPs were resuspended in the following amplification
reaction mixture:
[0246] 1.times.Phi29 DNA Polymerase Reaction Buffer, 0.1 mg/mL BSA,
1 uM ION Adaptor A PCR primer, 1 mM equimolar dNTP mix and 100 U
Phi29 enzyme (NEB #M0269L)
[0247] The amplification reaction mixture including the SNAPPs were
then incubated in a thermomixer with 1000 RPM shaking as follows:
[0248] 30.degree. C. for 6 hours [0249] 65.degree. C. for 30
minutes
[0250] The SNAPPS were pelleted and washed 2.times. in Life TEX
Buffer
[0251] The double-stranded amplified DNA on the SNAPPs was then
denatured to remove complementary strands that were not covalently
attached to the SNAPP, by performing 2 washes in ION Denaturing
Solution (125 mM NaOH, 0.1% Tween-20) with a 5 minute incubation at
room temperature for each wash.
[0252] The SNAPPS were then washed 2.times. in Life TEX Buffer (10
mM Tris, 1 mM EDTA, 0.01% Triton X-100). The SNAPPs were then
subjected to an ion-based sequencing reaction using the Ion Torrent
PGM.TM. sequencer, as described below:
[0253] Sequencing Using the Ion Torrent PGM.TM. Sequencer
[0254] The SNAPPs including the amplified DNA were washed 2.times.
in Annealing Buffer, which includes Gibco brand PBS, pH 7.2 (1.54
mM Potassium Phosphate monobasic, 155.17 mM sodium chloride, 2.71
mM sodium phosphate dibasic) with 0.2% Tween-20.
[0255] 5 ul of ION sequencing primer (Part number 602-1021-03, as
provided in the Nucleotides, Enzyme and Controls Kit, Part number
4462916, of the Ion PGM.TM. 314 Sequencing Kit, Part No. 4462909;
Ion Torrent Systems, which is a subsidiary of Life Technologies
Corp., Carlsbad, Calif.) was added to the washed SNAPPs and
hybridized in a thermocycler as follows: [0256] 95.degree. C. for 2
minutes [0257] 37.degree. C. for 2 minutes
[0258] Annealed SNAPPs were washed 2.times. in Annealing Buffer,
with approximately 7 ul of buffer left on the SNAPP pellet after
the final wash.
[0259] 1 ul of Ion Sequencing Polymerase (Part number 602-1023-01,
as provided in the Nucleotides, Enzyme and Controls Kit, Part
number 4462916, of the Ion PGM.TM. 314 Sequencing Kit, Part No.
4462909; Ion Torrent Systems, a subsidiary of Life Technologies,
Carlsbad, Calif.) was added to the SNAPPs and the mixture was
incubated for 5 minutes at room temperature.
[0260] SNAPPs were gently sonicated for 10 seconds in a water bath
to disperse any clumps.
[0261] 2 ul of 50% glycerol (diluted in Annealing Buffer) was added
and the sample was loaded on an Ion Torrent PGM.TM. 314 sequencing
chip from the Ion Sequencing 314 Kit (part number 4462909, Ion
Torrent Systems, Life Technologies. Briefly, the chip was primed by
addition of 50 ul Annealing Buffer (part number 603-1047-01,
included in the PGM Reagents Kit, part number 4465455, Ion Torrent
Systems). 10 ul of sample was injected into the port of the chip,
and the chip was then centrifuged for 10 minutes. The PGM.TM.
sequencing run was performed using standard nucleotide flow order
for 55 cycles.
[0262] Using this method, a total of 21 alignments with a minimum
of Q17 quality (allows up to 2% error) were generated, representing
1391 mapped bases of DH10b sequence. Included in these sequencing
reads were 18 alignments that meet the criteria for Q20 quality
(allows only 1% error), representing 956 mapped bases. The longest
alignment obtained was 109 bases with Q20 quality (1% error, in
this case 1 base mismatch). The longest perfectly mapped read (i.e.
error-free read) was 95 bases long.
Example 7
"Double" Walking
[0263] In this example, both the forward and reverse primers have a
breathable PBS prone to local denaturation at the isothermal
amplification conditions of choice. Each PBS thus has a
corresponding tendency to reassociate with a new unextended primer.
The forward primer is immobilized on a support, and thus the
forward PBS, after dissociating from a first forward primer, can
rehybridize with a second different forward primer that is
immobilized close to the first forward primer. Similarly, the
reverse PBS tends to dissociate from a first soluble reverse primer
and re-hybridize to a second soluble reverse primer, which is then
extended.
[0264] The following amplification reaction was done:
[0265] Hybridization: template at 100 pM total concentration in
1.times.SSPEt buffer, and pre-denatured by heat (95.degree. C. for
3 minutes) and held on ice. 12 ul was added to each lane. The mix
was subjected to 75.degree. C. for 2 min, 50.degree. C. for 1 min,
40.degree. C. for 1 min, 30.degree. C. for 1 min, 25.degree. C. for
2 minutes. The lanes were washed with 0.1.times.SSPEt (2.times.1
ml).
[0266] Primer Extension: The following master mix was prepared: 5
.mu.l of 10.times.NEB Buffer 2; 0.5 .mu.l of 100 mM dNTP; 1 .mu.l
of Klenow (NEB 5 u/ul, M0212L); 43.5 .mu.l of dH2O (Total of 50
.mu.l). 12 uL of reaction mix was added to all lanes, and incubated
at 37 C for 30 min. Lanes were washed with 2.times.SSC+0.1% SDS,
1.times.TMAX (1 ml) and incubated with 1.times.NEB ThermoPol buffer
at 60.degree. C. for 40 min and washed again with 2.times.SSC+0.1%
SDS, 1.times.TMAX (1 ml).
[0267] Template walking: The following master mix was prepared: 10
.mu.l of 10.times.NEB ThermoPol Buffer; 2 .mu.l of 100 mM dNTP; 50
.mu.l of soluble primer PE; 40 .mu.l of Bst (NEB 120 u/ul); 47
.mu.l of dH2O (Total of 100 .mu.l). 12 .mu.L master mix was added
to each lane and incubated at 60.degree. C. for 30 min. 12 .mu.l of
the supernatant was taken out and diluted 1:100, with 5 .mu.l used
for Taqman assays. Lanes were washed with 2.times.SSC+0.1% SDS,
1.times.TMAX (1 ml). In a HiDi wash (2.times.12 uL for 5 min) the
lanes were washed with 1.times.TMAX (2.times.1 ml).
[0268] For the TaqMan reactions, a reaction nmix was prepared with
10 .mu.l of TaqMan Fast Universal Master Mix, No UNG; 1 .mu.l of
20.times. primer probe mix; 4 .mu.l of H2O; 5 .mu.l of Stock
solution (Total: 20 .mu.l). The mix was subjected to 95.degree. C.
(20 sec) and 40 thermocycles (95.degree. C. 3 sec, 60.degree. C. 30
sec), followed by data collection.
[0269] Amplification using two breathable primers (an immobilized
"forward" primer and a soluble distal "reverse" primer, termed Pe
or Pd in Table 2 below) was more efficient than using one
breathable primer alone.
TABLE-US-00010 TABLE 2 Average Cq Supernatant Release
Copy/.mu.m.sup.2 Pc Conc. (uM) 0.8 20.2 14.9 334 Pd conc. (uM) 0.8
20.0 14.3 517 Pe Conc. (uM) 0.2 10.0 11.4 3357 0.3 8.3 11.4 3413
0.4 7.9 11.3 3601 0.5 7.8 10.9 4526
[0270] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description.
[0271] Unless otherwise apparent from the context, any feature can
be claimed in combination with any other, or be claimed as not
present in combination with another feature. A feature can be any
piece of information that can characterize an invention or can
limit the scope of a claim, for example any variation, step,
feature, property, composition, method, step, degree, level,
component, material, substance, element, mode, variable, aspect,
measure, amount, option, embodiment, clause, descriptive term,
claim element or limitation.
[0272] Generally, features described herein are intended to be
optional unless explicitly indicated to be necessary in the
specification. Non-limiting examples of language indicating that a
feature is regarded as optional in the specification include terms
such as "variation," "where," "while," "when," "optionally,"
"include," "preferred," "especial," "recommended," "advisable,"
"particular," "should," "alternative," "typical," "representative,"
"various," "such as," "the like," "can," "may," "example,"
"embodiment," or "aspect," "in some," "example," "exemplary",
"instance", "if" or any combination and/or variation of such
terms.
[0273] Any indication that a feature is optional is intended
provide adequate support (e.g., under 35 U.S.C. 112 or Art. 83 and
84 of EPC) for claims that include closed or exclusive or negative
language with reference to the optional feature. Exclusive language
specifically excludes the particular recited feature from including
any additional subject matter. For example, if it is indicated that
A can be drug X, such language is intended to provide support for a
claim that explicitly specifies that A consists of X alone, or that
A does not include any other drugs besides X. "Negative" language
explicitly excludes the optional feature itself from the scope of
the claims. For example, if it is indicated that element A can
include X, such language is intended to provide support for a claim
that explicitly specifies that A does not include X.
[0274] Non-limiting examples of exclusive or negative terms include
"only," "solely," "consisting of," "consisting essentially of,"
"alone," "without", "in the absence of (e.g., other items of the
same type, structure and/or function)" "excluding," "not
including", "not", "doesn't", "cannot," or any combination and/or
variation of such language.
[0275] Similarly, referents such as "a," "an," "said," or "the,"
are intended to support both single and/or plural occurrences
unless the context indicates otherwise. For example "a dog" is
intended to include support for one dog, no more than one dog, at
least one dog, a plurality of dogs, etc. Non-limiting examples of
qualifying terms that indicate singularity include "a single",
"one," "alone", "only one," "not more than one", etc. Non-limiting
examples of qualifying terms that indicate (potential or actual)
plurality include "at least one," "one or more," "more than one,"
"two or more," "a multiplicity," "a plurality," "any combination
of," "any permutation of," "any one or more of," etc. Claims or
descriptions that include "or" between one or more members of a
group are considered satisfied if one, more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process unless indicated to the contrary or
otherwise evident from the context.
[0276] Furthermore, it is to be understood that the inventions
encompass all variations, combinations, and permutations of any one
or more features described herein. Any one or more features may be
explicitly excluded from the claims even if the specific exclusion
is not set forth explicitly herein. It should also be understood
that disclosure of a reagent for use in a method is intended to be
synonymous with (and provide support for) that method involving the
use of that reagent, according either to the specific methods
disclosed herein, or other methods known in the art unless one of
ordinary skill in the art would understand otherwise. In addition,
where the specification and/or claims disclose a method, any one or
more of the reagents disclosed herein may be used in the method,
unless one of ordinary skill in the art would understand
otherwise.
[0277] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference. Genbank records referenced by GID or
accession number, particularly any polypeptide sequence,
polynucleotide sequences or annotation thereof, are incorporated by
reference herein. The citation of any publication is for its
disclosure prior to the filing date and should not be construed as
an admission that the present invention is not entitled to antedate
such publication by virtue of prior invention.
[0278] Where ranges are given herein, the endpoints are included.
Furthermore, it is to be understood that unless otherwise indicated
or otherwise evident from the context and understanding of one of
ordinary skill in the art, values that are expressed as ranges can
assume any specific value or subrange within the stated ranges in
different embodiments of the invention, to the tenth of the unit of
the lower limit of the range, unless the context clearly dictates
otherwise.
Sequence CWU 1
1
22130DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
30230DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 2tttttttttt tttttttttt tttttttttt
30320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 3tttttttttt tttttttttt 204330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
4naaaaaaaaa anaaaaaaaa aanaaaaaaa aaanaaaaaa aaaanaaaaa aaaaanaaaa
60aaaaaanaaa aaaaaaanaa aaaaaaaana aaaaaaaaan aaaaaaaaaa naaaaaaaaa
120anaaaaaaaa aanaaaaaaa aaanaaaaaa aaaanaaaaa aaaaanaaaa
aaaaaanaaa 180aaaaaaanaa aaaaaaaana aaaaaaaaan aaaaaaaaaa
naaaaaaaaa anaaaaaaaa 240aanaaaaaaa aaanaaaaaa aaaanaaaaa
aaaaanaaaa aaaaaanaaa aaaaaaanaa 300aaaaaaaana aaaaaaaaan
aaaaaaaaaa 330590DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 5caacaacaac aacaacaaca acaacaacaa
caacaacaac aacaacaaca acaacaacaa 60caacaacaac aacaacaaca acaacaacaa
90660DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 6cacacacaca cacacacaca cacacacaca cacacacaca
cacacacaca cacacacaca 607120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7caaacaaaca aacaaacaaa
caaacaaaca aacaaacaaa caaacaaaca aacaaacaaa 60caaacaaaca aacaaacaaa
caaacaaaca aacaaacaaa caaacaaaca aacaaacaaa 120890DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8gaagaagaag aagaagaaga agaagaagaa gaagaagaag aagaagaaga agaagaagaa
60gaagaagaag aagaagaaga agaagaagaa 90935DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9tttttttttt tttttttttt tttttttttt ttttt
3510144DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 10tttttttttt tttttttttt ccactacgcc
tccgctttcc tctctatggg cagtcggtga 60ttcgtggaag acgggggcag tctatacccc
tgtggcgacc actgcgcggt ggtttgctag 120gagagaatga ggaacccggg gcag
1441121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11ctgccccggg ttcctcattc t 211221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12agtcggtgat tcgtggaaga c 211323DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 13ctcattctct cctagcaaac cac
231416DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 14cccctgtggc gaccac 1615163DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
15tttttttttt tttttttttt ccactacgcc tccgctttcc tctctatggg cagtcggtga
60tgaaacagtt gatcatggac aaccatattc tgctgtacgg ccaaggcgga tgtacggtac
120agcagatact aagatgatga agagaatgag gaacccgggg cag
1631625DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16gaaacagttg atcatggaca accat 251728DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17tcatcttagt atctgctgta ccgtacat 281817DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
18ccgccttggc cgtacag 171935DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 19aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaa 3520144DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
20aaaaaaaaaa aaaaaaaaaa ccactacgcc tccgctttcc tctctatggg cagtcggtga
60ttcgtggaag acgggggcag tctatacccc tgtggcgacc actgcgcggt ggtttgctag
120gagagaatga ggaacccggg gcag 1442120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21aaaaaaaaaa aaaaaaaaaa 202232DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22tttttttttt tttttttttt tttttttttt tt 32
* * * * *