U.S. patent application number 14/705150 was filed with the patent office on 2015-08-27 for recombinase polymerase amplification reagents and kits.
The applicant listed for this patent is Alere San Diego Inc.. Invention is credited to Niall A. Armes, Olaf Piepenburg.
Application Number | 20150240298 14/705150 |
Document ID | / |
Family ID | 43298213 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150240298 |
Kind Code |
A1 |
Piepenburg; Olaf ; et
al. |
August 27, 2015 |
RECOMBINASE POLYMERASE AMPLIFICATION REAGENTS AND KITS
Abstract
This disclosure describes kits, reagents and methods for
Recombinase Polymerase Amplification (RPA) of a target DNA that
exploit the properties of recombinase and related proteins, to
invade double-stranded DNA with single stranded homologous DNA
permitting sequence specific priming of DNA polymerase reactions.
The disclosed kits, reagents and methods have the advantage of not
requiring thermocycling or thermophilic enzymes, thus offering easy
and affordable implementation and portability relative to other
amplification methods.
Inventors: |
Piepenburg; Olaf;
(Cambridge, GB) ; Armes; Niall A.; (Essex,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alere San Diego Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
43298213 |
Appl. No.: |
14/705150 |
Filed: |
May 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13375264 |
Feb 9, 2012 |
9057097 |
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PCT/US2010/037611 |
Jun 7, 2010 |
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14705150 |
|
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61184397 |
Jun 5, 2009 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12; 536/123.13 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 1/6848 20130101; C12Q 2527/137 20130101; C12Q 2527/125
20130101; C12Q 2521/507 20130101; C12Q 2527/137 20130101; C12Q
2521/507 20130101; C12Q 2549/101 20130101; C12Q 2549/101 20130101;
C12Q 1/6848 20130101; C12Q 2527/125 20130101; C12Q 1/6848 20130101;
C12Q 1/6806 20130101; C12Q 1/6806 20130101; C12Q 2521/507 20130101;
C12Q 1/6844 20130101; C12Q 2521/507 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-39. (canceled)
40. A frieze dried pellet for a recombinase polymerase
amplification process of DNA amplification of a target nucleic acid
molecule, comprising: trehalose, and wherein said pellet does not
contain polyethylene glycol.
41. The frieze dried pellet of claim 40, wherein said pellet
comprises 2.5%-7.5% weight/lyophilization mixture volume of
trehalose.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
patent application Ser. No. 13/375,264, filed Feb. 9, 2012, which
is a US National of PCT/US2010/037611, filed Jun. 7, 2010, which
claims the benefit of U.S. Provisional Patent Application No.
61/184,397 filed Jun. 5, 2009, which is hereby incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to reagents and kits, and the
use of such reagents and kits, for the amplification of nucleic
acids. More specifically, the present invention relates to the use
of reagents and kits in recombinase polymerase amplification
processes.
BACKGROUND OF THE INVENTION
[0003] Recombinase Polymerase Amplification (RPA) is a process in
which recombinase-mediated targeting of oligonucleotides to DNA
targets is coupled to DNA synthesis by a polymerase (U.S. Pat. No.
7,270,981 filed Feb. 21, 2003; U.S. Pat. No. 7,399,590 filed Sep.
1, 2004; U.S. Pat. No. 7,435,561 filed Jul. 25, 2006 and U.S. Pat.
No. 7,485,428 filed Aug. 13, 2007, as well as, U.S. application
Ser. No. 11/628,179, filed Aug. 30, 2007; Ser. No. 11/800,318 filed
May 4, 2007 and 61/179,793 filed May 20, 2009; the disclosures of
the foregoing patents and patent applications are each hereby
incorporated by reference in its entirety). RPA depends upon
components of the cellular DNA replication and repair machinery.
The notion of employing some of this machinery for in vitro DNA
amplification has existed for some time (Zarling et al., U.S. Pat.
No. 5,223,414), however the concept has not transformed to a
working technology until recently as, despite a long history of
research in the area of recombinase function involving principally
the E. coli RecA protein, in vitro conditions permitting sensitive
amplification of DNA have only recently been determined (Piepenburg
et al. U.S. Pat. No. 7,399,590, also Piepenburg et al., PlosBiology
2006). Development of a `dynamic` recombination environment having
adequate rates of both recombinase loading and unloading that
maintains high levels of recombination activity for over an hour in
the presence of polymerase activity proved technically challenging
and needed specific crowding agents, notably PEG molecules of high
molecular weight (e.g., Carbowax 20M molecular weight 15-20,000 and
PEG molecular weight 35,000), in combination with the use of
recombinase-loading factors, specific strand-displacing polymerases
and a robust energy regeneration system.
[0004] The RPA technology depended critically on the empirical
finding that high molecular weight polyethylene glycol species
(particularly >10,000 Daltons or more) very profoundly
influenced the reaction behavior. It has previously been discovered
that polyethylene glycol species ranging in size from at least
molecular weight 12,000 to 100,000 stimulate RPA reactions
strongly. While it is unclear how crowding agents influence
processes within an amplification reaction, a large variety of
biochemical consequences are attributed to crowding agents and are
probably key to their influence on RPA reactions.
[0005] Crowding agents have been reported to enhance the
interaction of polymerase enzymes with DNA (Zimmerman and Harrison,
1987), to improve the activity of polymerases (Chan E. W. et al.,
1980), to influence the kinetics of RecA binding to DNA in the
presence of SSB (Lavery P E, Kowalczykowski S C. J Biol Chem. 1992
May 5; 267(13):9307-14). Crowding agents are reported to have
marked influence on systems in which co-operative binding of
monomers is known to occur such as during rod and filament
formation (Rivas et al., 2003) by increasing association constants
by potentially several orders of magnitude (see Minton, 2001). In
the RPA system multiple components rely on co-operative binding to
nucleic acids, including the formation of SSB filaments,
recombinase filaments, and possibly the condensation of loading
agents such as UvsY. Crowding agents are also well known to enhance
the hybridization of nucleic acids (Amasino, 1986), and this is a
process that is also necessary within RPA reactions. Finally, and
not least, PEG is known to drive the condensation of DNA molecules
in which they change from elongated structures to compact globular
or toroidal forms, thus mimicking structures more common in many in
vivo contexts (see Lerman, 1971; also see Vasilevskaya. et. al.,
1995; also see Zinchenko and Anatoly, 2005) and also to affect the
supercoiling free energy of DNA (Naimushin et al., 2001).
[0006] Without intending to be bound by theory, it is likely that
crowding agents influence the kinetics of multiple protein-protein,
protein-nucleic acid, and nucleic acid-nucleic acid interactions
within the reaction. The dependence on large molecular weight
crowding agents for the most substantial reaction improvement
(probably greater than about 10,000 Daltons in size) may reflect a
need to restrict the crowding effect to reaction components over a
certain size (for example oligonucleotides, oligonucleotide:protein
filaments, duplex products, protein components) while permitting
efficient diffusion of others (say nucleotides, smaller peptides
such as UvsY). Further, it may also be that the high molecular
weight preference might reflect findings elsewhere that as PEG
molecular weight increases the concentration of metal ions required
to promote DNA condensation decreases. In any case it is an
empirical finding that RPA is made effective by the use of high
molecular weight polyethylene glycols.
[0007] In addition to a need for specific type of `crowded`
reaction conditions as described above (reaction in the presence of
crowding agents), effective RPA reaction kinetics depend on a high
degree of `dynamic` activity within the reaction with respect to
recombinase-DNA interactions. In other words, the available data
which includes (i) reaction inhibition by ATP-.gamma.-S, or removal
of the acidic C terminus of RecA or UvsX, and (ii) inhibition by
excessive ATP (Piepenburg et al., 2006) suggest that not only is it
important that recombinase filaments can be formed rapidly, but
also important that they can disassemble quickly. This data is
consistent with predictions made in earlier U.S. Pat. No.
7,270,981. Rapid filament formation ensures that at any given
moment there will be a high steady state level of functional
recombinase-DNA filaments, while rapid disassembly ensures that
completed strand exchange complexes can be accessed by
polymerases.
SUMMARY OF THE INVENTION
[0008] The invention provides a kit and reagents for, as well as
methods of, DNA amplification, termed RPA. RPA comprises the
following steps (See FIG. 1): First, a recombinase agent is
contacted with a first and a second nucleic acid primer to form a
first and a second nucleoprotein primer. Second, the first and
second nucleoprotein primers are contacted to a double stranded
target sequence to form a first double stranded structure at a
first portion of said first strand and form a double stranded
structure at a second portion of said second strand so the 3' ends
of said first nucleic acid primer and said second nucleic acid
primer are oriented towards each other on a given template DNA
molecule. Third, the 3' end of said first and second nucleoprotein
primers are extended by DNA polymerases to generate first and
second double stranded nucleic acids, and first and second
displaced strands of nucleic acid. Finally, the second and third
steps are repeated until a desired degree of amplification is
reached.
[0009] In one aspect, embodiments of the present invention provide
compositions and kits for recombinase polymerase amplification
processes of DNA amplification of a target nucleic acid molecule,
which include one or more freeze dried pellets. For example, each
freeze dried pellet includes a combination of the following
reagents in the following concentrations (which unless otherwise
indicated can be the concentration either when reconstituted or
when freeze dried): (1) 1.5%-5% (weight/lyophilization mixture
volume) of polyethylene glycol (e.g., 2.28% (weight/lyophilization
mixture volume) of polyethylene glycol with a molecular weight of
35 kilodaltons); (2) 2.5%-7.5% weight/volume of trehalose (e.g.,
5.7%); (3) 0-60 mM Tris buffer; (4) 1-10 mM DTT; (5) 150-400 .mu.M
dNTPs; (6) 1.5-3.5 mM ATP; (7) 100-350 ng/.mu.L uvsX recombinase;
(8) optionally 50-200 ng/.mu.L uvsY; (9) 150-800 ng/.mu.L gp32;
(10) 30-150 ng/.mu.L Bacillus subtilis Pol I (Bsu) polymerase or S.
aureus Pol I large fragment (Sau polymerase); (11) 20-75 mM
phosphocreatine; and (12) 10-200 ng/.mu.L creatine kinase.
[0010] In another aspect, rehydration buffers for reconstituting
freeze dried pellets for nucleic acid amplification are provided.
In some embodiments, the rehydration buffer for reconstituting the
freeze dried pellets are included with the kits described herein
and, the rehydration buffer includes 0-60 mM Tris buffer, 50-150 mM
Potassium Acetate, and 2.5%-7.5% weight/volume of polyethylene
glycol. In certain embodiments, the kits further include a 160-320
mM Magnesium Acetate solution.
[0011] In certain embodiments of the compositions and kits
described herein, the freeze dried pellets also include the first
and/or the second nucleic acid primers for the RPA process. In
certain embodiments of the foregoing kits, the freeze dried pellets
also include a nuclease. For example, the nuclesase is exonuclease
III (exoIII), endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase
(fpg).
[0012] In certain embodiments of the compositions and kits
described herein, the kits or compositions may further include
positive control primers and target DNA to test the activity of the
kit components. For example, the kit can include a positive control
DNA (e.g., human genomic DNA) and first and second primers specific
for the positive control DNA.
[0013] In another aspect, methods of recombinase polymerase
amplification are provided comprising the following steps: First,
one of the kits or compositions described herein that include one
or more freeze dried pellets and rehydration buffer is provided.
Second, at least one of the freeze dried pellets is reconstituted,
in any order, with the rehydration buffer, the first and the second
nucleic acid primers for the RPA process, the target nucleic acid,
and optionally water to a desired volume. Third, Magnesium (e.g.,
Magnesium Acetate solution) is added to initiate the reaction.
Finally, the reaction is incubated until a desired degree of
amplification is achieved. In some embodiments, this last step
comprises mixing the sample several minutes after the reaction is
initiated.
[0014] In yet another aspect, embodiments of the present invention
also provide methods to control RPA reactions, achieved by
initiating the RPA reaction with the addition of Magnesium (e.g.,
with Magnesium Acetate). For example, the methods include at least
three steps. In the first step, the following reagents are combined
in a solution in the absence of Magnesium: (1) at least one
recombinase; (2) at least one single stranded DNA binding protein;
(3) at least one DNA polymerase; (4) dNTPs or a mixture of dNTPs
and ddNTPs; (5) a crowding agent (e.g., polyethylene glycol); (6) a
buffer; (7) a reducing agent; (8) ATP or ATP analog; (9) optionally
at least one recombinase loading protein; (10) a first primer and
optionally a second primer; and (11) a target nucleic acid
molecule. In the second step, Magnesium is added to initiate the
reaction. In the third step, the reaction is incubated until a
desired degree of amplification is achieved. In certain
embodiments, one or more of the reagents are freeze dried before
the first step.
[0015] In yet another aspect, embodiments of the present invention
also include nucleic acid amplification mixtures for isothermal
nucleic acid amplification. For example, the mixtures include at
least: (1) at least one recombinase; (2) at least one single
stranded DNA binding protein; (3) at least one strand displacing
polymerase DNA polymerase; (4) dNTPs or a mixture of dNTPs and
ddNTPs; (5) ATP or ATP analog; (6) trehalose; (7) optionally at
least one recombinase loading protein; (8) optionally polyethylene
glycol (9) optionally a first primer and optionally a second
primer; and (10) optionally a target nucleic acid molecule.
[0016] In another aspect, embodiments of the present invention
include kits for nucleic acid amplification processes, such as
isothermal nucleic acid amplification processes (e.g., RPA
amplification of DNA) a target nucleic acid molecule, which include
one or more freeze dried pellets. In some embodiments, the freeze
dried pellets comprise polyethylene glycol. For example, the amount
of polyethylene glycol in the freeze dried pellets is an amount to
allow the amplification process to proceed (0.3%-7.5%
weight/lyophilization mixture volume of PEG). In some embodiments,
the freeze dried pellets comprise trehalose. For example, the
amount of trehalose in the freeze dried pellets is 2.5%-7.5%
weight/lyophilization mixture volume of trehalose.
[0017] In yet another aspect, embodiments of the present invention
include any of the freeze dried pellets described herein. In some
embodiments, the freeze dried pellets comprise polyethylene glycol.
For example, the amount of polyethylene glycol in the freeze dried
pellets is an amount to allow the amplification process to proceed
(0.3%-7.5% weight/lyophilization mixture volume of PEG). In some
embodiments, the freeze dried pellets comprise trehalose. For
example, the amount of trehalose in the freeze dried pellets is
2.5%-7.5% weight/lyophilization mixture volume of trehalose.
[0018] In yet another aspect, embodiments of the present invention
include rehydration buffers for reconstituting the freeze dried
pellets described herein. In some embodiments, the rehydration
buffer comprises polyethylene glycol (e.g., 0.3%-7.5% weight/volume
of PEG). In some embodiments, a kit comprising any of the foregoing
rehydration buffers is provided.
[0019] Other embodiments, objects, aspects, features, and
advantages of the invention will be apparent from the accompanying
description and claims. It is contemplated that whenever
appropriate, any embodiment of the present invention can be
combined with one or more other embodiments of the present
invention, even though the embodiments are described under
different aspects of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 schematically depicts an RPA reaction.
[0021] FIG. 2 depicts the structure of an annealed Exo-probe. The
abasic THF residue is cleaved by exonuclease only when the probe is
bound. Cleavage by exonuclease separates the fluorophore and
quencher and generates fluorescent signal.
[0022] FIG. 3 depicts the structure of an annealed LF-probe. The
abasic THF residue is cleaved by Nfo only when the probe is
bound.
[0023] FIG. 4 depicts the structure of an annealed Fpg-probe. The
abasic dR residue is cleaved by fpg only when the probe is bound.
Cleavage by fpg releases the fluorophore from the probe and
generates fluorescent signal.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Brief Description of RPA
[0025] RPA is a method (process) for amplifying DNA fragments. RPA
employs enzymes, known as recombinases, that are capable of pairing
oligonucleotide primers with homologous sequence in duplex DNA. In
this way, DNA synthesis is directed to defined points in a sample
DNA. Using two gene-specific primers, an exponential amplification
reaction is initiated if the target sequence is present. The
reaction progresses rapidly and results in specific amplification
from just a few target copies (such as less than 10,000 copies,
less than 1000 copies, less than 100 copies or less than 10 copies)
to detectable levels within as little as 20-40 minutes.
[0026] RPA reactions contain a blend of proteins and other factors
that are required to support both the activity of the recombination
element of the system, as well as those which support DNA synthesis
from the 3' ends of olignucleotides paired to complementary
substrates. The key protein component of the recombination system
is the recombinase itself, which may originate from prokaryotic,
viral or eukaryotic origin. Additionally, however, there is a
requirement for single-stranded DNA binding proteins to stabilize
nucleic acids during the various exchange transactions that are
ongoing in the reaction. A polymerase with strand-displacing
character is required specifically as many substrates are still
partially duplex in character. Reduction to practice has
established that in order to make the reaction capable of
amplifying from trace levels of nucleic acids precise in vitro
conditions are required that include the use of crowding agents and
loading proteins. A system comprising a bacteriophage T6 UvsX
recombinase (e.g., T6UvsXH66S), a bacteriophage Rb69 UvsY loading
agent, a bacteriophage Rb69 gp32 and a S. aureus Pol I large
fragment has proven to be effective.
[0027] Embodiments of the present invention provide for Recombinase
Polymerase Amplification (RPA)--a method for the amplification of
target nucleic acid polymers. They also provide for a general in
vitro environment in which high recombinase activity is maintained
in a highly dynamic recombination environment, supported by ATP.
One benefit of RPA is that it may be performed without the need for
thermal melting of double-stranded templates. Therefore, the need
for expensive thermocyclers is also eliminated.
[0028] Throughout this specification, various patents, published
patent applications and scientific references are cited to describe
the state and content of the art. Those disclosures, in their
entireties, are hereby incorporated into the present specification
by reference.
[0029] In Recombinase Polymerase Amplification single-stranded, or
partially single-stranded, nucleic acid primers are targeted to
homologous double-stranded, or partially double-stranded, sequences
using recombinase agents, which form D-loop structures. The
invading single-stranded primers, which are part of the D-loops,
are used to initiate polymerase synthesis reactions. A single
primer species will amplify a target nucleic acid sequence through
multiple rounds of double-stranded invasion followed by synthesis.
If two opposing primers are used, amplification of a fragment--the
target sequence--can be achieved.
[0030] The target sequence to be amplified, in any of the
embodiments of the present invention, is preferably a double
stranded DNA. However, the embodiments of the present invention are
not limited to double stranded DNA because other nucleic acid
molecules, such as a single stranded DNA or RNA can be turned into
double stranded DNA by one of skill in the art using known methods.
Suitable double stranded target DNA may be a genomic DNA or a cDNA.
An RPA of the invention may amplify a target nucleic acid at least
10 fold, preferably at least 100 fold, more preferably at least
1,000 fold, even more preferably at least 10,000 fold, and most
preferably at least 1,000,000 fold.
[0031] The terms `nucleic acid polymer` or `nucleic acids` as used
in this description can be interpreted broadly and include DNA and
RNA as well as other hybridizing nucleic-acid-like molecules such
as those with substituted backbones e.g. peptide nucleic acids
(PNAs), morpholino backboned nucleic acids, locked nucleic acid or
other nucleic acids with modified bases and sugars.
[0032] In addition, nucleic acids of embodiments of the present
invention may be labeled with a detectable label. A detectable
label includes, for example, a fluorochrome, an enzyme, a
fluorescence quencher, an enzyme inhibitor, a radioactive label and
a combination thereof.
[0033] Lyophilization of the RPA Reaction
[0034] One advantage of RPA is that the reagents for RPA, may be
freeze dried (i.e., lyophilized) before use. Freeze dried reagents
offer the advantage of not requiring refrigeration to maintain
activity. For example, a tube of RPA reagents may be stored at room
temperature. This advantage is especially useful in field
conditions where access to refrigeration is limited. Freeze dried
reagents also offer the advantage of long term storage without
significant activity loss. For example, a tube of RPA reagents may
be stored at -20.degree. C. for up to six months without
significant activity loss.
[0035] While lyophilization is a well-established process there is
no guarantee that all components of a reaction system will
successfully be co-lyophilized and reconstituted under the same
conditions. We have attempted to lyophilize RPA reactions with and
without various of the final reaction components. The disaccharide
sugar trehalose proves in these experiments to be required to
stabilize the lyophilisate, permitting room temperature storage for
at least 10 days. We have also found that it is preferable to
exclude the salt (e.g., Potassium Acetate) and reduce the buffer
concentration to 25 mM of Tris or less from the lyophilisate, to
maximize its stability--particularly for storage above 0.degree.
C.
[0036] We have also found that, if salt is present in the
lyophilisate, polyethylene glycol is required to stabilize the
lyophilisate. By contrast, if salt is not present, then PEG is not
required to stabilize the lyophilizate, and need only be provided
in the rehydration buffer. A typical RPA reaction will have a final
PEG concentration in the reaction of 5%-6% (w/v).
[0037] In addition trehalose and PEG, the reagents that can be
freeze dried before use can include, at least, the recombinase, the
single stranded DNA binding protein, the DNA polymerase, the dNTPs
or the mixture of dNTPs and ddNTPs, the reducing agent, the ATP or
ATP analog, the recombinase loading protein, and the first primer
and optionally a second primer or a combination of any of
these.
[0038] In some embodiments, the RPA reagents may be freeze dried
onto the bottom of a tube, or on a bead (or another type of solid
support). In use, the reagents are reconstituted with buffer (a)
Tris-Acetate buffer at a concentration of between 0 mM to 60 mM;
(b) 50 mM to 150 mM Potassium Acetate and (c) polyethylene glycol
at a concentration of between 2.5% to 7.5% by weight/volume. If the
primers were not added before freeze drying, they can be added at
this stage. Finally, a target nucleic acid, or a sample suspected
of containing a target nucleic acid is added to begin the reaction.
The target, or sample, nucleic acid may be contained within the
reconstitution buffer as a consequence of earlier extraction or
processing steps. The reaction is incubated until a desired degree
of amplification is achieved.
[0039] We have found that it is possible to increase the
sensitivity of the RPA reaction by agitating or mixing the sample
several minutes (e.g., two, three, four, five or six minutes) after
reconstituting and initiating the reaction. For example, after
reconstituting and initiating the RPA reaction, the tube containing
the RPA reaction is placed into an incubator block set to a
temperature of 37.degree. C. and is incubated for 4 minutes. The
sample is then taken out of the incubator, vortexed and spun down.
The sample is then returned to the incubator block and incubated
for an additional 15-40 minutes.
[0040] In one aspect, embodiments of the present invention comprise
kits for performing RPA reactions. In certain embodiments, the kits
include one or more freeze dried pellets each including a
combination of reagents for performing RPA reactions. In certain
embodiments, the kits comprise 8 freeze dried pellets. In some
embodiments, the kits comprise 96 freeze dried pellets. If desired,
the freeze dried reagents may be stored for 1 day, 1 week, 1 month
or 1 year or more before use.
[0041] In certain embodiments, the pellets can be assembled by
combining each reagent in the following concentrations (which
unless otherwise indicated can be the concentration either when
reconstituted or when freeze dried): (1) 1.5%-5%
(weight/lyophilization mixture volume) of polyethylene glycol; (2)
2.5%-7.5% weight/volume of trehalose; (3) 0-60 mM Tris buffer; (4)
1-10 mM DTT; (5) 150-400 .mu.M dNTPs; (6) 1.5-3.5 mM ATP; (7)
100-350 ng/.mu.L uvsX recombinase; (8) optionally 50-200 ng/.mu.L
uvsY; (9) 150-800 ng/.mu.L gp32; (10) 30-150 ng/.mu.L Bsu
polymerase or Sau polymerase; (11) 20-75 mM phosphocreatine; and
(12) 10-200 ng/.mu.L creatine kinase. For example, the reagents in
the solution mixture frozen for lyophilization can have
approximately the following concentrations: (1) 2.28% weight/volume
of polyethylene glycol with a molecular weight of 35 kilodaltons;
(2) 5.7% weight/volume of trehalose; (3) 25 mM Tris buffer; (4) 5
mM DTT; (5) 240 .mu.M dNTPs; (6) 2.5 mM ATP; (7) 260 ng/.mu.L uvsX
recombinase; (8) 88 ng/.mu.L uvsY; (9) 254 ng/.mu.L gp32; (10) 90
ng/.mu.L Bsu polymerase or Sau polymerase; (11) 50 mM
phosphocreatine; and (12) 100 ng/.mu.L creatine kinase. The
reagents may be freeze dried onto the bottom of a tube or in a well
of a multi-well container. The reagents may be dried or attached
onto a mobile solid support such as a bead or a strip, or a
well.
[0042] While it is often preferred that the volume of the reagent
mixture that is frozen and lyophilized is the same as the final
volume of the RPA reaction after rehydration, this is not
necessary. For example, an 80 .mu.L volume of reagents can be
freeze dried, which can then be reconstituted to a final RPA
reaction volume of 50 .mu.L.
[0043] In certain embodiments, the kits further include a
rehydration buffer for reconstituting the freeze dried pellets,
where the rehydration buffer includes 0-60 mM Tris buffer, 50-150
mM Potassium Acetate, and 0.3%-7.5% weight/volume of polyethylene
glycol. For example, the rehydration buffer includes approximately
25 mM Tris buffer, 100 mM Potassium Acetate, and 5.46%
weight/volume of polyethylene glycol with a molecular weight of 35
kilodaltons. In certain embodiments, the kit will comprise 4 mL of
rehydration buffer.
[0044] In certain embodiments, the kits further include a 160-320
mM Magnesium Acetate solution (e.g., about 280 mM Magnesium Acetate
solution). In some embodiments, the kit will comprise 250 .mu.L of
the Magnesium Acetate solution. In other embodiments, the
rehydration buffer itself will comprise 8-16 mM Magnesium Acetate
(e.g., about 14 mM Magnesium Acetate).
[0045] In certain embodiments of the foregoing kits, the freeze
dried pellets also include the first and/or the second nucleic acid
primers for the RPA process. In certain embodiments of the
foregoing kits, the freeze dried pellets also include 50-200
ng/.mu.L of either exonuclease III (exoIII), endonuclease IV (Nfo)
or 8-oxoguanine DNA glycosylase (fpg).
[0046] In any of the foregoing embodiments, the kit may further
include positive control primers and target DNA to test the
activity of the kit components. For example, the kit can include a
positive control DNA (e.g., human genomic DNA) and first and second
primers specific for the positive control DNA.
[0047] In yet another aspect, embodiments of the present invention
also include nucleic acid amplification mixtures for isothermal
nucleic acid amplification. For example, the mixtures include at
least: (1) at least one recombinase; (2) at least one single
stranded DNA binding protein; (3) at least one strand displacing
polymerase DNA polymerase; (4) dNTPs or a mixture of dNTPs and
ddNTPs; (5) ATP or ATP analog; (6) trehalose; (7) optionally at
least one recombinase loading protein; (8) optionally polyethylene
glycol (9) optionally a first primer and optionally a second
primer; and (10) optionally a target nucleic acid molecule.
[0048] In another aspect, embodiments of the present invention
include kits for nucleic acid amplification processes, such as
isothermal nucleic acid amplification processes (e.g., RPA
amplification of DNA) a target nucleic acid molecule, which include
one or more freeze dried pellets. In some embodiments, the freeze
dried pellets comprise polyethylene glycol. For example, the amount
of polyethylene glycol in the freeze dried pellets is an amount to
allow the amplification process to proceed (0.3%-7.5%
weight/lyophilization mixture volume of PEG). In some embodiments,
the freeze dried pellets comprise trehalose. For example, the
amount of trehalose in the freeze dried pellets is 2.5%-7.5%
weight/lyophilization mixture volume of trehalose.
[0049] In yet another aspect, embodiments of the present invention
include any of the freeze dried pellets described herein. In some
embodiments, the freeze dried pellets comprise polyethylene glycol.
For example, the amount of polyethylene glycol in the freeze dried
pellets is an amount to allow the amplification process to proceed
(0.3%-7.5% weight/lyophilization mixture volume of PEG). In some
embodiments, the freeze dried pellets comprise trehalose. For
example, the amount of trehalose in the freeze dried pellets is
2.5%-7.5% weight/lyophilization mixture volume of trehalose.
[0050] In yet another aspect, embodiments of the present invention
include rehydration buffers for reconstituting the freeze dried
pellets described herein. In some embodiments, the rehydration
buffer comprises polyethylene glycol (e.g., 0.3%-7.5% weight/volume
of PEG). In some embodiments, a kit comprising any of the foregoing
rehydration buffers is provided.
[0051] RPA Initiation by Magnesium
[0052] In another aspect, methods of recombinase polymerase
amplification are provided comprising the following steps: First,
one of the foregoing kits that include one or more freeze dried
pellets and rehydration buffer is provided. Second, at least one of
the freeze dried pellets is reconstituted, in any order, with the
rehydration buffer, the first and the second nucleic acid primers
for the RPA process, the target nucleic acid, and optionally water
to a desired volume. Third, Magnesium (e.g., Magnesium Acetate
solution) is added to initiate the reaction. Finally, the reaction
is incubated until a desired degree of amplification is
achieved.
[0053] RPA is a versatile method, but it can be improved by
incorporation of features to control the RPA reaction. Embodiments
of the present invention also provide methods to control RPA
reactions, achieved by initiating the RPA reaction with the
addition of Magnesium (e.g., with Magnesium Acetate). For example,
the method includes at least three steps. In the first step, the
following reagents are combined in a solution in the absence of
Magnesium: (1) at least one recombinase; (2) at least one single
stranded DNA binding protein; (3) at least one DNA polymerase; (4)
dNTPs or a mixture of dNTPs and ddNTPs; (5) a crowding agent (e.g.,
polyethylene glycol); (6) a buffer; (7) a reducing agent; (8) ATP
or ATP analog; (9) optionally at least one recombinase loading
protein; (10) a first primer and optionally a second primer; and
(11) a target nucleic acid molecule. In the second step, Magnesium
is added to initiate the reaction. In the third step, the reaction
is incubated until a desired degree of amplification is achieved.
In certain embodiments, one or more of the reagents are freeze
dried before the first step. Furthermore, it is possible to
initiate a plurality of RPA reactions simultaneously by the
simultaneous addition of Magnesium to each reaction.
EXAMPLES
[0054] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various uses and conditions.
Example 1
Reagents for RPA Reactions
[0055] To form a freeze dried reaction pellet for a typical single
basic RPA reaction, the following RPA reagents with the indicated
concentrations are freeze dried (lyophilized) onto the bottom of a
tube:
BASIC RPA FREEZE DRIED REACTION PELLET
TABLE-US-00001 [0056] Component Concentration PEG 35,000 2.28%
(w/v) Trehalose 5.7% (w/v) UvsX recombinase 260 ng/.mu.L UvsY 88
ng/.mu.L Gp32 254 ng/.mu.L Sau polymerase 90 ng/.mu.L ATP 2.5 mM
dNTPs 240 nM Tris buffer 25 mM DTT 5 mM Phosphocreatine 50 mM
Creatine kinase 100 ng/.mu.L
[0057] For reconstituting the freeze dried reaction pellet, a
rehydration solution is prepared from the following rehydration
buffer:
REHYDRATION BUFFER
TABLE-US-00002 [0058] Component Concentration Tris buffer 25 mM
Potassium Acetate 100 mM PEG 35,000 5.46% (w/v)
[0059] Unlike PCR, which requires small volumes for rapid
temperature change, there is no limit to the reaction volume of
RPA. Reaction volumes of 25 .mu.L, 50 .mu.L, 100 .mu.L, 1 mL, 10 mL
and 100 mL or larger may be performed in one vessel. For the
examples given below, a reaction volume of 50 .mu.L is used.
[0060] To permit monitoring of the RPA reaction, a nuclease may
also be added to each freeze dried reaction pellet. For example,
the "Exo RPA Freeze Dried Reaction Pellet" is the basic RPA
freeze-dried reaction pellet plus 96 ng/.mu.L exonuclease III
(exoIII). Similarly, the "Nfo RPA Freeze Dried Reaction Pellet" is
the basic RPA freeze-dried reaction pellet plus 62 ng/.mu.L
endonuclease IV (Nfo). Finally, the "Fpg RPA Freeze Dried Reaction
Pellet" is the basic RPA freeze-dried reaction pellet plus 114
ng/.mu.L 8-oxoguanine DNA glycosylase (fpg).
[0061] The tubes with the freeze dried pellets can be vacuum-sealed
in pouches, for example in 12 strips of 8 pouches/strip for a total
of 96 RPA reactions. While the vacuum-sealed pouches can be stored
at room temperature for days without loss of activity, long term
storage (up to at least about six months) at -20.degree. C. is
preferred. The rehydration buffer can be stored as frozen aliquots,
for example 4.times.1.2 mL aliquots. For long term storage (up to
at least about six months), storage at -20.degree. C. is preferred.
Unused rehydration buffer can be refrozen, or stored at 4.degree.
C. for up to 1 week. However, excessive freeze-thaw cycles should
be avoided.
Example 2
Basic RPA Reaction
[0062] A basic RPA reaction for each sample is established by
reconstituting the basic RPA freeze-dried reaction pellet of
Example 1 with a suitable rehydration solution. The rehydration
solution is prepared from the rehydration buffer of Example 1,
amplification primers, and template (and water to a total volume of
47.5 .mu.L per sample).
[0063] The components of the rehydration solution can be combined
in a master-mix for the number of samples required. In some
circumstances, for example when performing a primer screen, a
number of different rehydration solutions are to be made (here
according to the number of primer pairs being tested). In that case
components common to all reactions (e.g., template, rehydration
buffer, water) is prepared as a master-mix, distributed in a
corresponding volume into fresh tubes, and is combined with the
required volume of the different primer pairs. The different
rehydration solutions are then used as normal according to the
protocol below.
[0064] The reaction is initiated by the addition of 2.5 .mu.L of a
280 mM Magnesium-Acetate solution, bringing the final reaction
volume to 50 .mu.L per sample.
[0065] For each sample, the rehydration solution is prepared by
adding 2.4 .mu.L of the first primer (10 .mu.M), 2.4 .mu.L of the
second primer (10 .mu.M), the Template and H.sub.2O to a total
volume of 18 .mu.L. 29.5 .mu.L of the rehydration buffer of Example
1 is added. The rehydration solution is then vortexed and is spun
briefly.
[0066] For each sample, the 47.5 .mu.L of rehydration solution is
transferred to a basic RPA freeze-dried reaction pellet of Example
1. The sample is mixed by pipetting up and down until the entire
pellet has been resuspended.
[0067] For each sample, 2.5 .mu.L of 280 mM Magnesium-Acetate is
added and is mixed well. One way to do this simultaneously for many
samples is to place the Magnesium-Acetate into the lid of the
reaction tubes and then spin it down into the tubes to initiate the
reactions. The reaction mixture is vortexed briefly and is spun
down once again.
[0068] The tubes are place into a suitable incubator block (e.g.,
set to a temperature of 37-39.degree. C.) and are incubated for 4
minutes. For ultra-high sensitivity, after 4 minutes, the samples
are taken out of the incubator, vortexed, spun down and returned to
the incubator block. The total incubation time is 20-40 minutes. If
a timecourse of the reaction is desired the incubation time is
adjusted as required. After the reaction is completed, the outcome
of each reaction is typically analyzed by an endpoint method, such
as agarose-gel-electrophoresis.
Example 3
Detection Probes for Use with RPA Reactions
[0069] A detection probe can be used to monitor RPA reactions. The
probe is a third oligonucleotide primer which recognizes the target
amplicon and is typically homologous to sequences between the main
amplification primers. The use of fluorophore/quencher with probes
in real-time detection formats is a very convenient way to monitor
amplification events in RPA reactions.
[0070] RPA technology is compatible with a variety of different
types of oligonucleotide probes. The structures of three
types--Exo-probes, LF-probes, and Fpg-probes--are each discussed
below.
[0071] Exo-Probes
[0072] Exo-probes are generally 46-52 oligonucleotides long. Signal
is generated by an internal dT fluorophore (Fluorescein or TAMRA)
and quenched by an internal dT quencher (typically Black Hole
Quencher (BHQ) 1 or 2) located 1-5 bases 3' to the fluorophore. In
this case, probes are restricted to contain sequences where two
thymines can be found with <6 intervening nucleotides. One of
the bases between the fluorophore and quencher is the abasic
nucleotide analog, tetrahydrofuran (THF--sometimes referred to as a
`dSpacer`). There should be at least 30 nucleotides placed 5' to
the THF site, and at least a further 15 located 3' to it. When the
probe has hybridized to the target sequence, Exonuclease III will
recognize and cleave the THF, thereby separating the fluorophore
and quencher and generating a fluorescent signal. The THF should be
at least 31 bases from the 5' end of the probe and 16 bases from
the 3' end. Finally, the probe is blocked from polymerase extension
by a 3'-blocking group (e.g., Biotin-TEG). FIG. 2 depicts a typical
annealed Exo-probe.
[0073] While there is no fixed rule describing the best position of
a given probe relative to its corresponding amplification primers,
care must be taken to avoid the possibility that primer artefacts
can be detected by the probe. Although primers that have the same
direction as the probe can even overlap its 5' part, this overlap
must not extend up to the fluorophore/abasic-site/quencher portion
of the probe (i.e., the overlap of the primer should be restricted
to the 5'-most 27 nucleotides of the probe or so). This design will
prevent the inadvertent generation of hybridization targets for the
`sensitive` sequence element of the probe by primer artefacts.
Primers opposing the direction of the probe should not overlap to
avoid the occurrence of primer-probe dimers.
[0074] LF-Probes
[0075] LF-probes are often 46-52 oligonucleotides long and intended
for detection of RPA reactions in simple sandwich assays such as
lateral flow strips. The probe is blocked from polymerase extension
by making the last nucleotide a dideoxy nucleotide. As in an
Exo-probe, a THF is typically positioned about 30 bases from the 5'
end of the probe and 16 bases from the 3' end. When the probe has
annealed to the target sequence, Nfo nuclease will recognize and
cleave the THF. This allows the 5' portion of the cut probe to then
act as a primer, ultimately leading to an amplicon containing the
5' portion of the probe conjoined to the opposing primer. The
amplicon is detected by virtue of labels attached to the 5' end of
the opposing primer (usually biotin) and to the 5' end of the probe
(usually FAM). The duplex formed is captured on a surface coated
with the appropriate capture molecule (e.g., streptavidin for
biotin or an anti-FAM antibody for FAM). RPA products are run on
lateral flow strips, such as available from Milenia Biotec. FIG. 3
depicts a typical annealed LF-probe.
[0076] While there is no fixed rule describing the best position of
a given probe relative to its corresponding amplification primers,
care must be taken to avoid the possibility that primer artefacts
can be detected by the probe. Although primers that have the same
direction as the probe can even overlap its 5' part, this overlap
must not extend up to the abasic-site portion of the probe (i.e.,
the overlap of the primer should be restricted to the 5'-most 27
nucleotides of the probe or so). This design will prevent the
inadvertent generation of hybridization targets for the `sensitive`
sequence element of the probe by primer artefacts. Primers opposing
the direction of the probe should not overlap to avoid the
occurrence of primer-probe dimers. The opposing amplification
primer is usually labelled with biotin.
[0077] Fpg-Probes
[0078] Fpg-probes are generally 35 oligonucleotides long. At the 5'
end of the probe is a quencher (typically Black Hole Quencher (BHQ)
1 or 2). Signal is generated by a fluorophore (typically FAM or
Texas Red) attached to the ribose of a base-less nucleotide analog
(a so-called dR residue; a fluorophore/O-linker effectively
replaces the base at the Cl position of the ribose) 4-6 bases
downstream of the 5' end. When the probe has annealed to the target
sequence, fpg will recognize and cleave the dR, thereby releasing
the fluorophore from the probe and generating a fluorescent signal.
Finally, the probe is blocked from polymerase extension by a
3'-blocking group (e.g., Biotin-TEG). FIG. 4 is a schematic of a
typical annealed Fpg-probe. FIG. 7 depicts the structure of an
annealed Fpg-probe. The abasic dR residue is cleaved by fpg only
when the probe is bound. This releases the fluorophore from the
probe and generates fluorescent signal.
[0079] While there is no fixed rule describing the best position of
a given Fpg-probe relative to the amplification primers with which
it is used, care must be taken to avoid the possibility that primer
artefacts can be detected by the probe. As a result any overlap
between primers and the probe should be avoided.
Example 4
RPA Reaction with Real Time Monitoring Using Exonuclease III
[0080] A RPA reaction using exonuclease III is performed using a
modified protocol of Example 2. Each sample is established by
reconstituting the Exo RPA Freeze Dried Reaction Pellet of Example
1 with a suitable rehydration solution. The rehydration solution is
prepared from the rehydration buffer of Example 1, amplification
primers, template and an Exo-probe (and water to a total volume of
47.5 .mu.L per sample). The reaction is initiated by the addition
of 2.5 .mu.L of a 280 mM Magnesium-Acetate solution, bringing the
final reaction volume to 50 .mu.L per sample.
[0081] For each sample, the rehydration solution is prepared by
adding 2.4 .mu.L of the first primer (10 .mu.M), 2.4 .mu.L of the
second primer (10 .mu.M), the Template and 0.6 .mu.L of an
Exo-probe (10 .mu.M) as described in Example 3. H.sub.2O is added
to bring the total volume of the foregoing components to 18 .mu.L.
29.5 .mu.L of the rehydration buffer of Example 1 is added. The
rehydration solution is then vortexed and is spun briefly.
[0082] For each sample, the 47.5 .mu.L of rehydration solution is
transferred to an Exo RPA Freeze Dried Reaction Pellet of Example
1. The sample is mixed by pipetting up and down until the entire
pellet has been resuspended. For each sample, 2.5 .mu.L of 280 mM
Magnesium-Acetate is added and is mixed well to initiate the
reaction.
[0083] The tubes are place into a suitable thermal
incubator/fluorometer (e.g., isothermally set to a temperature of
37-39.degree. C.) and are incubated while fluorescence measurements
are periodically taken. After 4 minutes, the samples are taken out
of the incubator, vortexed, spun down and returned to the
incubator/fluorometer. The total incubation/detection time is 20
minutes.
Example 5
RPA Reaction Using Nfo
[0084] A RPA reaction using Nfo is performed using a modified
protocol of Example 2. Each sample is established by reconstituting
the Nfo RPA Freeze Dried Reaction Pellet of Example 1 with a
suitable rehydration solution. The rehydration solution is prepared
from the rehydration buffer of Example 1, amplification primers,
template and an LF-probe (and water to a total volume of 47.5 .mu.L
per sample). The reaction is initiated by the addition of 2.5 .mu.L
of a 280 mM Magnesium-Acetate solution, bringing the final reaction
volume to 50 .mu.L per sample.
[0085] For each sample, the rehydration solution is prepared by
adding 2.4 .mu.L of the first primer (10 .mu.M), 2.4 .mu.L of the
second primer (10 .mu.M), the Template and 0.6 .mu.L of an LF-probe
(10 .mu.M) as described in Example 3. H.sub.2O is added to bring
the total volume of the foregoing components to 18 .mu.L. 29.5
.mu.L of the rehydration buffer of Example 1 is added. The
rehydration solution is then vortexed and is spun briefly.
[0086] For each sample, the 47.5 .mu.L of rehydration solution is
transferred to an Nfo RPA Freeze Dried Reaction Pellet of Example
1. The sample is mixed by pipetting up and down until the entire
pellet has been resuspended. For each sample, 2.5 .mu.L of 280 mM
Magnesium-Acetate is added and is mixed well to initiate the
reaction.
[0087] The tubes are place into a suitable incubator block (e.g.,
set to a temperature of 37-39.degree. C.) and are incubated for 4
minutes. For ultra-high sensitivity after 4 minutes, the samples
are taken out of the incubator, vortexed, spun down and returned to
the incubator block. The total incubation time is 15-30 minutes.
After the reaction is completed, the outcome of each reaction is
typically analyzed by an endpoint method, such as a sandwich assay
technique.
Example 6
RPA Reaction with Real Time Monitoring Using Fpg
[0088] A RPA reaction using fpg is performed using a modified
protocol of Example 2. Each sample is established by reconstituting
the Fpg RPA Freeze Dried Reaction Pellet of Example 1 with a
suitable rehydration solution. The rehydration solution is prepared
from the rehydration buffer of Example 1, amplification primers,
template and an Fpg-probe (and water to a total volume of 47.5
.mu.L per sample). The reaction is initiated by the addition of 2.5
.mu.L of a 280 mM Magnesium-Acetate solution, bringing the final
reaction volume to 50 .mu.L per sample.
[0089] For each sample, the rehydration solution is prepared by
adding 2.4 .mu.L of the first primer (10 .mu.M), 2.4 .mu.L of the
second primer (10 .mu.M), the Template and 0.6 .mu.L of an
Fpg-probe (10 .mu.M) as described in Example 3. H.sub.2O is added
to bring the total volume of the foregoing components to 18 .mu.L.
29.5 .mu.L of the rehydration buffer of Example 1 is added. The
rehydration solution is then vortexed and is spun briefly.
[0090] For each sample, the 47.5 .mu.L of rehydration solution is
transferred to an Fpg RPA Freeze Dried Reaction Pellet of Example
1. The sample is mixed by pipetting up and down until the entire
pellet has been resuspended. For each sample, 2.5 .mu.L of 280 mM
Magnesium-Acetate is added and is mixed well to initiate the
reaction.
[0091] The tubes are place into a suitable thermal
incubator/fluorometer (e.g., isothermally set to a temperature of
37-39.degree. C.) and are incubated while fluorescence measurements
are periodically taken. After 4 minutes, the samples are taken out
of the incubator, vortexed, spun down and returned to the
incubator/fluorometer. The total incubation/detection time is 20
minutes.
[0092] The details of one or more embodiments of the invention have
been set forth in the accompanying description above. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
Other features, objects, and advantages of the invention will be
apparent from the description and from the claims.
[0093] In the specification and the appended claims, the singular
forms include plural referents unless the context clearly dictates
otherwise. Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
Unless expressly stated otherwise, the techniques employed or
contemplated herein are standard methodologies well known to one of
ordinary skill in the art. All sequence citations, patents, patent
applications and publications cited in this specification are
hereby incorporated by reference herein, including the disclosures
provided by U.S. Pat. No. 7,270,981 filed Feb. 21, 2003; U.S. Pat.
No. 7,399,590 filed Sep. 1, 2004; U.S. Pat. No. 7,435,561 filed
Jul. 25, 2006 and U.S. Pat. No. 7,485,428 filed Aug. 13, 2007, as
well as, U.S. application Ser. No. 11/628,179, filed Aug. 30, 2007;
Ser. No. 11/800,318 filed May 4, 2007 and 61/179,793 filed May 20,
2009.
* * * * *