(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2005/0142559 A1
`Makrigiorgos
`(43) Pub. Date:
`Jun. 30, 2005
`
`US 2005O142559A1
`
`(54) AMPLIFICATION OF DNA IN A HAIRPIN
`STRUCTURE, AND APPLICATIONS
`(75) Inventor: G. Mike Makrigiorgos, Brookline, MA
`(US)
`Correspondence Address:
`Ronald I. Eisenstein
`NXON PEABODY LLP
`100 Summer Street
`Boston, MA 02110 (US)
`(73) Assignee: Dana-Farber Cancer Institute, Inc.,
`Boston, MA (US)
`
`(21) Appl. No.:
`(22) Filed:
`
`10/758,401
`Jan. 15, 2004
`
`Related U.S. Application Data
`(60) Provisional application No. 60/440,184, filed on Jan.
`15, 2003.
`Publication Classification
`
`(51) Int. Cl. .............................. C12Q 1/68; C12P 19/34
`(52) U.S. Cl. ............................................... 435/6; 435/91.2
`
`(57)
`
`ABSTRACT
`
`The present invention is directed to a hairpin nucleic acid
`Structure and its use. In a preferred embodiment, the hairpin
`nucleic acid Structure can be used in a method of amplifi
`cation of a template nucleic acid Sequence that Substantially
`reduces polymerase-induced errors.
`
`Oxford, Exh. 1006, p. 1
`
`

`

`Patent Application Publication Jun. 30, 2005 Sheet 1 of 6
`
`US 2005/0142559 A1
`
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`Oxford, Exh. 1006, p. 2
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`

`

`Patent Application Publication Jun. 30, 2005 Sheet 2 of 6
`
`US 2005/0142559 A1
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`Oxford, Exh. 1006, p. 3
`
`

`

`Patent Application Publication Jun. 30, 2005 Sheet 3 of 6
`
`US 2005/0142559 A1
`
`3A
`
`Taq Site
`
`Alu site
`
`W
`Ligate Cap1 and Cap2
`in a single reaction
`Cap2
`
`v
`Cap1
`Treat W. uracil glycosylase.
`Add primers, start hairpin PCR
`
`
`
`DURING PCR OPO w
`
`Heating generates
`a strand break at the abasic site
`
`HARPIN SAMPLIFED
`
`Oxford, Exh. 1006, p. 4
`
`

`

`Patent Application Publication Jun. 30, 2005 Sheet 4 of 6
`
`US 2005/0142559 A1
`
`Figure 4A
`primer-binding site
`
`TN
`
`primer-binding site T
`
`Figure 4B
`primer-binding site
`
`primer-binding site
`
`Oxford, Exh. 1006, p. 5
`
`

`

`Patent Application Publication Jun. 30, 2005 Sheet 5 of 6
`
`US 2005/0142559 A1
`
`
`
`
`
`Oxford, Exh. 1006, p. 6
`
`

`

`Patent Application Publication Jun. 30, 2005 Sheet 6 of 6
`
`US 2005/0142559 A1
`
`A)
`S/
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`1A
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`
`Oxford, Exh. 1006, p. 7
`
`

`

`US 2005/0142559 A1
`
`Jun. 30, 2005
`
`AMPLIFICATION OF DNA IN A HARPIN
`STRUCTURE, AND APPLICATIONS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`0001) This application claims benefit under 35 U.S.C. S
`119(e) of U.S. Provisional Application No. 60/440,184, filed
`Jan. 15, 2003, which is incorporated herein by reference.
`
`FIELD OF THE INVENTION
`0002 The present invention is directed to a hairpin
`nucleic acid structures and its use. In a preferred embodi
`ment, the hairpin nucleic acid structure can be used in a
`method of amplification of a template nucleic acid Sequence
`that Substantially reduces polymerase-induced errors.
`
`BACKGROUND OF THE INVENTION
`Substantial interest has been directed to the detec
`0.003
`tion of changes in nucleic acid Sequences, Such as caused by
`mutation and methylation. For example, mutation in certain
`genes have been associated with a variety of disorders
`ranging from blood disorders to cancers. Genetic testing is
`one way to find this information out. However, our ability to
`detect Such mutations is limited by certain problems with a
`key component in these tests, namely the polymerase chain
`reaction (PCR).
`0004. A major problem with PCR is that polymerases
`invariably generate errors during amplification. Such poly
`merase misincorporations can be indistinguishable from
`genuine mutations, and lower the quality of DNA cloning
`and protein functional analysis by in Vitro translation. Poly
`merase misincorporations Set a limit for molecular mutation
`detection methods: the most Selective technologies invari
`ably rely on PCR, but PCR also poses a final selectivity
`limit, typically 1 mutant in 10-10° alleles, since all DNA
`polymerases generate errors during DNA Synthesis which
`can be misinterpreted as mutations (false positives). Thus,
`high Selectivity mutation detection technologies often fall
`Short of the enormous Selectivity needed to address issues
`like the generation of Spontaneous mutations in Somatic
`tissues '', the early detection of genomic instability, the
`mutation screening of single cells" or the reliable detection
`of minimal residual disease .
`. Both unknown and known
`mutation detection methods are affected by PCR errors and
`the most Selective methods are affected most.
`0005 For example, the principal limitation for mutation
`Scanning via constant denaturant capillary electrophoresis
`(CDCE) is the fidelity of the polymerase used 7 . High
`selectivity mutation scanning via DGGE and dHPLC is
`ultimately hindered by polymerase error rate ' ' '. Some of
`the highest sensitivity assays for RFLP-based known muta
`tion detection, including PCR/RE/LCR '', MutEx-ACB
`PCR , Radioactivity-based PCR-RFLP, RSM ' ',
`APRIL-ATM , and others reviewed in Parsons et al. 7,
`utilize PCR in at least one stage prior to RFLP-selection, and
`are therefore also limited by PCR errors .
`0006 Accordingly, it would be desirable if one had a
`means of amplifying DNA free of polymerase-induced mis
`incorporations, to detect mutations without being limited by
`polymerase-induced errors. This could significantly impact
`mutation detection, disease diagnosis, and cancer diagnosis.
`
`SUMMARY OF THE INVENTION
`0007 We have now discovered compositions and meth
`ods to amplify a target nucleic acid Sequence, Sometimes
`referred to as the template, that Substantially reduces poly
`merase induced errors in a Sequence of interest, and which
`can Supply existing technologies with the necessary Selec
`tivity leap. The first step of this method involves converting
`the Sequence of interest into a hairpin, which contains a
`double Stranded region linked at one end through a single
`Stranded loop, and performing PCR on the hairpin-Structure.
`In the second step, the amplified PCR products are heat
`denatured and rapidly cooled, to convert each amplified
`PCR product into a hairpin: genuine polymorphisms or
`mutations will remain fully matched in the hairpin, whereas
`PCR products which contain a PCR induced error will form
`a hairpin that contains a mismatch in the double-Stranded
`region. Thereafter, one removes those amplified nucleic
`acids which contain a mismatch by Standard means. This
`method results in an amplified target nucleic acid which is
`Substantially free of polymerase induced errors.
`0008. In an alternative embodiment, amplification of the
`hairpin Structure is performed using isothermal rolling circle
`amplification (RCA).
`0009 True nucleic acid changes such as from a mutation
`can be separated from polymerase-generated Single nucle
`otide changes, insertions, deletions, or Slippage thereby
`providing practically error-leSS nucleic acid, preferably
`DNA. By using a hairpin sequence one can obtain a sample
`(template) from a range of Sources Such as from genomic
`DNA. Large fractions of the human genome can be ampli
`fied via hairpin PCR to provide faithfully-replicated
`genomic DNA for extensive, genome-wide Screening for
`differences from a standard. This is particularly desirable
`when Starting from limited amounts of biopsy material, i.e.
`from a few cells obtained via laser capture microdissection.
`0010 Additional technical factors limit the overall selec
`tivity of mutation detection (e.g. amount of DNA, mis
`priming; heteroduplex formation; incomplete enzymatic
`digestion'); however, with appropriate selection of condi
`tions these problems can often be overcome. In contrast,
`PCR errors have been regarded as a glass ceiling for
`mutation detection Selectivity. The present method of using
`hairpin PCR will allow a boost to almost every existing
`method for highly Selective mutation detection and lead to
`Studies and diagnostic tests that were impossible with pre
`vious technology by Substantially reducing the number of
`errors that are an artifact of PCR from the sample. This
`method will also improve microSatellite analysis by elimi
`nating polymerase slippage artifacts 19 and will also have
`application in other areas Such as molecular beacons ''f''
`and real time PCR, DNA cloning for protein functional
`analysis by in vitro translation ".
`0011. In one embodiment of the present invention, a
`hairpin with non-complementary ends can be efficiently
`PCR-amplified. In this embodiment, a target DNA sequence
`which needs to be PCR-amplified is first converted to a
`hairpin following ligation of an oligonucleotide cap on one
`end and a pair of non-complementary linkers on the other
`end (See FIG. 1A). Next, primers corresponding to the two
`non-complementary linkers are used in a PCR reaction that
`proceeds by displacing the opposite Strand and amplifying
`the entire complement of the hairpin.
`
`Oxford, Exh. 1006, p. 8
`
`

`

`US 2005/0142559 A1
`
`Jun. 30, 2005
`
`0012. In one preferred embodiment, these primers corre
`sponding to the non-complementary linkers can overlap the
`Sequence of interest, thus conferring Sequence Specificity. In
`this embodiment, exponential PCR amplification of the
`hairpin is enabled and Sequences can be amplified directly
`from human genomic DNA. Following hairpin amplifica
`tion, the PCR product is heat-denatured to allow the hairpins
`to Separate from their complementary Strand, and placed
`rapidly on ice. Because of the Sudden cooling, cross-hybrid
`ization of different hairpins is minimal, and thus the original
`hairpins are reformed, following their amplification.
`0013 By amplifying DNA in a hairpin-formation, poly
`merase-errors practically always end-up forming a mis
`match. Genuine mutations, however, remain fully-matched.
`For example, if the polymerase introduces an A>G mutation
`on the upper Strand of the original Sequence, it is very
`unlikely that, during Synthesis of the bottom Strand of a
`Single hairpin it will perform the exact opposite error (TYC
`mutation) at exactly the complementary-Strand position.
`This can be seen when one looks at the normal probability
`for Such a double-error. Even for a polymerase with a large
`error rate of 10"/base the odds for a double-error event are
`10'x10'x0.25=2.5x10, i.e. less than the expected spon
`taneous mutation rate in somatic tissues '''. On the other
`hand, practically all genuine mutations remain fully matched
`following hairpin-PCR, as these reside in both strands from
`the beginning (FIG. 1A).
`0014 Preferably, the amplified hairpins that contain mis
`matches are efficiently Separated from those that do not,
`using any procedure that recognizes mismatch. Preferred
`methods include dHPLC-mediated fraction collection and
`enzymatic based Separation. Preferably, the hairpin caps are
`removed Subsequent to the Separation of hairpins containing
`mismatches from mismatch-free hairpins, thus allowing the
`original DNA sequence to be recovered. While the amplified
`DNA will have PCR-induced errors Such errors can be
`removed from the amplified Sample, which can now be
`processed for mutation detection without Sensitivity being
`limited by polymerase errors.
`0015. In a further preferred embodiment, DGGE,
`dHPLC, as well as methods based on the mismatch-binding
`protein MutS or Ce1I or resolvases (endo V) or exomu
`cleases are used to separate the fraction of PCR-amplified
`sequences containing polymerase errors 7 '
`'. These
`methods utilize the conversion of homoduplexes to hetero
`duplexes via cross-hybridization of PCR amplified products.
`Previously, both mutations and PCR errors are simulta
`neously converted to mismatches. When mutations are at a
`low frequency, practically all of them are converted to
`mismatches. Thus, Such a means did not discriminate them
`from PCR errors. By the present method mutations and other
`preexisting changes do not appear as mismatches. The
`present method of using a hairpin Structure takes advantage
`of the fact that genuine mutations are witnessed in both
`upper and lower DNA strands while PCR errors occur on
`one Strand at a time. Forcing DNA polymerase to copy both
`Strands in one pass creates a double record of the Sequence.
`Thus, effectively the method boosts the replication fidelity
`and converts PCR errors, but not other changes to mis
`matches.
`0016. The method of the present invention has wide
`applicability. For example, polymerase Slippage errors pro
`
`duce Stutter banding that complicate microSatellite analysis
`of single ', or pooled samples . Scanning for very low
`frequency changes occurring naturally in Somatic tissues (<1
`mutant in 107 alleles, ) or at early stages of carcinogenesis
`will enable identification of tumor Signatures as markers for
`early tumor detection. Identification of low level mutations
`in Somatic tissues will also facilitate elucidation of carcino
`gen-Specific mutational fingerprints following environmen
`tal exposures '. Reliable Screening for traces of 'onco
`mutations' can enhance the clinical and diagnostic
`utility of minimal residual disease detection
`and the
`identification of mutations in bodily excretions
`. For
`investigating the mechanisms of carcinogenesis, determina
`tion of carcinogen-induced mutational spectra in disease
`related genes in non-tumorous tissues can provide evidence
`as to whether a specific mutagenic agent or pathway is
`involved in a particular disease or cancer. This high-Selec
`tivity mutational spectrometry will also help determine
`whether or not a mutator phenotype must be invoked to
`explain the acquisition of multiple mutations in tumor cells
`18.32
`
`0017 Most previous studies of mutational spectra were
`based on phenotypic selection methods (e.g. HPRT, lacz
`assays). These methods preclude analysis of genes and
`human tissues for which Selective conditions cannot be
`devised in in-vitro Single cell Systems. Molecular methods
`with Selectivity comparable to the Spontaneous mutation
`frequency (107-10) that can be applied to all tissues are
`highly desirable
`7. However, the onset of PCR errors
`limits several approaches, such as CDCE, which would
`otherwise have the Sensitivity needed to measure the Spon
`taneous mutation frequency .
`0018) Mutation scanning methods such as DGGE or
`dHPLC are particularly hampered by PCR errors since, by
`detecting all possible mutations, they are more likely than
`RFLP-based methods to encounter misincorporation
`hotspots which result in false positives. Particularly for
`mutation detection from limited Starting material, Such as
`micrometastatic cells or laser capture microdissected
`Samples, very large DNA amplification is required. The error
`rate of conventional PCR is then particularly problematic
`as error containing Sequences can comprise >30% of the
`overall population ', making it almost impossible to iden
`tify genuine mutations. The present method changes that and
`it allows, for example dHPLC to overcome PCR errors and
`to perform reliable mutation analysis when Starting from a
`few cells or from minute, laser capture microdissected
`specimens. RFLP-based methods can now be used to exam
`ine few sites for mutations relative to mutation Scanning
`methods.
`0019. When a sample is limited, such as in minute
`LCM-dissected Samples, it previously was often not possible
`to perform more than a single PCR amplification towards the
`detection of mutations in one gene. With the present method,
`one can now perform mutation Screening in Several genes
`Simultaneously from a single Sample, for disease gene
`discovery or diagnostic applications. This whole genome
`amplification method permits amplification of genomic
`DNA from small tissue samples in an error-free manner. This
`allows repeated multi-gene mutation Screening from large
`collections of minute fresh or paraffin-embedded Samples
`without being limited by available starting material or PCR
`COS.
`
`Oxford, Exh. 1006, p. 9
`
`

`

`US 2005/0142559 A1
`
`Jun. 30, 2005
`
`0020) By removing PCR errors from amplified
`sequences, the present hairpin-PCR permits the use of
`well-established techniques such as dHPLC, CDCE, RFLP
`and microsatellite analysis for detecting traces of mutations
`in minute biopsies and for investigating the origins of cancer
`in human tissues without the introduction of polymerase
`induced errors.
`BRIEF DESCRIPTION OF THE DRAWINGS
`0021 FIGS. 1A-1C outline the generation of error-free
`amplified DNA via hairpin PCR. In FIG. 1A, the scheme for
`removing PCR errors following amplification of DNA in a
`hairpin structure is shown. FIG. 1B shows the expected
`structure and sequence of hairpin A. FIG. 1C shows the
`expected structure and sequence of hairpin D, an oligonucle
`otide encompassing both top and bottom Strands of p53 exon
`9.
`0022 FIGS. 2A-2H show PCR amplification and
`dHPLC separation of hairpin-shaped oligonucleotides. FIG.
`2A, lanes 1-5 show the PCR product of hairpins A,B,C,E,
`and D, respectively. Lanes 6 and 7 of FIG. 2A show
`amplification of hairpin D with only forward or only reverse
`primer. FIG. 2B shows amplification of hairpin C using
`Advantage Titanium(8) (lane 1), Pfu Turbo6 (lane 3) or
`Advantage HF2(R) (lane 5) polymerases respectively; lanes
`2, 4 and 6 are water-controls (no template) in each case.
`FIG. 2C shows quantitative real time PCR of hairpin D:
`curves 1-4, starting material of 1 ng, 100 pg, 10 pg and 1 pg
`respectively. FIG. 2D shows hairpin PCR (lanes 1 and 2, in
`duplicate) followed by denaturation and rapid cooling of the
`product (lanes 3 and 4, in duplicate). FIG.2E shows hairpin
`D amplified with primers that bind the non-complementary
`ends, and either not extending (lane 1) or extending 9 bases
`into the hairpin sequence (lane 2). FIG.2F shows spiking of
`p53 exon 9-containing hairpin D into 100 ng p53-negative
`HL-60 genome, followed by hairpin PCR using Advantage
`Titanium(R) polymerase. Spiking of 0.01 pg hairpin D cor
`responds to adding a single p53 exon 9 allele in the genome.
`Lanes 1-6, hairpin D addition of 0, 0.1, 1, 10, 100, 1000 pg
`respectively. FIG. 2G is similar to FIG. 2F, but using
`Advantage HF2(R) polymerase. Lanes 1-5, hairpin Daddition
`of 0, 0.01, 0.1, 1, 10 pg. FIG. 2H shows dHPLC-based
`separation of 1:1 mixtures of homoduplex and heteroduplex
`hairpins. The threshold of the fraction collector is set on the
`trailing (slowest) portion of the homoduplex.
`0023 FIGS. 3A-3B show conversion of a DNA sequence
`to a hairpin and PCR amplification. FIG. 3A shows the
`procedure used to convert a native DNA sequence, flanked
`by two different restriction sites, into a hairpin with non
`complementary ends that can be amplified. The hairpin
`shaped oligonucleotides Cap1 and Cap2 are ligated to the 5'
`and 3' of both sequence ends. During hairpin PCR, primers
`extending into the sequence are used to confer sequence
`specificity. FIG. 3B shows conversion of a native p53
`sequence flanked by Taq I/Alu I Sites to a hairpin, followed
`by hairpin-PCR. Lane 1: Hairpin-PCR product obtained by
`applying the scheme in FIG. 1A for an isolated p53
`sequence. Lane 2: Hairpin-PCR product obtained by apply
`ing the scheme in FIG. 1A to human genomic DNA, in order
`to directly amplify the same Alu I/Taq I target Sequence
`depicted in Lane 1. Lane 3: AS in lane 2, but omitting the
`addition of ligase from scheme FIG. 1A. Lanes 4 and 5: AS
`in lane 2, but omitting the forward or reverse primer,
`respectively, from PCR.
`
`0024 FIGS. 4A and 4B depict two preferred DNA
`structures. FIG. 4A depicts a DNA structure with a hairpin
`at one and non-complementary ends at the other end. FIG.
`4B depicts a DNA structure with hairpins at both ends of the
`double-stranded DNA.
`0.025 FIG.5 depicts the use of hairpin-shaped DNA as a
`detector for radiation and/or chemical exposures. The DNA
`strand breaks off following a strand break anywhere in the
`shaded area (target), thereby allowing the primers to bind
`and to PCR amplify the DNA segment. The amount of PCR
`amplification is proportional to how many DNA molecules
`undergo strand breaks and therefore it can be used to
`quantify the amount of radiation or chemical agent interact
`ing with the DNA. Finally, the fraction of DNA molecules
`that remain intact can be re-amplified by using primers
`binding to the non-complementary linkers, thereby regen
`erating the original DNA detector molecule.
`0026 FIG. 6 shows amplification of hairpins using roll
`ing-circle amplification (RCA). The hairpin-shaped oligo
`nucleotide of FIG. 6A was self-ligated to form a closed
`“dumbbell-like structure resembling the structures used for
`RNA-interference. The dumbbell was then amplified in an
`isothermal rolling-circle amplification reaction using Phi29
`polymerase (from New England Biolabs) and random prim
`ers. Following digestion of the RCA product with Alu, the
`amplified hairpin-dimer DNA was recovered. FIG. 6B
`shows in lane 1, no Alu digestion; in lane 2, digestion with
`Alu. The amplification is about 1000-fold. In another
`example, the hairpin-shaped oligonucleotide of FIG. 6C
`was self-ligated to form a closed 'dumbbell-like structure,
`and then amplified in an isothermal rolling-circle amplifi
`cation reaction using Phi29 polymerase (from New England
`Biolabs) and random primers. Following digestion of the
`RCA product with Nla-III, the amplified hairpin-dimer DNA
`was recovered. FIG. 6D shows in lane 1, no digestion
`Nla-III; lane 1: with Nla-III digestion). The amplification is
`about 500-fold.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`0027) We have discovered compositions and a method to
`amplify a target nucleic acid sequence, Sometimes referred
`to as the template, that Substantially reduces polymerase
`induced errors in a sequence of interest, and which can
`supply existing technologies with the necessary selectivity
`leap'. The first step of the method involves converting the
`sequence of interest into a hairpin, which contains a double
`stranded region linked at one end through a single Stranded
`loop, and performing PCR on the hairpin-structure. In the
`second step, the amplified PCR products are heat denatured
`and rapidly cooled, to convert each amplified PCR product
`into a hairpin: genuine polymorphisms or mutations will
`remain fully matched in the hairpin, whereas PCR products
`which contain a PCR induced error will form a hairpin that
`contains a mismatch in the double-stranded region. There
`after, one removes those amplified nucleic acids which
`contain a mismatch by standard means. This method results
`in an amplified target nucleic acid which is Substantially free
`of polymerase induced errors.
`0028) Any method of converting the nucleic acid to a
`hairpin with non-complementary ends can be used. AS used
`herein, hairpin structures include hairpins and dumbbells.
`
`Oxford, Exh. 1006, p. 10
`
`

`

`US 2005/0142559 A1
`
`Jun. 30, 2005
`
`Preferably, one uses oligonucleotide caps which, in a
`Single ligation Step allow the conversion of a native DNA
`Sequence to a hairpin with non-complementary ends. For
`example, one transforms the template nucleic acid, prefer
`ably DNA, into a hairpin by capping it at one end, Cap1.
`Cap1 is Sometimes referred to as a joining Structure,
`because once it is ligated to the nucleic acid Sequence of
`interest it joins the upper Strand of the nucleic acid Sequence
`of interest to the lower Strand of the Same nucleic acid
`molecule. Preferably, one caps the template at the other end,
`Cap2. Cap 2 is Sometimes referred to as a priming structure,
`because it contains regions of Single-Stranded nucleic acid to
`which primers can bind to initiate the polymerization reac
`tion. CapS1 and 2 naturally form hairpins on their own, to
`allow their respective ligation to the double stranded DNA
`ends of the template DNA. In addition, Cap2 contains a
`region with two non-complementary Sequences to allow
`Subsequent primer binding. Finally, Cap2 contains a poly
`merase block approximately at the center. This block can
`be one or more Synthetic abasic Sites, or a deoxynucleotide
`analogue that does not allow polymerase Synthesis, or a
`uracil that, upon treatment with uracil glycosylase and
`heating is converted to a Strand break, thus effectively
`providing the polymerase block. Any of the above men
`tioned polymerase blocks will enable the formation of a
`hairpin with non-complementary ends during the Subse
`quent PCR amplification. See FIG. 4. Alternatively, Cap2 or
`the priming Structure can be a pair of oligonucleotides with
`are complementary to each other at the ends ligated to the
`nucleic acid of interest, and non-to each other at their other
`ends.
`0029. In one preferred embodiment of the present inven
`tion, unbalanced concentrations of primers are used during
`PCR (asymmetric PCR) such that the result of amplifica
`tion is a single Stranded nucleic acid product (i.e. monomer
`hairpins) instead of a double Stranded product (dimer hair
`pins). In this embodiment, denaturation-renaturation of the
`DNA is unnecessary.
`0.030. In contrast to the method developed by Jones et al.
`(Jones and Winistorfer, 1992) (“panhandle PCR) where the
`overall Structure is in a Stem-loop shape but the template
`DNA is not in a hairpin formation, the present hairpin PCR
`has the template DNA itself in a hairpin formation. This
`allows replication of both top and bottom strands of the
`template in a Single pass of the DNA polymerase and
`Subsequent conversion of polymerase errors to mismatches.
`0031 Gupte et al., U.S. Pat. Nos. 6,251,610; 6,258,544;
`and 6,087,099, describe the generation of a DNA hairpin
`during PCR, by joining top and bottom DNA strands, in
`order to allow DNA sequencing of both Strands in one pass.
`However, because their procedure requires polymerase
`extension (i.e. PCR) to generate the DNA strand-joining, it
`cannot be used to eliminate PCR errors since by the time the
`two Strands are joined together Some of the errors can have
`already occurred. (i.e. Since they start by a regular PCR
`reaction they have already lost the game in Step 1).
`0.032
`Oligonucleotide primers useful in the present
`invention can be Synthesized using established oligonucle
`otide Synthesis methods. Methods of Synthesizing oligo
`nucleotides are well known in the art. Such methods can
`range from Standard enzymatic digestion followed by nucle
`otide fragment isolation (see for example, Sambrook, et al.,
`
`Molecular Cloning: A Laboratory Manual, Second Edition,
`Cold Spring Harbor, N.Y., (1989), Wu et al, Methods in
`Gene Biotechnology (CRC Press, New York, N.Y., 1997),
`and Recombinant Gene Expression Protocols, in Methods in
`Molecular Biology, Vol. 62, (Tuan, ed., Humana Press,
`Totowa, N.J., 1997), the disclosures of which are hereby
`incorporated by reference) to purely Synthetic methods, for
`example, by the cyanoethyl phosphoramidite method using
`a Milligen or Beckman System 1 Plus DNA synthesizer (for
`example, Model 8700 automated synthesizer of Milligen
`Biosearch, Burlington, Mass. or ABI Model 380B). Syn
`thetic methods useful for making oligonucleotides are also
`described by Ikuta et al., Ann. Rev. Biochem. 53:323-356
`(1984), (phosphotriester and phosphite-triester methods),
`and Narang et al., Methods Enzymol., 65:610-620 (1980),
`(phosphotriester method). Protein nucleic acid molecules
`can be made using known methods Such as those described
`by Nielsen et al., Bioconjug. Chem. 5:3-7 (1994).
`0033 AS used herein, the term “primer' has the conven
`tional meaning associated with it in Standard nucleic acid
`procedures, i.e., an oligonucleotide that can hybridize to a
`polynucleotide template and act as a point of initiation for
`the Synthesis of a primer extension product that is comple
`mentary to the template Strand.
`0034. Many of the oligonucleotides described herein are
`designed to be complementary to certain portions of other
`oligonucleotides or nucleic acids Such that Stable hybrids
`can be formed between them. The Stability of these hybrids
`can be calculated using known methods Such as those
`described in Lesnick and Freier, Biochemistry 34:10807
`10815 (1995), McGraw et al., Biotechniques 8:674-678
`(1990), and Rychlik et al., Nucleic Acids Res. 18:6409-6412
`(1990).
`0035. The template nucleic acid that is to be amplified in
`a hairpin formation is preferably DNA, but it can also be
`RNA or a synthetic nucleic acid. The template can be of any
`size, but preferably of a size that can be replicated by DNA
`or RNA polymerases; most preferably the template in the
`region 50 bp-1000 base pairs.
`0036) The nucleic acid target can be any double stranded
`nucleic acid which is capable of being amplified.
`0037. The target nucleic acid can be from any source,
`Such as a PCR product of a known gene or a preparation of
`genomic DNA. The preferred target nucleic acid is DNA,
`but MRNA can also be used. The DNA can be any mixture
`containing one or various sizes of DNA, such as cDNA
`synthesized from the whole MRNA collected from cells that
`need to be Screened for mutation/polymorphism; or fractions
`thereof; or the whole genomic DNA collected from cells that
`need to be Screened for mutation/polymorphism; or fractions
`thereof; or any combination of the above digested into
`Smaller pieces by enzymes.
`0038 Any method of amplifying a nucleic acid target can
`be used. The amplification reaction can be polymerase chain
`reaction (PCR), ligase chain reaction (LCR), Strand dis
`placement amplification (SDA), transcription mediated
`amplification (TMA), O?3-replicase amplification (Q-beta),
`or rolling circle amplification (RCA).
`0039) Preferably, PCR is used to amplify the nucleic acid
`target.
`
`Oxford, Exh. 1006, p. 11
`
`

`

`US 2005/0142559 A1
`
`Jun. 30, 2005
`
`0040 Any polymerase which can synthesize the desired
`nucleic acid may be used. Preferred polymerases include but
`are not limited to Sequenase, Vent, and Taq polymerase.
`Preferably, one uses a high fidelity polymerase (Such as
`Clontech HF-2) to minimize polymerase-introduced muta
`tions.
`0041. In one preferred embodiment, rolling circle ampli
`fication (RCA) is used to amplify the nucleic acid template.
`Rolling circle amplification (RCA) is an isothermal process
`for generating multiple copies of a Sequence. In rolling circle
`DNA replication in vivo, a DNA polymerase extends a
`primer on a circular template (Komberg, A. and Baker, T. A.
`DNA Replication, W. H. Freeman, New York, 1991). The
`product consists of tandemly linked copies of the comple
`mentary Sequence of the template. RCA is a method that has
`been adapted for use in vitro for DNA amplification (Fire, A.
`and Si-Oun Xu, Proc. Natl. Acad Sci. USA, 1995, 92:4641
`4645; Lui, D., et al., J. Am. Chem. Soc., 1996, 118:1587
`1594; Lizardi, P. M., et al., Nature Genetics, 1998, 19:225
`232; U.S. Pat. No. 5,714,320 to Kool).
`0042. In RCA techniques a primer sequence having a
`region complementary to an amplification target circle
`(ATC) is combined with an ATC. Following hybridization,
`enzyme, dNTPs, etc. allow extension of the primer along the
`ATC template, with DNA polymerase displacing the earlier
`Segment, generating a Single Stranded DNA product which
`consists of repeated tandem units of the original ATC
`Sequence. RCA techniques are well known in the art, includ
`ing linear RCA (LRCA). Any such RCA technique can be
`used in the present invention.
`0043. When RCA is used to amplify the hairpin structure,
`Cap2 should not contain a polymerase block in order to
`allow the enzyme to continuously perform DNA synthesis
`on the circularized DNA template. In this approach, follow
`ing ligation of Cap 1 and Cap 2 a polymerase reaction is
`initiated by addition of a single primer that binds to the Cap
`2 non-complementary region. The polymerase then extends
`the primer by performing numerous circles around the
`original template, and resulting in a DNA amplification that
`copies both DNA strands every time it performs a full circle.
`Similar to non-isothermal amplification, during isothermal
`amplification too, every time there is a polymerase error
`during amplification it will form a mismatch while genuine
`changes such as mutations will be fully matched. Follow
`ing amplification, the original DNA sequence can be recov
`ered with a restriction digestion which separates the DNA
`caps introduced in the first Step of the procedure.
`0044) It is possible that instead of ligating Cap1 and Cap2
`to the template DNA for the purpose of generating a hairpin
`Structure with non complementary ends, the same result can
`be achieved via utilization of the first few steps described in
`t