Patent Application: US-49589504-A

Abstract:
this invention relates to methods , reagents and kits for enriching nucleic acid sequences . more particularly , the present invention relates to methods , reagents and kits for sample preparation including sample modification , sample enrichment and amplification .

Description:
the terms “ nucleic acid ”, “ nucleic acid sequence ”, “ nucleic acid fragment ”, “ nucleic acid segment ”, “ nucleic acid probe ”, “ oligonucleotide ”, “ target nucleic acid sequence ” or “ target sequence ” describe interchangeably and without preference , a plurality of nucleotides , covalently linked as such to form linear molecules of dna or rna . the term “ variant ” describes interchangeably and without preference a nucleic acid encoding a variant , which may for example be selected from the group including any one or more of the following ; a single nucleotide sequence variant , deletion sequence variant , insertion sequence variant , sequence length variants , and sequence variation among paralogous or orthologous nucleic acid sequence , or among edited sequences or splice variants the first approach , described in part in fig1 , is based on cleavage of dna at any predetermined site through the use of so called nucleic acid adapters , hereafter called adapters , that are targets or part of targets for restriction enzymes preferably type ii or type iis restriction enzymes [ 7 , 8 ]. adapters and sample are mixed , denatured and subsequently allowed to cool . the adapters hybridise to their complementary regions in the sample nucleic acid . one of the adapters is positioned so that the resulting cleaved sample dna contains a variant position at the 5 ′ position ( a ). added restriction enzymes cleave the sample and , through addition of a ligation template that anneals to both the 5 ′ and 3 ′ end of the cleaved sample dna , circular molecules are obtained by ligation of the ends that are brought next to each other ( b ). this circularisation is driven by the higher relative concentration of two ends belonging to the same molecule compared to those of two different copies of the same or similar molecules . if the added template is complementary to the sample dna - ends , juxtaposing these , then ligation of the two ends can occur . if a mismatch between the sample dna and the ligation template exists at the variant position used for selection , or if there are no free ends at the site intended for ligation , then ligation will not occur . circularised molecules can then be enriched for through the use of exonucleases that degrade uncircularised dna , and / or amplification of the circularised dna , for example with rolling circle amplification ( rca ) can be performed ([ 9 , 10 ]). alternatively , the adapter could be positioned upstream of the variant position used for selection . optionally this adapter could be completely omitted . after cleavage , as described earlier , one or a plurality of oligonucleotides is added , ( template ), which hybridises to both the 3 ′ end and to an upstream sequence around the variant position , as shown in fig2 a . this provides a specificity step . the structure is then cleaved by chemical , enzyme or other means to generate a structure , as shown in fig2 b . where an enzyme is used , any enzyme capable of cleaving such a structure may be used [ 11 ]. the enzyme is preferably selected from , fen nuclease , mja nuclease , native or recombinant polymerase from thermus aquatiqus , thermus thermophilus , or thermus flavus , or any enzyme selected according to the teachings of lyamichev et al [ 11 ] or u . s . pat . no . 5 , 846 , 717 , which are incorporated herein by reference . the variant position used for selection can either be removed by cleavage , or the cleavage can be performed so that the variant position is the 5 ′- most nucleotide of the sequence . hence the major selective step is in the subsequent ligation reaction . the use of nucleic acid ligation for allele distinction is well described in the literature , for example [ 12 , 13 ]. to ensure that the cleaved substrate is eligible for ligation the 3 ′ sample nucleotide must be complementary to the added template . this can be achieved directly from cleavage of the sample , in which case it is possible to ligate the dna directly . another approach , which confers increased specificity , is to construct the added template so that it contains one extra nucleotide , giving a gap between the hybridised 3 ′ and 5 ′ sequences , similar to that observed for the snp . by adding only the complementary nucleotide to the cleavage reaction a substrate for cleavage will only be generated from nucleic acid sequences that contain the complementary nucleic acid sequence . yet another approach is to construct the added template so that there will be a gap . this gap may be filled in by the addition of a complementary oligonucleotide , as shown in fig2 c . optionally , this gap filling oligonucleotide can be labelled with an affinity tag , for example a specific sequence or specific molecule for subsequent affinity purification . the gap filling oligonucleotide can also be of a specific sequence to be used for circular dna amplification as described in co - pending application pct / se02 / 01378 . cleavage of the sample dna can also be achieved with restriction enzymes through the addition of oligonucleotides that hybridise to the selected sequence . the 5 ′ cleavage site may or may not be influenced by the variable sequence . circularisation and selection is then conducted via any of the above - mentioned approaches . instead of circularising the dna , the nucleic acid fragment ends can be protected via addition of protecting adapters to one or both ends based on selective addition at a variant position at at least one of the ends , as shown in fig3 . generation of the 3 ′ or 5 ′ sample ends could be achieved either through cleavage at the variable position or upstream at a generic site , as previously described . in the latter case cleavage will be performed via structure - specific cleavage as previously described . this protected linear substrate can now be enriched for , through degradation of unprotected sample using exonucleases . selective amplification of the protected allele can be performed based on the presence of the added sequence / sequences . it is not necessary to generate restriction sites in the sample or to denature double stranded dna . any number of restriction enzymes having recognition sequences located on either side but not within the sequence of interest , can be used . double stranded dna can be digested at a multitude of sites with one or several different restriction enzymes . digestion at one or several of the sites may or may not be affected by a sequence variant . if one specific sequence variant affects digestion by a restriction enzyme at a given site , only one of the alleles will become circularised upon ligation with a ligation template in the form a ligation casette . a ligation cassette consists of a pair of prehybridized complementary oligonucleotides with single stranded sequences protruding at one or both ends to form a correct ligation site for the chosen sequences to be ligated . only the circularised allele becomes a template for circular amplification by e . g . rolling circle amplification . if the sample is kept double stranded throughout the process the rca amplified allele will be the only single stranded dna in the sample . this single stranded dna can then be genotpyed by a single strand specific genotyping method such as , including by way of example only , padlock probes , oligonucleotide ligation assay or invader assay . the principle of specifically generate only a subset of a sample single stranded can be utilized with any method capable of performing such an action and is not to be limited to the one mentioned . subsequent analysis with single strand specific methods reveals the genotype of only the selected , and thus single stranded sequence . in one version an exonuclease is added to make one or both ends of a restriction enzyme digested double stranded sample partially single stranded before circularization . a chosen specific sequence is circularized , templated by an added oligonucleotide or pairs of oligonucleotides either directly or via a structure - specific enzyme cut , as described above , followed by specific ligation . the strands are then gap - filled , followed by dna ligation . only the correct allele can be made into a complete circle possible to amplify with rca . a further variant to generate single stranded dna from a restriction enzyme digested of a double stranded sample is to specifically degrade only one of the strands with exonucleases . this can be achieved , by way of example only , through making a proper choice of restriction enzymes that will produce a sticky end that is not a substrate for the chosen exonuclease or exonucleases ; or via dna ligation adding a protecting sequence to one or both of the ends ; or via dna ligation add a chemically modified and protected sequence to one or both of the ends . the single stranded dna can then be circularized , either directly via specific ligation or via a structure - specific enzyme cut followed by specific ligation as previously described . it is also possible to circularise double stranded dna with or without the addition of a ligation cassette , as described earlier , to make one of the strands so it can prime an rca with the intact circularised strand as template . after a predefined time the polymerase is inactivated and an oligonucleotide complementary to a specific part of the amplification product is added so that it creates a restriction enzyme site in one of the alleles . after restriction enzyme digestion the digested allele is recircularised , according to the description in co - pending application pct / se02 / 01378 , making it resistant to exonuclease degradation . after exonuclease treatment , serving to degrade all linear nucleic acids and thereby avoiding branched amplification of the non - circularized allele , a second generation rca is conducted , primed with a second oligonucleotide , effectively amplifying only the circularised allele . the enriched sample can be subjected to genotyping through any method and compared to results from genotyping of the total sample . examples of methods which may be used are oligonucleotide ligation assays [ 12 ], padlock probes [ 13 ], primer extension assays [ 14 ], pyrosequencing [ 15 ], invader technology [ 16 ], mass - spectroscopy [ 17 ] or homogenous pcr methods e . g . taqman [ 18 ] or molecular beacons [ 19 ]. however , other methods may be employed with equal utility . by using the enriched sample instead of a whole sample as the test sample it is also feasible to use any suitable method -, to find new / unknown mutations or polymorphisms . thereby all possible mutations in the enriched segment may be detected , also unknown ones , for example by sanger sequencing or by hybridising the enriched sample to an array in order to resequence the sample and in this respect also find new or unknown mutations . the methods could be , but are not limited to the use of , mismatch recognising enzymes for example t4 endo vii [ 20 ], dhplc resequencing , sanger or array , or pyrosequencing [ 15 ]. however , other methods may be employed with equal utility . the resulting genotypes will reveal the specific haplotype of the sample . accordingly , the present invention provides one or several sets of probes . a first set of probes / probe direct site specific cleavage at predetermined sites of the sample upon hybridisation . a second set of probes / probe is used to specifically modify the sample based upon a sequence variant . a third set of probes is used for amplification of the sample and a fourth set of probes is used for scoring the genotypes . instead of investigating the genotypes all along the selected nucleic acid one can use the same principle for genotyping the variant position used for selection . upon cleavage of sample dna an oligonucleotide can be added that anneals to the 3 ′ end of a generated fragment and to a stretch upstream , around the variant position to be scored , so that a probe with a hybridising region at its 5 ′ end is formed , ( as shown in fig4 a ), or a probe with a non - hybridising region at its 5 ′ end is formed , ( as shown in fig4 b ). if necessary this structure can then be cleaved as previously described . the use of ligase will complete the nucleic acid circle . the circle can then be enriched for , using exonuclease treatment and nucleic acid amplification , preferably rolling circle amplification . preferentially the oligonucleotide added contains a sequence between the 3 ′ and the 5 ′ hybridising end that consist of a selected sequence used for later hybridisation that can be rendered double stranded through the addition of a second oligonucleotide , shown in fig4 a as object 1 . the added oligonucleotide could contain a recognition sequence for a type iis restriction enzyme and preferably a sequence as dissimilar as possible compared to other oligonucleotides used for other loci , as described in co - pending application pct / se02 / 01378 , the contents of which are incorporated herein by reference . detection of the circularised nucleic acid or amplification products templated by the circularised nucleic acid is used to score the genotype of the selected position . due to the intramolecular nature of the ligation reaction it is feasible to perform many reactions at the same time ( from one to several tens of thousands ). at any practical concentration the fragments will circularise intramolecularly in preference to intermolecular reactions . accordingly , the present invention further provides one or a set of probes . a first set of probes / probe directs site - specific cleavage at predetermined sites of the sample upon hybridisation . a second set of probes / probe is used to specifically modify the sample based upon a sequence variant . a third set of probes is used for amplification of the enriched sample . the variant position could be , but is not limited to a sequence variant polymorphism which may be selected from the group including any one or more ; deletion variant , insertion variant , sequence length variant , single nucleotide polymorphism , substitution variant , paralogous or orthologous nucleic acid sequences , edited sequences or splice variants . the present invention is also to be used as a mean to isolate and enrich for a specific sequence or sequences among a multitude of sequences , with the intention of further manipulation of the enriched sequence / sequences . the methods could be any , sole or a combination of but not limited to , amplification , quantification , sequencing , variant scoring , using the enriched sequence / sequences as probes or to compare different enriched samples on the basis of for example amount of sample . accordingly , the present invention further provides one or a set of probes . a first set of probes / probe directs site specific cleavage at predetermined sites of the sample upon hybridisation . a second set of probes / probe is used to specifically modify the sample based upon a nucleotide sequence . a third set of probes is used for amplification of the enriched sample . in all of the above - mentioned methods where dna samples are mentioned they could be exchanged with rna or cdna samples . an added oligonucleotide probe can also be treated by the same principles and to be used for subsequent genotyping , as shown in fig5 , if the added oligonucleotide anneals forming a non - hybridising region at the 5 ′ end . cleavage of this structure will generate a molecule that can be circularised with a ligase . ligation will depend on whether the 5 ′ nucleotide is matched or not with the sample . this circularised probe can then be detected either directly or via the presence of amplification products ( based on the presence of the circle or amplification products of the circle ). the presence of such a product describes the nature of the variant position . the added oligonucleotide could preferentially contain a molecule or sequence in the 5 ′ part that is used as an affinity tag for removal of unmodified circles before amplification of the circularised probes . accordingly , the present invention provides one or a set of probes . a first set of probes to be specifically modified based on the nature of a nucleotide in the target nucleic acid . a second set of probes could be used for purification of the sample . a third set of probes is used for amplification of the modified probes . embodiments of the invention will now be described in greater detail , by way of example only not in any way to limit the invention , with reference to the accompanying drawings , of which ; fig1 is a schematic representation of cleavage and circularisation of sample nucleic acid through the use of adapters ; fig2 is a schematic representation of structure specific cleavage for circularisation of sample nucleic acids ; fig3 is a schematic representation of addition of protecting ends to a linear nucleic acid sample ; fig4 is a schematic representation of the use of gap - oligonucleotides for circularisation of sample nucleic acids ; fig5 is a schematic representation of scoring snps through circularisation of nucleic acid probes ; fig6 shows ( a ) the result from a real - time pcr experiment and ( b ) the gel of the same amplification reactions from an experiment of cleaving , ligating and rolling circle amplification of bac dna ; fig7 is a schematic representation ( a ) of the experimental set - up for detection of circularisation of nucleic acids via inverse pcr and ( b ) a photo of an agarose gel showing the result of such an experiment where bac dna cut with foki adapters , circularised with ligase , circular molecules enriched for via exonucleases and finally used for template in an inverse pcr reaction ; fig8 is showing an image of a poly acrylamide gel of radioactive labelled nucleic acids showing cleavage and ligation of structure specific cleaved nucleic acids with native dna taq polymerase and tth ligase ; and fig9 is showing a photo of an ethidium bromide stained gel of amplification products obtained from an experiment with cleaved bac dna that had been circularised via cleavage by a structure specific enzyme and the two ends joined by a ligase . circularisation of dna after cleavage with restriction enzymes followed by enrichment through exonuclease treatment and rolling circle amplification . ( see fig4 ) a bac clone ( rp11 - 381l18 , bacpac resources , children &# 39 ; s hospital , oakland ) with a genomic fragment containing the gene atp7b was used . dna was isolated by the rapid alkaline lysis miniprep method and the dna concentration was determined measuring uv a 260 . hpaii 5 u ( new england biolabs ) was used to cleave a double stranded ( ds ) template in buffer ( 10 mm tris - hcl ph 7 . 5 , 10 mm mgcl 2 , 1 mm dtt ) for 2 hours at 37 ° c . before heat - inactivation of the enzyme . two pmol of the ds template was cleaved with hpaii . after the cleavage , the reaction was diluted to different concentrations ( 10 4 - 10 8 molecules / μl ). the template was ligated into a circle using 0 . 5 units of t4 dna ligase , 1 × t4 dna ligase bf ( 66 mm tris - hcl ph 7 . 6 , 6 . 6 mm mgcl 2 , 10 mm dtt , 66 μm atp ) and 10 nm ligation template , 5 ′ biotin - tt ttt ttt ttt ttt gtc tgg aaa gca aac cgg tgc cca ccc atg a 3 ′ seq id no1 , in each reaction . after denaturation and subsequent addition of ligase to half of the reactions ( see below ), the samples were incubated at 37 ° c . for 30 min and then the ligase was heat - inactivated at 65 ° c . for 20 minutes after ligation , the samples were treated with exonucleases . exonuclease v ( 5 units ) was used for 30 min 37 ° c . before heat - inactivation . the result was detected by performing a pcr with the following primers , 5 acg ccc acg gct gtc at 3 ′ seq id no2 and 5 ′ tgg acg tct gga aag caa a 3 ′ seq id no3 , ( 1 μm ) located on both sides of the ligation junction . in 50 mm tris hcl ph 8 . 3 , 50 mm kcl , 200 μm dntp , 0 . 125 u taq gold polymerase ( perkin elmer ), 0 . 08 × sybr green ( molecular probes ) as reporter molecule , and 1 × rox ( molecular probes ) as standard , temperature cycles as follows 95 ° c . 10 min activation of taq polymerase followed by 40 cycles of 95 ° c . 20 sec , 52 ° c . 1 min , 72 ° c . 20 sec . the experiments yield a cycle threshold value , ct which is inversely proportional to the amount of starting material in the sample . after the pcr amplification the reactions products were electrophoresed in a 3 % agarose gel to ensure that a product of the correct length had been produced . the results are shown in fig4 , where a ) graph showing the fluorescence readings from a real - time pcr experiment read in an abi 7700 . the figures to the left corresponds to the numberings in b . reactions were as follows ; # 2 , 3 — no template control , # 4 sample + ligase , # 5 sample − ligase , # 6 sample + ligase + rca , # 7 sample − ligase + rca b ) a 3 % agarose gel of the pcr reactions shown in a . lane 1 in b is loaded with a 100 bp - ladder ( lowest band around 50 bp ). lane 2 - 7 corresponds to the same reactions . the arrow denotes the size for a correct length product . enrichment of circular dna over non - circular dna through the use of different exonucleases . bac dna as described in example 1 were cleaved and ligated as described in example 1 . half of the sample was ligated with t4 dna ligase and half of the sample was not . the two reactions were further divided into five different reactions of each (+/− ligase ) treated as follows . 1 5 u exov and 1 mm atp , 2 . 5 u exoi , 50 u exoiii and 25 u t7gene6 3 . 5 u exoi 50 u exoiii and 2 . 45 u lambda exo 4 . 50 u exoiii , 0 . 5 u exovii and 2 . 45 u lambda exo 5 . 5 u exoi , 0 . 5 u exovii in 1 × tris buffer all reactions were incubated at 37 ° c . for 30 minutes before heat inactivation of the nucleases at 80 ° c . for 20 minutes . the results were determined as described in example 1 . after the pcr amplification , the reaction products were electrophoresed in a 3 % agarose gel , and the nucleic acid visualised to ensure that a product of the correct length had been produced ( not shown ). the results are shown in table 1 . circularisation of dna after denaturation of dsdna , hybridisation of foki adapters , cleavage of the dna at predetermined sites , specific circularisation of the cleaved fragment based on an snp at the 5 ′ prime end and enrichment of the circularised dna . ( see fig7 ) bac dna was diluted in a series and denatured by heat . after denaturation the samples were directly put on ice . different amounts ( 10 1 - 10 10 molecules ) of bac dna were cleaved with 2 units foki and 2 fmol foki adapters ( foki adapter 5 ′ utr 5 ′ cgc atc cca cgt ggg atg cga aag caa aca ggg gt 3 ′ seq id no4 , foki adapter c2930t c - allele 5 ′ gcc atc cgt gca cgg atg gct gca cag cac cgt gat 3 ′ seq id no5 , foki adapter c2930t t - allele 5 ′ gcc atc cgt gca cgg atg gct gca cag cac cat gat 3 ′ seq id no6 ) in 10 mm tris - hcl ph 7 . 5 , 10 mm mgcl 2 , 1 mm dtt , 50 mm nacl , 1 × bsa1 for 2 hours 37 ° c . before heat - inactivation of the enzyme . the ends of the generated fragment nucleic acid were ligated into a circle using 8 fmol of the correct / incorrect ligation template ( 20 + 20 wdgdna 5 ′ utr - ex13 c - allele , 5 ′ ctc ggc tct aaa gca aac agg tga tgg acg tct gga aag ctt t 3 ′ seq id no7 , 20 + 20 wdgdna 5 ′ utr - ex13 t - allele 5 ′ ctc ggc tct aaa gca aac aga tga tgg acg tct gga aag ctt t 3 ′ seq id no8 ). one unit t4 dna ligase and 1 × t4 dna ligase buffer was used , and the reactions were incubated for approximately 30 minutes at 37 ° c . before heat - inactivation of the dna ligase . the circles were exonuclease treated with 5 units exov and 1 mm atp and the samples were incubated in 37 ° c . for 30 min before heat - inactivation at 80 ° c . for 20 minutes . pcr amplification was performed with primers ( frw wdgdna 5 ′ utr - ex13 5 ′ cag agg tga tca tcc ggt ttg 3 ′ seq id no9 , rew wdgdna 5 ′ utr - ex13 5 ′ gga gag gag gcg cag agt gt 3 ′ seq id no10 ), 0 . 5 μm of each , located on both sides of the ligation junction . with a total volume of 50 μl , 200 μm dntp , 1 unit taq gold polymerase , 1 × pcr buffer ( 10 mm tris - hcl ph 8 . 3 , 50 mm kcl , 1 . 5 mm mgcl 2 , 0 . 001 % ( w / v ) gelatine ). 40 amplification cycles were run after activation of the polymerase : 95 ° c . 15 sec , 58 ° c . 1 min and 72 ° c . 20 sec . the amplified nucleic acids were detected by electrophoresis in a 3 % agarose gel and visualisation by staining with ethidium bromide . the results are shown in fig6 b . the following samples were loaded into the different lanes ; 1 — marker , 2 no template control , 3 - 8 samples from a 10 - fold dilution series ( 10 10 - 10 1 ) of bac dna with correct ligation template and ligase , 9 sample with correct ligation template but minus ligase , 10 - 12 samples from a 10 - fold dilution series ( 10e10 to 10e9 ) with a ligation template corresponding to the wrong allele , t instead of c ). the arrow denotes the size of a correct length product . selective ligation of oligonucleotides cleaved with a structure specific enzyme . ( see fig8 ) the reactions were performed in 1 × tth buffer ( 1 mm nad , 10 mm dtt and 0 . 1 % triton x - 100 ). 20 μl reactions containing 0 . 5 pmol of the upstream , downstream and target oligonucleotides respectively ( primer22 + 1 5 ′ gta ttt gct ggg cac tca ctg ca 3 ′ seq id no11 , armc 5 ′ tcc aga cgt cca tca cgg tgc tgt gca ttg cct g 3 ′ seq id no12 or armt 5 ′ tcc aga cgt cca tca tgg tgc tgt gca ttg cct g 3 ′ seq id no13 , template2930 5 ′ cag gca atg cac agc acc gtg cag tga gtg ccc agc aaa tac3 ′ seq id no14 ), 1 unit of tth ligase and native taq polymerase . the reactions were prepared on ice and initiated by transfer to a thermal cycle where the following program was run : 95 ° c . 20 sec , 72 ° c . 30 min for 2 cycles . the upstream or downstream oligonucleotide was radio labelled and the samples were analysed on a 10 % denaturing polyacrylamide gel . ten pmol target dna was end - labelled with 1 . 65 pmol γ - 32 p datp ( nen ). 4 . 9 u t4 pnk enzyme and 1 × t4 pnk buffer ( 0 . 05 m tris - hcl ph 7 . 6 , 10 mm mgcl 2 , 10 mm 2 - mercaptoethanol ) was added to each labelling reaction and the tubes were incubated for 45 min in 37 ° c . edta ( 1 mm ) was added and the samples were boiled for 5 min in a water bath . the unincorporated nucleotides were removed from the labelling reaction with a microspin ™ g - 50 column ( amersham pharmacia biotech ). the experiments with the radio labelled oligonucleotides were detected on a 10 % polyacrylamide gel containing 7 m urea . the gel was run with 0 . 5 × tris borat edta buffer at 30 w for approximately 30 min and was dried in a gel dryer for 2 hours 80 ° c . the dried gel was exposed to a phosphorimager screen overnight . the results are shown in fig8 . oligonucleotides yielding structure a was used in experiments 1 - 6 and oligonucleotides yielding structure b was used in experiments 7 - 12 . ( i ) denotes the size of un - reacted oligonucleotide in experiments 1 - 6 , ( ii ) the size for ligated product in reactions 1 - 6 , ( iii ) uncleaved oligonucleotide used in reactions 7 - 12 and ( iv ) cleaved oligonucleotide in reactions 7 - 12 . 32p denotes a radioactive label on respective oligonucleotide . lanes 1 - 6 shows the results from experiments with oligonucleotide 1 labelled with 32 p . lane 1 , t - allel ( wrong )— taq polymerase , lane 2 c - allel ( correct )— taq polymerase , lane 3 t - allele — tth ligase , lane 4 c - allel — tth ligase , lane 5 t - allel , lane 6 c - allele . lanes 7 - 12 show the results from experiments with oligonucleotide 2 radio labelled with p 32 lanes 7 , 9 , 11 is with the t - allele ( incorrect ) and lane 8 , 10 , 12 is with the c - allele ( correct ). lanes 7 - 8 minus taq polymerase , lanes 9 - 10 minus tth ligase . circularisation of bac dna after cleavage with restriction enzymes , intramolecular hybridisation and cleavage with a structure specific enzyme followed by ligation , as shown in fig9 . denatured , ss bac dna ( 1 × 10 10 molecules ) was cleaved with 10 units draiii in buffer ( 10 mm nacl , 5 mm tris - hcl , 1 mm mgcl 2 , 0 . 1 mm dtt ph 7 . 9 ) was used . draiii was allowed to cleave the dna for 1 hour 37 ° c . before heat - inactivation . this experiment was also done with genomic dna . 10 10 molecules were cleaved by 1 pmol of each adapter ( cleave draiii up2930 5 ′ act gga cac aac gtg acg aac ttg ggt 3 ′ seq id no15 and cleave draii down2930 5 ′ cag ggc tca cac gca gtg agt gcc c 3 ′ seq id no16 ) designed to hybridise to sequences in exon 13 . the subsequent concerted structure - specific cleavage and ligation reaction contained the same reagents as above and 2 pmol of ligation any of two different templates ( 20 + 20 draiii - c 5 ′ taa acg acc cgt gag tga cgc aca ggt cac ggg ggg ac 3 ′ seq id no17 or 20 + 20 draiii - g 5 ′ taa acg acc cgt gag tga cgg aca ggt cac ggg ggg ac 3 ′ seq id no18 ). the samples were divided into two parts and on one half was subjected to a rca . a real - time pcr was performed on the samples with primers located on both sides of the ligation junction . in a total volume of 50 μl the following reagents were included : 2 . 5 μl sample , 1 × pcr bf , 100 μm dntp , 1 unit taq gold polymerase , 0 . 5 μm of each primer , 1 × rox and 0 . 08 × sybr . after activating the polymerase 95 ° c . for 10 min , 40 cycles of the following program was run in a thermal cycler : 95 ° c . 20 sec , 58 ° c . 1 min and 72 ° c . 30 sec . draiii and specific adapters designed to hybridise to sequences in exon 13 of atp7b cleaved bac dna at predetermined sites . the target dna was denatured to become single - 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