(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2005/0112631 A1
`(43) Pub. Date:
`May 26, 2005
`Piepenburg et al.
`
`US 20050112631A1
`
`(54) RECOMBINASE POLYMERASE
`AMPLIFICATION
`
`(76) Inventors: Olaf Piepenburg, St. Albans (GB);
`Colin H. Williams, St. Albans (GB);
`Niall A. Armes, Fulbourn (GB); Derek
`L. Stemple, St. Albans (GB)
`
`Correspondence Address:
`MINTZ LEVIN COHN FERRIS GLOVSKY &
`POPEO
`666 THIRD AVENUE
`NEW YORK, NY 10017 (US)
`
`(21) Appl. No.:
`
`10/931,916
`
`(22) Filed:
`
`Sep. 1, 2004
`
`Related US. Application Data
`
`(63) Continuation-in-part of application No. 10/371,641,
`?led on Feb. 21, 2003.
`
`(60) Provisional application No. 60/553,999, ?led on Mar.
`16, 2004. Provisional application No. 60/358,563,
`?led on Feb. 21, 2002.
`
`Publication Classi?cation
`
`(51) Int. Cl? .......................... .. C12Q 1/68; c12P 19/34;
`C12N 9/22
`(52) Us. 01. ............................ .. 435/6; 435/91.2; 435/199
`
`(57)
`
`ABSTRACT
`
`This disclosure describe three related novel methods for
`Recombinase-Polymerase Ampli?cation (RPA) of a target
`DNA that exploit the properties of recornbinase and related
`proteins, to invade double-stranded DNA With single
`stranded homologous DNA permitting sequence speci?c
`priming of DNA polymerase reactions. The disclosed meth
`ods have the advantage of not requiring thermocycling or
`thermophilic enzymes. Further, the improved processivity of
`the disclosed methods may alloW ampli?cation of DNA up
`to hundreds of megabases in length.
`
`1
`
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`Patent Application Publication May 26, 2005 Sheet 44 0f 44
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`US 2005/0112631 A1
`
`May 26, 2005
`
`RECOMBINASE POLYMERASE AMPLIFICATION
`
`RELATED APPLICATIONS
`
`[0001] This application claims the benefit of US. appli-
`cation 60/553,999 filed Mar. 16, 2004, and is a continuation-
`in-part of US. application Ser. No. 10/371,641 filed Feb. 21,
`2003, which claims the benefit of US. Application 60/358,
`563 filed Feb. 21, 2002. All patents and patent applications
`cited in this specification are hereby incorporated by refer-
`ence herein in their entirety.
`
`BACKGROUND OF THE INVENTION
`
`[0002] The ability to amplify DNA lies at the heart of
`modem biological and medical research. This is because
`most molecular biology techniques rely on samples contain-
`ing many identical molecules to increase the sensitivity of an
`assay or to prepare enough material for further processing.
`Among the various nucleic acid amplification techniques,
`polymerase chain reaction (PCR)
`is the most common
`because of its sensitivity and efficiency at amplifying short
`nucleic acid sequences.
`
`[0003] While PCR is of great utility, it is also limited in a
`number of ways. The first limitation of PCR is that it relies
`on multiple cycles of thermal melting (denaturing) at high
`temperatures followed by hybridization and elongation at a
`reduced temperature. To maximize efficiency and to mini-
`mize noise, complex temperature control of multiple reac-
`tions is required. This necessitates the use of a thermocycler
`controllable rapid heating/cooling block made with exotic
`material (e. g., gold plated silver blocks), or a robotic mecha-
`nism to move samples between temperature-controlled
`zones. Because of the high-temperature required to melt
`DNA in physiological salt conditions, PCR technology
`requires either the addition of fresh polymerase per cycle or
`the use of thermostable polymerases. The approach of
`adding fresh polymerase has not been automated and is thus
`labor intensive and prone to errors (e.g., contamination,
`dropped tubes, labeling errors). Furthermore, the need to add
`enzymes and to mix each reaction individually presents
`serious drawbacks that have limited adaptation of enzyme-
`addition PCR methods to the small scale.
`
`[0004] Compared to methods involving the addition of
`fresh polymerase, the use of thermostable polymerases in
`PCR is the most widely practiced. This approach suffers
`from the fact that thermostable polymerases are found in a
`limited number of organisms, and the replication mecha-
`nisms used by thermophilic organisms are poorly under-
`stood. The available repertoire of thermostable polymerases
`is
`limited to single polypeptide polymerase enzymes
`involved in DNA repair, and/or lagging strand synthesis.
`DNA repair and/or lagging strand polymerases are poor
`choices for DNA amplification because they exhibit poor
`processivity (distributive synthesis). In part as a conse-
`quence of using repair and/or lagging strand polymerases
`(e.g. Taq, Pfu, Vent polymerases), and due to the formation
`of inhibitory secondary or tertiary nucleic acid structures
`following thermal melting, current PCR protocols do not
`readily amplify sequences longer than several thousands of
`base pairs. Reliable synthesis (and amplification) of longer
`templates will rely on polymerases and auxiliary enzymatic
`complexes collectively exhibiting much higher levels of
`processivity, strand displacement, and secondary structure
`
`resolution, as well as limiting the formation of inhibitory
`higher order nucleic acid structures that may form on
`cooling heat-denatured DNA.
`
`[0005] A second limitation of PCR is that it relies on
`solution hybridization between oligonucleotides (PCR prim-
`ers) and denatured template DNA (i.e.,
`the DNA to be
`amplified) in an aqueous environment. To be effective, PCR
`reactions are performed in a short time because the thermo-
`stable polymerases have a rapidly declining activity at PCR
`temperatures. Further, for effective hybridization in a short
`time, a feature critical to rapid turnaround, it is necessary to
`perform PCR in an environment with high concentrations of
`oligonucleotides. The high oligonucleotide concentration
`also ensures rapid interaction of target sequences with the
`oligonucleotides in competition with the heat-denatured
`complementary strand still present in solution. High oligo-
`nucleotide primer concentrations can cause problems, par-
`ticularly when the copy number of the target sequence is low
`and present in a complex mixture of DNA molecules. This
`would be the case, for example, in a PCR of a genome to
`determine the genetic polymorphism in one locus.
`
`[0006] One problem with using high oligonucleotide con-
`centrations is that it enhances the degree of false priming at
`only partly matched sequences in the complex DNA mix-
`ture. False priming refers to the hybridization of a primer to
`a template DNA in PCR even when the primer sequence is
`not completely complementary to the template nucleic acid,
`which can lead to non-specific amplification of nucleic
`acids. Noise, due to false priming, increases with the oligo-
`nucleotide concentration and the complexity of total starting
`DNA. In addition, the possibility of false priming increases
`as the copy number of target sequences decreases. Where the
`conditions for false priming are favorable (i.e., high oligo-
`nucleotide concentration, high complexity, low copy num-
`ber), errant amplified sequences can become a dominant
`reaction product. Consequently it can be difficult to identify
`conditions, and oligonucleotides, for clean amplification of
`target sequences from a sample DNA without an excess of
`false priming background. Thus a further disadvantage of
`using PCR is the limited success at cleanly amplifying rare
`target DNAs from complex sequences mixtures.
`
`[0007] One solution to the problems of specificity and
`template-melting problem incurred by PCR is to employ
`methods that rely on the biological properties of the bacterial
`RecA recombinase protein, or its prokaryotic and eukaryotic
`relatives. These proteins coat single-stranded DNA (ssDNA)
`to form filaments, which then scan double-stranded DNA
`(dsDNA) for regions of sequence homology. When homolo-
`gous sequences are located,
`the nucleoprotein filament
`strand invades the dsDNA creating a short hybrid and a
`displaced strand bubble known as a D-loop. The free 3'-end
`of the filament strand in the D-loop can be extended by DNA
`polymerases to synthesize a new complementary strand. The
`complementary strand displaces the originally paired strand
`as it elongates. By utilizing pairs of oligonucleotides in a
`manner similar to that used in PCR it should be possible to
`amplify target DNA sequences in an analogous fashion but
`without any requirement for thermal melting (thermocy-
`cling). This has the advantage both of allowing the use of
`heat labile polymerases previously unusable in PCR, and
`increasing the fidelity and sensitivity by template scanning
`and strand invasion instead of hybridization.
`
`46
`
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`

`

`US 2005/0112631 A1
`
`May 26, 2005
`
`[0008] Although the use of RecA and its homologues for
`in vitro amplification of nucleic acids has been previously
`described (US. Pat. No. 5,223,414 to Zarling et a1., referred
`to herein as “Zarling”), the method and results are limited.
`Zarling’s method has critical failings that limit its ability to
`achieve exponential amplification of double-stranded DNA.
`The failure of the Zarling method to achieve exponential
`amplification may be due to its specification for the use of
`ATPyS rather than ATP. The Zarling method urges the use of
`ATPyS, instead of ATP, in the assembly of RecA nucleopro-
`tein filaments because it results in a more stable RecA/
`ssDNA filament
`structure. Normally,
`filaments
`are
`assembled in a 5'
`to 3' direction and will spontaneously
`disassemble in the same 5' to 3' direction as RecA hydro-
`lyzes ATP. This process is dynamic in that assembly and
`disassembly occurs at the same time and the amount of
`assembled filaments is at equilibrium. If the non-hydolyz-
`able ATP analog, ATPyS, is used, hydrolysis of the ATPyS
`and the 5' to 3' disassembly of the filaments are inhibited.
`The great stability of RecA/ATPyS filaments, both before
`and after strand exchange, while helpful in the method of
`targeting (i.e.,
`the Zarling method)
`is detrimental and
`unpractical for DNA amplification.
`
`In the Zarling method, RecA protein involved in
`[0009]
`strand invasion will remain associated with the double-
`
`stranded portion of the exchanged material after strand
`exchange. This interaction occurs because the newly formed
`duplex is bound in the high-affinity site of RecA. The
`displaced strand occupies a different low-affinity site, unless
`it is bound to another single-stranded DNA binding protein
`(SSB), such as E. coli SSB. If ATP had been utilized to
`generate the exchange structure, spontaneous 5' to 3' disas-
`sembly might occur, although the exchange complex can be
`quite stable and may require additional factors to stimulate
`ATP-dependent disassembly. Regardless of whether sponta-
`neous or stimulated,
`in the presence of ATPyS, 5'
`to 3'
`disassembly of the RecA filament is inhibited (Paulus, B. F.
`and Bryant, F. R. (1997). Biochemistry 36, 7832-8; Rosselli,
`W. and Stasiak, A. (1990). J Mol Biol 216, 335-52; Shan, Q.
`et a1., (1997). J Mol Biol 265, 519-40).
`
`[0010] These RecA/dsDNA complexes are precisely the
`sites targeted by the RecA/ssDNA primer complexes used to
`initiate subsequent rounds of invasion and synthesis. Indeed,
`with the RecAbound, the intermediate may not be accessible
`to polymerase, and certainly the dsDNAs can no longer be
`invaded by RecA/ssDNA primer complexes and are there-
`fore not amplifiable from this point. Further synthesis from
`these templates might occur if initiated at the other end of
`the template, which is free of RecA, and this might even-
`tually lead to physical displacement of the bound RecA. It
`is not clear, however, whether many polymerases can dis-
`place RecA in this manner. Moreover, the initiation site for
`that synthetic round will now be ‘blocked’ instead. In such
`a situation, amplification is only linear with time, and wil