Patent Application: US-99913809-A

Abstract:
the disclosed invention teaches processes to amplify oligonucleotides by contacting templates and primers with dna polymerases and triphosphates of non - standard nucleotides , which form nucleobase pairs fitting the standard watson - crick geometry , but joined by hydrogen bonding patterns different from those that join standard a : t and g : c pairs . thus , this invention relates to nucleotide analogs and their derivatives that , when incorporated into dna and rna , expand the number of replicatable nucleotides beyond the four found in standard dna and rna . the invention further relates to polymerases that incorporate those non - standard nucleotide analogs into oligonucleotide products using the corresponding triphosphate derivatives , and more specifically , polymerases and non - standard nucleoside triphosphates that support the polymerase chain reaction , including pcr where the products contain more than one non - standard nucleotide unit .

Description:
6 - amino - 5 - nitro - 3 -( 1 ′- beta - d - 2 ′- deoxyribofuranosyl )- 2 ( 1h )- pyridone ( dz ), its protected phosphoramidite derivatives suitable for chemical oligonucleotides synthesis , its triphosphate and its thiotriphosphate are reported in u . s . patent application ser . no . 11 / 372 , 400 and in [ hut03 ]; both are incorporated herein by reference . its complement , 2 - amino - 8 -( 1 ′- β - d - 2 ′- deoxyribofuranosyl )- imidazo [ 1 , 2 - α ]- 1 , 3 , 5 - triazin - 4 ( 8h )- one ( implementing puaad , as dp ) and these derivative are reported in [ hut03 ][ yan06 ], the synthetic procedures therein being incorporated herein by reference . these are preferred as non - standard nucleotides in the instant invention , although any non - standard nucleotide shown in fig1 might be used in an eternal primer in a pcr . it is known in art published less than a year before the priority date of the instant application [ yan07 ] that pcr amplification is possible for oligonucleotides that contain a single dp or a single dz in the amplicon . however , the prior art shows that upon multiple pcr cycles , dp and dz are removed from the amplicon . the instant invention was made following first the recognition it was possible to place dp and / or dz in the pcr primers themselves . to amplify standard oligonucleotides , the dp and dz would not be in the region of the primer that contacts that target , but rather within an oligonucleotide tag ( or tail ) appended to the 5 ′- end of the pcr primer that does contact the standard oligonucleotide target . pcr primers having two parts , a 5 ′- end containing dp and / or dz ( as well as standard nucleotides , optionally ) and a 3 ′- end that is complementary to the target , are called “ chimeric primers ”. pcr amplification with chimeric primers is then done in a nested fashion [ bro97 ]. any loss of the non - standard nucleotide is restored in a subsequence pcr cycle , solving the loss problem described in the literature . after the chimeric primers are consumed in the first cycles , “ external primers ” having the sequence of only the 5 ′- tags continue the pcr . not known in any art published before the priority date of the instant application , and not anticipatable from the prior art given the idiosyncrasies of polymerases when challenged with unnatural nucleoside triphosphate substrates [ hor95 ], it was then discovered that pcr could succeed with certain polymerases even for amplicons that contain multiple dps and / or multiple dzs . further , we disclose here for the first time that certain polymerases support incorporation of adjacent dzs and adjacent dps in prime extension reactions a third discovery was also unanticipated by us or the art . it turned out that by doing nested pcr with dz and / or dp in the external primers , it was possible to obtain cleaner pcr products . given this result , it became evident that pcr with dz and dp in the primers was a useful , patentable invention , as is herein claimed . accordingly , the invention together with its associated discoveries provides a process for amplifying an oligonucleotide sequence in a pcr format , where a forward chimeric primer and a reverse chimeric primer are contacted with that target sequence . the forward chimeric primer is complementary to a region at the 3 ′- end of said sequence ( as in any pcr ), the reverse chimeric primer is identical in sequence to a region at the 5 ′- end of said sequence ( as in any pcr ), and the forward and reverse primers are joined at their 5 ′- prime ends to oligonucleotide tags having independently selected sequences that contain at least one non - standard nucleotide ( see fig1 ), where the presently preferred non - standard nucleotide is dz or dp , most preferably dp . then , as with standard ocr , the mixture is incubated the mixture with a dna polymerase , rna polymerase , or a reverse transcriptase , depending on the nature of the oligonucleotide to be amplified , together with the triphosphates needed to complement the nucleotides in the primers and sequence . the presently preferred polymerases are phusion and vent or deep vent having exonuclease activity , as described further in the examples . this process , of course , comprises a simpler pcr process that amplifies an oligonucleotide sequence that contains one or more non - standard nucleotides , preferably dz or dp . pcr amplification of an oligonucleotide containing just one was disclosed less than a year before the priority date in [ yan07 ], and therefore is patentable under united states law . pcr amplification of oligonucleotides containing more than one non - standard nucleotide and adjacent non - standard nucleotides was not in the prior art prior to the priority date , and is not rendered obvious by [ tan07 ], and is therefore internationally patentable . this example demonstrates that chimeric primers containing dz and dp in their external segments support pcr . the following oligonucleotides were prepared by phosphoramidite synthesis . these include two reverse chimeric primers , identical except that in r - 36 - nest , some of the g &# 39 ; s were replaced by p &# 39 ; s in the segment not complementary to the template : the underlined portions of the primers are complementary to the template . the portions not underlined are the tags that contain non - standard nucleotides . in separate experiments the chimeric and non chimeric primers were incubated under the following conditions : the products were resolved by gel electrophoresis ( fig3 ). these results show that primers containing multiple dp &# 39 ; s support pcr works . the experiment does not show , however , that primers containing consecutive dps effectively support pcr . this experiment demonstrated the use of dz and dp pairing in the external segments of a nested pcr [ bro97 ], shown schematically in fig2 . the following oligonucleotides were prepared by phosphoramidite synthesis . these were set up in three set of nested pcr experiments . the first used external primers , one containing dp , the other not : applied in a direct pcr experiment for a longer template that included temp - r - 47 in its middle : and two reverse external primers , one containing dp , the other not : the standard primers f - 17 - std and r - 17 - std should amplify the temp - r - 81 target , leading to a band in a gel electrophoresis resolution that migrates as an 81 - mer . this is in fact the case ( fig4 , a - std lane ). if dp does not bind to dc , then the aegis dp - containing primers should not yield and 81 - mer band . this is also the case ( fig4 , a - aegis ). in another experiment , the following recipe was used in a nested pcr experiment : 1 × standard taq reaction buffer ( 10 mm tris - hcl , 50 mm kcl , 1 . 5 mm mgcl 2 , ph 8 . 3 at 25 ° c .). 1 × thermopol reaction buffer ( 20 mm tris - hcl , 10 mm ( nh 4 ) 2 so 4 , 10 mm kcl , 2 mm mgso 4 , 0 . 1 % tritonx - 100 , ph 8 . 8 at 25 ° c .). experiment b used f - 34 - std and r - 36 - std as chimeric primers . these should generate products when amplified with external primers built without dp , but not when amplification was sought with external primers containing dp . this was in fact the case ( fig4 , b - std and b - aegis , respectively ). experiment c used f - 34 - nest and r - 36 - nest as chimeric primers . these should not generate products when amplified with external primers built with dp , but should generate when amplification was sought with external primers containing dp . this was in fact the case ( fig4 , c - std and c - aegis , respectively ). these experiments shows showed the ability of dna polymerase to support a six letter pcr with da , dt , dg , dc , dz , and dp as the six letters . they also demonstrate the orthogonality of the process . nested pcr works with aegis external primers when it should and not when it should not , and vice versa . given the well - known idiosyncrasies of polymerases and the possibility of strong neighbor effects [ hor95 ], it was not clear that these results would be extendable to pcr amplifications where dz or dp are adjacent in a template , requiring the incorporation of dp and dz adjacent in the template . the following experiments were done to screen thermophilic polymerases for their ability to incorporate dptp opposite dz in the template . this was done at the following concentrations : [ datp ]=[ dctp ]=[ dttp ]= 100 microm ), dgtp ( 5 microm to 100 microm ), or dptp ( 5 microm to 100 microm ) at ph7 . 0 or 7 . 5 , with the following oligonucleotides ( r - 19 - s was p - 32 labeled at its 5 ′- end ): 5 ′- 32 p - labeled primer r - 19 - s ( 2 pmole , final assay concentration 50 nm ) was annealed to a template sequence r - 36 - nest - 6z ( 3 pmole , final assay concentration 75 nm ) by heating ( 5 min 95 ° c .) and then slow cooling ( 0 . 5 h ) to room temperature . datp , dttp and dctp ( 4 nmole each , final 100 microm ) and dgtp ( final 10 microm ), or dptp ( final 10 microm ) were added at room temperature . the reaction mixture was pre - incubated at 72 ° c . for 30 seconds and followed by the addition of taq dna polymerase to give a final volume of 40 microl . the mixture was incubated at 72 ° c . for 4 minutes , and quenched by dilution into page loading / quench buffer ( 10 microl , 10 mm edta in formamide ). samples were resolved by electrophoresis using a 14 % page ( 7 m urea ). the gel was analyzed using molecularimager software . these results ( fig5 ) showed that vent and deep vent performed better than taq . without wishing to be bound by theory , this may be due to their exonuclease activities . to compare the efficiency and fidelity of dna polymerases ( taq , vent ( exo +), and dv ( exo +)) incorporating dztp opposite two consecutive dps in a template , 5 ′- 32 p - labeled primer t7 - y - rs - s16 ( 0 . 2 pmole of hot primer plus 4 pmole of cold primer , final assay concentration 70 nm ) was annealed to template t7 - pp - temp ( 6 pmole , final assay concentration 100 nm ) in 1 ×. thermopol polymerase reaction buffer ( 20 mm tris - hcl , 10 mm ( nh 4 ) 2 so 4 , 10 mm kcl , 2 mm mgso 4 , 0 . 1 % triton x - 100 , ph = 8 . 0 at room temperature ) by heating ( 5 min at 95 ° c .) and then slow cooling ( 0 . 5 h ) to room temperature . dntp ( each final 0 . 1 mm ) and dztp ( final 0 . 1 mm , with (+) or without (−)) were added at room temperature . the reaction mixture was cooled to 4 ° c . for 1 min and followed by the addition of taq ( 2 . 5 units ), vent ( exo +), or deep vent ( exo +) dna polymerase ( 2 units for vent and deep vent ) to give a final volume of 60 microl . the primer was extended at 65 ° c . and aliquots ( 7 microl ) were taken from each reaction at time intervals ( 1 , 2 , 4 , 8 , and 16 min ), quenched by page loading / quench buffer ( 7 microl , 10 mm edta in formamide ) and resolved by electrophoresis using a 16 % page ( 7 m urea ). the gel was analyzed using molecularimager . these oligonucleotides were used : the order of performance of the polymerases tested is deep vent ( exo +)& gt ; vent ( exo +)& gt ; taq . in the absence of dztp , deep vent and vent misincorporates only one dctp opposite the first dp . however , taq can misincorporate dctp opposite two consecutive dps , and then keep extending primer . all are better than exo (−) polymerases ( not reported in [ yan07 ]). to compare the outcome of pcr with templates containing one or two adjacent dps , the following oligonucleotides were prepared : thermopol reaction buffer ( 20 mm tris - hcl , 10 mm ( nh 4 ) 2 so 4 , 10 mm kcl , 2 mm mgso 4 , 0 . 1 % triton x - 100 , ph 8 . 0 at 25 ° c .). pcr : one cycle of 95 ° c . for 15 min ; 26 cycles of 95 ° c . for 20 s , ( 55 ° c . for 30 s , 72 ° c . for 1 min or 2 min ; 72 ° c . for 5 min ; then stored at 4 ° c . the results are shown in fig7 . all pcrs generates some degree of 51 - mer product . template a contained only standard nucleotides . template b contained a single dp . template c contained a series of dps , including two adjacent dps . to demonstrate that if dp or dz were incorporated into pcr primers in place of one or more dgs or dcs , then the synthetic primers containing dps or dzs would not find their perfectly matched complementary strands in a primer pool , a 17 - mer , 5 ′- caggaaggagcgatcgc - 3 ′ ( seq id 35 ) was deliberately designed to form a self - dimer with 8 base pairs at the 3 ′- end ( underline region , t m = 32 ° c . ), and subjected to pcr conditions . as expected , primer - dimer formed rapidly . in contrast , perfectly mismatched primers 5 ′- caggaaggagcpatcpc - 3 ′ ( seq id 36 ) and 5 ′- caggaaggagzgatzgc - 3 ′ ( seq id 37 ), which would form primer - dimers only by mismatching dp with dc ( in the first case ) and dz with dg ( in the second ) gave no detectable amplicon under the same conditions , even after 45 cycles . with several polymerases able to replicate efficiently dna fragments containing multiple dps and dzs , the preferred polymerase having the highest pcr efficiency to amplify target using a nested pcr architecture with aegis nucleotides in the external primers was determined . taq , 9 ° n , deep vent ( both exo + and exo − ), vent ( both exo + and exo − ), phusion , and herculase were tested ; the pcr efficiency was monitored by real - time pcr . the polymerase has the higher pcr efficiency generates more pcr amplicon and producing higher fluorescence signal at an earlier cycle of the pcr . phusion was found to have the highest pcr efficiency among the polymerases tested with proofreading activity ; deep vent ( exo − ) is the most efficient among all the polymerases without exonuclease activity . for all polymerases tested , dp - containing nested pcr , in general , has higher pcr efficiency than that of the dz - containing nested pcr . phusion dna polymerase generates long templates with an accuracy and speed previously unattainable with a single enzyme . in addition , the error rate of phusion is 50 - fold lower than that of taq , and about 6 - fold lower than that of vent and deep vent . therefore , phusion dna polymerase was further optimized as a presently preferred polymerase for nested pcr with dp - containing external primers . the major infidelity during the 6 - nucleotide pcr arises from misincorporation of dgtp opposite template dzs or dztp opposite template dgs . this infidelity is ph dependent , when the ph of the buffer is low , the rate of misincorporation decreases . to determine a preferred ph for pcr efficiency and fidelity , three types of nested pcr were conducted with phusion hf buffer at four different ph values ( 7 . 0 , 7 . 5 , 8 . 0 , and 8 . 5 ). amplification was monitored by the real - time pcr with sybr green . for “ type a ” nested pcr , standard external and chimeric primers and four standard nucleotide triphosphates ( dntps ) were used ; amplification curves in real - time show that pcr efficiency increases when the ph of the buffer decreases . after 30 cycles , the melting temperature of each pcr amplicon was measured and the size of each amplicon was analyzed by agarose ( 3 %) gel . the t m of each pcr amplicon generated under four different ph values is roughly the same ( about 91 . 49 ± 0 . 5 ° c .). the type b nested pcr is identical to the type a nested pcr , except that dztp was also included into the reaction . by comparing the melting temperature of each pcr amplicon in the type b reaction with that of the type a reaction , misincorporation of dztp at different phs was measured . the t m of the amplicon improves as the ph of the reaction buffer decreases . for example , at the highest ph value tested ( 8 . 5 ), the t m of the pcr amplicon is 3 . 75 ± 0 . 05 ° c . below than that of the control pcr ( type a ); at the lowest ph value ( 7 . 0 ), the δt m was to 0 . 29 ± 0 . 05 ° c . below the fully standard pcr . for the type c nested pcr , dp - containing primers were used instead of standard primers , and pcr amplifications were conducted under the same conditions as type b nested pcr . the t m of each pcr amplicon increases as the ph value of the reaction buffer decreases ; the effect of ph on pcr efficiency and misincorporation of dztp opposite template dg agreed with that with type b nested pcr . however , the t m of each pcr amplicon in the type c nested pcr is higher ( about 3 . 6 ° c .) than in type b nested pcr , this enhancement of the t m is mainly due to the higher thermostability of the z : p base pairs in the pcr amplicon . the pcr amplicons obtained at four different phs in type b nested pcr were cloned , and their sequences were verified by sanger sequencing . this shows that misincorporation of dztp opposite template g is insignificant and does not prevent the pcr amplicon of interest to be cloned and sequenced using the conventional sanger method . this too was not expected given [ yan07 ], and can be used as a restrictive element of a claim . to show whether the dp - containing nested pcr can enhance the capability of multiplexed pcr , this system was applied to human genomic dna , targeting the three genes associated with cancer : top1 , hbegf , and myc . the oligonucleotides used in this experiment were : the external primers were adopted from luminex &# 39 ; s 5 ′- universal tag sequences , which were designed by the company to have unique sequences to avoid cross - hybridization and have roughly equal melting temperatures . to design the chimeric primers , universal external primer was added to either forward or reverse gene - specific primers , the other three external primers were also attached to gene - specific primers , the combination of each external primer to a certain gene - specific primer was optimized using primer design software ( oligoanalyzer 3 . 1 ), and following the general principles of multiplex pcr primer design to avoid cross - hybridization and hairpin structure of primers . three cancer genes in human genomic dna were multiplexed amplified by standard nested pcr and dp - containing nested pcr using phusion under seven different annealing temperatures . as shown in fig8 , standard nested pcr with all annealing temperatures give messy pcr results ( left ): at the lower annealing temperatures ( from 53 . 4 ° c . to 58 . 8 ° c . ), significant amounts of primer dimer ( about 40 nucleotides in length ) were generated ; at the higher annealing temperatures ( from 61 . 5 ° c . to 65 . 5 ° c . ), non - specific pcr artifacts ( pcr amplicons longer than the desired length ) were produced along with the disappearance of the primer dimer . however , the dp - containing nested pcr generated the desired pcr amplicons with minimal pcr artifacts ( right ). for the two control reactions ( without genomic dna ), control a ( standard nested pcr ) gave some primer dimers ( a 40 - mer amplicon formed by standard external primers ) and significant amount of long pcr amplicons , which may caused by the further priming from the primer dimer . the control b ( dp - containing nested pcr ) gave one band , which was formed by the dimerization of the dp - containing chimeric primers , as the 3 ′- ends of the chimeric primers are the standard gene - specific oligonucleotides . this dimerization could be further eliminated by reducing the concentration of the dp - containing chimeric primers . we further verified that the dp - containing multiplexed nested pcr also performed better than the standard nested pcr under hf phusion buffer with other different ph values ( 8 . 5 , 8 . 0 , and 7 . 5 ). this result was entirely unanticipated . nested pcr using aegis external primers leads to cleaner multiplexed pcr . without wishing to be bound by theory , this may arise because even with standard primers not having exact matched in a genome , standard primers have sufficient mismatches to prime at off - target sites . 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