Patent Application: US-89964110-A

Abstract:
the present invention provides a process for the production of nucleic acid encoding a target protein , which comprises : providing an array of rna or dna molecules including one or more encoding the target protein ; generating a target protein from the array to form rna - protein or dna - protein complexes in which the rna or dna molecule is non - covalently or covalently bound to the complex ; separating the complexes into compartments wherein most or all of the compartments contain no more than one complex ; subjecting the complexes to reaction conditions which allow target protein activity ; and selecting nucleic acid encoding the target protein on the basis of the activity associated therewith , wherein when the complex is a dna - protein complex in which the dna is non - covalently bound , step b ) is performed in the absence of separate compartments for each complex .

Description:
the invention will now be described in further detail , by way of example only , with reference to the accompanying figures and appendices : fig1 . the experimental scheme of example 1 . two plasmids pet_his_mlv_pd ( encoding moloney murine leukemia virus ( m - mlv ) reverse transcriptase fused to protein d spacer ) and pet_his_del_pd ( encoding inactivated ( 57 amino acids deletion in pol domain ) moloney murine leukemia virus ( m - mlv ) reverse transcriptase fused to protein d spacer ) were used to synthesize pcr fragments . pcr fragments furtheron are used in transcription reaction and synthesis of mrna lacking stop codon at the 3 ′ end . purified mrna is mixed with ratio 1 : 50 = mlv ( active rt ): del ( inactive rt ) and used for in vitro translation reaction . during translation reaction ribosomal complex synthesizes protein and stops at the end of mrna lacking stop codon . mixture of ternary complexes ( tc ) is purified by ultracentrifugation on sucrose cushions . purified ternary complexes (& lt ; 3 * 10 9 molecules taken ) already containing mrna linked to in vitro translated mlv reverse transcriptase are used to prepare reverse transcription reaction mix supplemented with external dntp set and primer for rt reaction . ice - cold rt reaction mixture is emulsified giving ˜ 1 * 10 10 water in oil compartments ˜ 2 μm in size . emulsified rt reaction mixture ( less than one tc ( mrna + mlv rt ) per compartment is incubated for 1 hr at 42 ° c . in order to perform rt reaction . after the temperature of compartmentalized rt reaction mixture is raised most of tc dissociate releasing mrna and reverse transcriptase . successful rt reaction is performed only in compartments containing active mlv reverse transcriptase ( mlv_pd ) and no cdna is synthesized in compartments with inactive reverse transcriptase ( del_pd ). subsequent pcr amplifies cdna and enrichment of active reverse transcriptase ( mlv_pd ) genes over inactive reverse transcriptase ( del_pd ) is observed . fig3 . example 1 — the agarose gel electrophoresis of first pcr performed on cdna synthesized during crd selection . primers used : rd_nde ( seq id no : 9 ) and pd — 55 ( seq id no : 10 ). expected length of pcr fragments was 2185 bp for mlv_pd and 2014 bp for del_pd . amplification was analyzed on 1 % agarose gel loading 10 μl of pcr mix per well . fig4 . example 1 — the agarose gel electrophoresis of nested pcr for partial gene amplification performed on first pcr product . primers used : m_f ( seq id no : 11 ) and m — 2r ( seq id no : 12 ). expected length of pcr fragments was 907 bp for mlv_pda and 736 bp for del_pd . amplification was analyzed on 1 % agarose gel loading 10 μl of pcr mix per well . fig5 . example 1 — the agarose gel electrophoresis of nested pcr for full gene amplification performed on first pcr product . primers used : m_esp ( seq id no : 13 ) and m_eri ( seq id no : 14 ). expected length of pcr fragments was 2077 bp for mlv_pda and 1906 bp for del_pd . amplification was analyzed on 1 % agarose gel loading 10 μl of pcr mix per well . fig6 . the experimental scheme of crd selection in example 2 . pcr fragments encoding mutants library of reverse transcriptase ( in fusion with protein d ) mlv_pd was used to synthesize mrna . purified mrna was used for in vitro translation reaction . ternary complexes ( tc ) of mrna - ribosome - mlv_pd ( trna ) were formed in translation mixture and stabilized by low temperature and high concentration of mg 2 + ions . mixture of tc was purified by ultracentrifugation on sucrose cushions . precipitated tc was dissolved in ice - cold buffer ( 50 mm mg 2 + ) and used to prepare reverse transcription reaction mix supplemented with external dntp set and primer for rt reaction . ice - cold rt reaction mixture was emulsified giving ˜ 1 * 10 10 water in oil compartments ˜ 2 μm in size . optimal reaction temperature of mlv rt is ˜ 42 ° c . in order to select for reverse transcriptase variants , which are working better at higher temperatures emulsified rt reaction mixture ( less than one tc ( mrna + mlv rt ) per compartment ) was incubated for 1 hr at 50 ° c . at this temperature successful synthesis of full length cdna was performed better in compartments containing more active or thermostable mlv reverse transcriptase variants . subsequent pcr was used to amplify full length cdna and enrichment of more active and thermostable reverse transcriptase genes was performed . by pcr amplified genes were moved back to crd format restoring intact 5 ′ ( start fragment — t7 polymerase promoter , sd and his - tag coding sequences ) and 3 ′ ( end fragment — gs linker , protein d and second gs linker ) sequences by ligation pcr . reconstructed pcr fragment , containing enriched library of reverse transcriptase genes , was used for subsequent mrna transcription and next crd selection round . each selection round was performed at higher and higher temperatures of rt reaction : 50 ° c . ( 1 st round ); 52 . 5 ° c . ( 2 nd round ); 55 ° c . ( 3 rd round ); 57 . 5 ° c . ( 4 th round ) and 60 ° c . ( 5 th round ). fig7 . the scheme of pcr fragment reconstruction before new round of crd selection . mutated mlv rt library was digested with esp3i ( ncoi compatible end ) and ecori and ligated with start ( 244 bp ) and end ( 398 bp ) fragments in order to get pcr fragment suitable for crd selection . start fragment ( containing t7 polymerase promoter , sd and his - tag coding sequences ) was constructed by pcr amplification of initial 983 bp start fragment ( target — plasmid pet_his_del_pd ( seq id no : 2 ), primers — pro - pivex ( seq id no : 3 ) and m — 1r ( seq id no : 15 )) and subsequent digestion with ncoi ( recognition sequence c ↓ catgg ) giving 244 bp dna fragment . end fragment ( containing gs linker , protein d and second gs linker sequences ) was constructed by pcr amplification of initial 1039 bp end fragment ( target — plasmid pet_his_del_pd ( seq id no : 2 ), primers — m — 3f ( seq id no : 16 ) and pd - ter ( seq id no : 4 )) and subsequent digestion with ecori ( recognition sequence g ↓ aattc ) giving 398 bp dna fragment . fig8 . reverse transcriptase activities of mutant rt variants measured at 37 ° c ., 50 ° c . and residual activity at 37 ° c . after 5 min incubation at 50 ° c . reverse transcriptase activity at 37 ° c . is normalized to be always 100 % and is omitted . thus only two types of columns ( percents of rt activity at 50 ° c . and residual rt activity at 37 ° c . after 5 min incubation at 50 ° c .) are shown . as a control is given wt m - mulv reverse transcriptase used for mutants library construction . this primary enzyme is expressed in the same vector and purified in the same way as mutant variants of rt . an average value of mutant rt activity at 50 ° c . for all tested mutants is about ˜ 92 % and is more than 2 times higher comparing to wt enzyme ( 45 %). an average residual activity of mutant rt variants at 37 ° c . after 5 min preincubation at 50 ° c . is 12 % ( wt enzyme — 11 %). fig9 . specific activity ( u / mg of protein ) of partially purified wt and mutant rt variants measured 10 min at 37 ° c . fig1 . the proposed experimental scheme of crd selection using facs . protein of interest is displayed in ribosome display format . purified ( or just diluted many times ) ternary complex comprising mrna - ribosome - protein ( trna ) is mixed with non fluorescent substrate ( s ) in reaction buffer and subsequently emulsified producing double water - in - oil - in - water emulsions . active variants of compartmentalized enzymes will convert substrate ( s ) to fluorescent product ( p ) allowing facs to distinguish between fluorescent ( active enzyme inside ) and “ dark ” ( inactive enzyme inside ) droplets . fig1 . the proposed experimental scheme for selection and evolution of thermostable dna polymerases using crd . polymerase of interest has to be displayed in ribosome display format . optionally purified ( or just diluted many times ) ternary complex comprising mrna - ribosome - polymerase can be used to prepare reaction mixture with reverse transcriptase ( helper enzyme ), dntp &# 39 ; s and primer set in pcr buffer . reaction solution should be emulsified producing water - in - oil emulsion . in first rt step — reverse transcriptase has to synthesize cdna , which subsequently will serve as a target for second pcr step — cdna amplification by ribosome displayed dna polymerase . one of the primers used in pcr can have biotin for optional subsequent purification with streptavidin beads and non - complementary 5 ′ end . after rt - pcr emulsions should be broken , newly synthesized dna fragment can be purified via biotin and reamplified using new primer set , which will contain one primer with sequence non - complementary to cdna , but identical to 5 ′ part of primer used in first amplification reaction ( selective amplification of dna over cdna background ). more active variants of dna polymerase will be enriched over less active variants and can be used for further analysis or next selection round . fig1 . the experimental scheme of example 4 . in this experimental setup m - mulv reverse transcriptase is used as dna dependent dna polymerase . two plasmids pet_his_mlv_d583n_pd ( encoding rnase h minus moloney murine leukemia virus ( m - mlv ) reverse transcriptase fused to protein d spacer ) and pet_his_del_pd ( encoding inactivated reverse transcriptase fused to protein d spacer — 57 amino acids deletion in pol domain and point mutation d583n in rnase h domain ) were used to synthesize pcr fragments . pcr fragments furtheron are used in transcription reaction . purified mrna is mixed with ratio 1 : 20 = mlv_d583n_pd ( active rt ): del_pd ( inactive rt ) and used to prepare mrna / dsdna complex by dsdna ligation to mrna mix using t4 dna ligase . mrna / dsdna complex was used for in vitro translation reaction . during translation reaction ribosomal complex synthesizes protein and stops at the end of mrna ( at the beginning of mrna / dna hybrid ). mixture of ternary complexes ( tc ) is purified by ultracentrifugation on sucrose cushions . purified ternary complexes (& lt ; 3 * 10 9 molecules taken ) already containing mrna / dsdna linked to in vitro translated polymerase ( m - mulv reverse transcriptase ) are used to prepare elongation reaction mix supplemented with external biotin - dutp . ice - cold reaction mixture is emulsified giving ˜ 1 * 10 10 water in oil compartments ˜ 2 μm in size . emulsified elongation reaction mixture ( less than one tc ( mrna / dsdna + polymerase ) per compartment is incubated for 30 min at 37 ° c . in order to incorporate biotinylated nucleotide . after the temperature of compartmentalized reaction mixture is raised most of tc dissociate releasing mrna / dsdna complex and polymerase . successful incorporation reaction in to dsdna substrate is performed only in compartments containing active polymerase ( reverse transcriptase — mlv_d583n_pd ) and no cdna is synthesized in compartments with inactive polymerase ( del_pd ). after the emulsions are broken excess of biotin - dutp is removed using gel - filtration mini - column . biotinylated mrna / dsdna complex is purified on streptavidin beads and used to synthesize cdna . subsequent pcr amplifies cdna and enrichment of active polymerse ( reverse transcriptase — mlv_d583n_pd ) genes over inactive polymerase ( del_pd ) is observed . fig1 . determination of biotin - dutp incorporation efficiencies into mrna / dsdna complex and into self primed mrna . picture of rt - pcr performed on samples of mrna / dsdna ( mlv_d583n_pd ) and mrna ( del_pd ) after the incorporation of dttp or biotin - dutp . predicted amplicons size are 907 bp for mlv_d583n_pd and 736 bp for del_pd cdna . pcr products were analyzed on 1 % agarose gel loading 10 μl of pcr mix per well . fig1 . general control of mrna / dsdna complex existence by incorporation of dttp ( biotin - dutp ) and [ α - p 33 ] datp . radioactive datp should be introduced into dsdna substrate subsequentialy after the incorporation of initial dttp or biotin - dutp . a — ethidium bromide visualized agarose gel ( mrna or mrna / dsdna bands ˜ 2 . 5 kb ). b — the same gel as in a dried on filter paper and visualized using cyclone phosphor imager ( perkin - elmer , wellesley , mass .). labelled mrna / dsdna complex and / or only dsdna bands are observed . c — structure and sequence of dsdna counterpart in mrna / dsdna complex seq id nos . : 21 and 22 . fig1 . the resultant final rt - pcr fragments analysis of example 4 . predicted amplicons size are 907 bp for mlv_d583n_pd and 736 bp for del_pd cdna . pcr products were analyzed on 1 % agarose gel loading 10 μl of pcr mix per well . rt - pcr samples before streptavidin beads corresponds to the ratio of active and inactive polymerase genes 1 : 20 ( almost only del_pd fragment ˜ 736 bp is visible ). rt - pcr samples after the purification on streptavidin beads corresponds to the ratio of active and inactive polymerase genes after the single selection round — 1 : 1 . an enrichment factor of ˜ 20 is observed in this selection . fig1 . some examples of alkaline agarose gels used to determine highest temperature of 1 kb and 4 . 5 kb cdna synthesis reaction . pcr machine — eppendor mastercycle gradient . a - d — 1 kb cdna synthesis ( m - mulv ( wt ), d200n , l603w and q221r ); temperature gradient 41 . 9 ° c ., 43 . 6 ° c ., 45 . 5 ° c ., 47 . 8 ° c ., 50 . 4 ° c ., 53 . 1 ° c ., 55 . 8 ° c ., 58 . 1 ° c ., 60 . 1 ° c ., 62 . 1 ° c . ; size standart — dna fast ruler middle range ( fermentas ). e - g — 4 . 5 kb cdna synthesis ( m2 , m3 and m4 ); temperature gradient 49 . 8 ° c ., 51 . 5 ° c ., 53 . 4 ° c ., 55 . 7 ° c ., 58 . 3 ° c ., 61 . 0 ° c ., 63 . 7 ° c ., 66 . 1 ° c ., 68 . 0 ° c ., 70 . 0 ° c . ; size standart — zip ruler express dna ladder 2 ( fermentas ). appendix 1 . the general scheme of mutations found in initial mlv rt library ( sequence between ncoi and ecori restriction sites — seq id no . 24 ). 23 nucleotide mutations were found among 10 sequenced genes ( 1 transversion , 20 transitions — mutated positions are underlined , mutations indicated above the sequence , 2 deletions — underlined and indicated as dashed line above the sequence ) giving 15 amino acids exchanges , 6 silent mutations , 1 stop codon and 2 frame shifts of coding frame — on average 1 - 2 amino acids substitutions per gene . appendix 2 . the clustalw alignment of all 104 protein sequences without n terminal his tag in order to have the same numeration of amino acids as is usually used in literature . wild type sequence denoted as mlv ( seq id no : 25 ) is always given as first sequence ( mutated sequences represent seq id nos : 26 to 128 based on the order in which they are shown ). mutations are marked using white font color in black background amino acids positions , mutations of which somehow improve m - mulv reverse transcriptase properties and are described in different patent applications are marked in the alignment as columns of amino acids ( white font ) highlighted in grey . mutations originating from our selection and located in grey columns indicate that our selection procedure precisely targeted the beneficial hot spot or even exact amino acid mutations described elsewhere . sequences of analyzed proteins , activity of which at 50 ° c . was substantially better as compared to the primary wt m - mulv ( 70 % and more as compared to 45 % of wt activity ) are highlighted in grey . appendix 3 . list of mutations found in all selected rt variants . proteins are sorted by decreasing number of mutations . appendix 4 . mutations frequency ( in decreasing order ) of selected rt variants . names of analyzed proteins , which activity at 50 ° c . was substantially better as compared to primary wt m - mulv ( 70 % and more comparing to 45 % of wt activity ) are highlighted in grey . appendix 5 . summarized table of data on m - mulv ( wt ) reverse transcriptase and single mutants , which contains : name of protein ; selection frequency ( number of sequenced mutants , which had exact mutation and the number in the parentheses indicates total number of particular amino acid mutations found in selection ); protein concentration ( mg / ml ); reverse transcriptase specific activity at 37 ° c . ( u / mg ); relative activity at 50 ° c . (%); relative residual activity at 37 ° c . after 5 min enzyme incubation at 50 ° c . (%); specific rnase h activity of protein ( u / mol ); relative rnase h activity (%) and highest temperature of 1 kb cdna synthesis reaction . appendix 6 . summarized table of data on m - mulv ( wt ) reverse transcriptase and single mutants , which contains : name of protein ; protein concentration ( mg / ml ); reverse transcriptase specific activity at 37 ° c . ( u / mg ); relative activity at 50 ° c . (%) and highest temperature of 1 kb and 4 . 5 kb cdna synthesis reaction . appendix 7 . sequence and information relating to seq id nos : 1 to 23 . to provide proof of principle for compartmentalized ribosome display selection system test selection was performed . typical proof of principle experiment should give positive signal for active enzyme ( in our case rt - pcr fragment for original mlv reverse transcriptase ) and no signal for inactive enzyme ( no rt - pcr fragment for inactivated mlv reverse transcriptase ). more sophisticated experiment is to use mixture of genes with defined ratio encoding active and inactive enzymes . as a result of successful experiment genes encoding active enzyme should be enriched over genes encoding inactive enzyme . general scheme of experiment is shown in fig1 . two plasmids pet_his_mlv_pd ( encoding moloney murine leukemia virus ( m - mlv ) reverse transcriptase fused to protein d spacer ) and pet_his_del_pd ( encoding inactivated ( 57 amino acids deletion in pol domain ) moloney murine leukemia virus ( m - mlv ) reverse transcriptase fused to protein d spacer ) were used to synthesize pcr fragments . further pcr fragments were used in transcription reaction for synthesis of mrna , which lacks stop codon at the 3 ′ end . purified mrnas resulting from the two abovementioned pcr fragments were mixed with a ratio 1 : 50 = mlv ( active rt ): del ( inactive rt ) and used for in vitro translation reaction . during translation reaction ribosomal complex synthesizes protein and stops at the end of mrna lacking stop codon . translation reaction was stopped by dilution with ice - cold buffer containing 50 mm mg 2 + . low temperature , high concentration of mg 2 + ions and absence of stop codon at the end of mrna stabilize ternary complexes ( tc ) of mrna - ribosome - protein ( trna ). mixture of ternary complexes ( tc ) was purified by ultracentrifugation on sucrose cushions . ultracentrifugation was optimized in such a way that tc (˜ 3 . 5 mda ) were precipitated at the bottom of ultracentrifugation tube , meanwhile small molecular weight molecules , proteins and most of free mrna (˜ 0 . 9 mda ) remained in the supernatant . precipitated tcs were dissolved in the ice - cold buffer ( 50 mm mg 2 + ). purified ternary complexes (& lt ; 3 * 10 9 molecules taken ) already containing mrna linked to in vitro translated mlv reverse transcriptase were used to prepare reverse transcription reaction mix supplemented with external dntp set and primer for rt reaction . ice - cold rt reaction mixture was emulsified yielding ˜ 1 * 10 10 water in oil compartments ˜ 2 μm in size . emulsified rt reaction mixture ( less than one tc ( mrna + mlv rt ) per compartment was incubated for 1 hr at 42 ° c . in order to perform rt reaction . after the temperature of compartmentalized rt reaction mixture is raised most of tcs dissociate releasing mrna and reverse transcriptase . successful rt reaction is performed only in compartments containing active mlv reverse transcriptase ( mlv_pd ) and no cdna is synthesized in compartments with inactive reverse transcriptase ( del_pd ). subsequent pcr ensures the amplification of synthesized cdna and enrichment of active reverse transcriptase ( mlv_pd ) genes over inactive reverse transcriptase ( del_pd ) is observed . initial plasmid pet_his_mlv_pd ( seq id no : 1 and fig2 ) was constructed by modification of pet type plasmid in t7 polymerase promoter and shine - dalgarno sequences region and insertion of mlv h + reverse transcriptase coding sequence ( 306 - 2363 on seq id no : 1 ) with n - terminal his - tag ( 258 - 305 on seq id no : 1 ) and c - terminal fusion with glycine - serine ( gs ) linker ( 2364 - 2393 on seq id no : 1 ), part of protein d ( pd ) from phage lambda ( 2394 - 2669 on seq id no : 1 ) and second glycine - serine ( gs ) linker ( 2670 - 2759 on seq id no : 1 ). n - terminal his - tag is used for protein express purification . c - terminal fusion has to remain in ribosome tunnel during protein in vitro translation and formation of mrna - ribosome - mlv ( trna ) ternary complex . m - mulv reverse transcriptase has two main enzymatic activities : rna dependent dna polymerase and rnase h . reverse transcriptase rnase h activity was turned off introducing point mutation d583n ( single nucleotide g to a exchange at position 2055 in plasmid pet_his_mlv_pd , seq id no : 1 ). aspartate 583 is located in rnase h active site , is involved in mg ion binding and is crucial for rnase h activity . new plasmid is identified as pet_his_mlv_d583n_pd and was used for further construction of next plasmid pet_his_del_pd ( seq id no : 2 ), which encodes inactivated reverse transcriptase . plasmid pet_his_mlv_d583n_pd was digested with restriction endonuclease xmaji ( recognition sequence c ↓ ctagg — positions 1047 and 1218 on seq id no : 1 ). gene fragment 171 bp in length was removed and digested plasmid was self - ligated , yielding plasmid pet_his_del_pd ( seq id no : 2 ), which encodes reverse transcriptase gene shorter by 171 nucleotides or 57 amino acids , without shift in protein translation frame . it was important to have the same reverse transcriptase gene : 1 ) shorter in length ( for easy pcr detection ); 2 ) inactive ( it was confirmed experimentally that deletion of 57 amino acids in polymerase domain completely inactivated polymerase activity and mutation d583n inactivated rnase h activity ); and 3 ) without frameshift ( any frameshift will result in appearance of stop codons , which are not compatible with ribosome display format ). preparation of pcr fragments for in vitro transcription . pcr mixture was prepared on ice : 20 μl - 10 × taq buffer with kcl ( fermentas ); 20 μl - 2 mm of each dntp ( fermentas ); 12 μl - 25 mm mgcl 2 ( fermentas ); 16 μl - dmso ( d8418 - sigma ); 4 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 1 μl - 100 μm pro - pivex primer ( seq id no : 3 ); 1 μl - 100 μm pd - ter primer ( seq id no : 4 ); 122 μl water - mixture divided into two tubes 2 × 98 μl . to 2 × 98 μl of pcr master mix were added either 2 μl of pet_his_mlv_pd ( diluted to ˜ 1 ng / μl ) or 2 μl of pet_his_del_pd ( diluted to ˜ 1 ng / μl ). the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 30 cycles ( 45 sec at 94 ° c ., 45 sec at 53 ° c ., and 2 min at 72 ° c .) and final elongation 5 min at 72 ° c . amplification was ˜ 7000 fold from 2 ng of plasmid ( 7873 bp ) target to ˜ 5 μg ( 50 ng / μl ) of amplified product ( 2702 bp pcr fragment for pet_his_mlv_pd ; 2531 bp pcr fragment for pet_his_del_pd ). transcription mixture was prepared : 40 μl - 5 × t7 transcription buffer ( 1 m hepes - koh ph 7 . 6 ; 150 mm mg acetate ; 10 mm spermidin ; 0 . 2 m dtt ); 56 μl - 25 mm of each ntp ( fermentas ); 8 μl - 20 u / μl t7 rna polymerase ( fermentas ); 4 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 52 μl nuclease - free water - mixture divided into two tubes 2 × 80 μl and add 20 μl - 50 ng / μl of mlv_pd ( pro - pivex // pd - ter ) or 20 μl - 50 ng / μl of del_pd ( pro - pivex // pd - ter ) pcr mixture . transcription was performed 3 hr at 37 ° c . both transcription mixtures were diluted to 200 μl with ice - cold nuclease - free water and 200 μl of 6 m licl solution were added . mixtures incubated 30 min at + 4 ° c . and centrifuged for 30 min at + 4 ° c . in cooling centrifuge at max speed ( 25 , 000 g ). supernatant was discarded and rna pellet washed with 500 μl of ice - cold 75 % ethanol . tubes again were centrifuged for 5 min at + 4 ° c . in cooling centrifuge at max speed and supernatant was discarded . rna pellet was dried for 12 min at room temperature and subsequently resuspended in 200 μl of nuclease - free ice - cold water by shaking for 15 min at + 4 ° c . and 1400 rpm . tubes again were centrifuged for 5 min at + 4 ° c . in cooling centrifuge at max speed in order to separate not dissolved rna . about 180 μl of supernatant were moved to new tube with 20 μl of 10 × dnase i buffer ( mg 2 + ) ( fermentas ); 1 μl - 1 u / μl dnasei ( rnase - free ) ( fermentas ) and incubated for 20 min at + 37 ° c . in order to degrade dna . to each tube were added 20 μl of 3 m sodium acetate ph 5 . 0 solution and 500 μl of ice - cold 96 % ethanol . finally rna was precipitated by incubation for 30 min at − 20 ° c . and centrifugation for 30 min at + 4 ° c . in cooling centrifuge at max speed ( 25 , 000 g ). supernatant was discarded and rna pellet washed with 500 μl of ice - cold 75 % ethanol . tubes again were centrifuged for 5 min at + 4 ° c . in cooling centrifuge at max speed and supernatant was discarded . rna pellet was dried for 12 min at room temperature and subsequently resuspended in 43 μl of nuclease - free ice - cold water by shaking for 15 min at + 4 ° c . and 1400 rpm . rna solution was aliqouted 4 × 10 μl and liquid nitrogen frozen . concentration of mrna was measured spectrophotometrically and double checked on agarose gel using riboruler ™ rna ladder , high range ( fermentas )— mlv_pd mrna ˜ 1 . 2 μg / μl ; del_pd mrna ˜ 1 . 2 μg / μl . purified mrna is mixed with ratio 1 : 50 = mlv ( active rt ): del ( inactive rt ). mlv_pd mrna was diluted 25 times to ˜ 48 ng / μl and 1 μl (˜ 48 ng ) was mixed with 2 μl ˜ 1 . 2 μg / μl del_pd mrna ( 2 . 4 μg ) giving mrna mixture ˜ 0 . 8 μg / μl with ratio 1 : 50 . in vitro translation was performed using two translation systems rts 100 e . coli hy kit ( 03 186 148 001 — roche ) and synthetic wakopure ( 295 - 59503 — wako ). proteins translation sequences are given in seq id no : 6 for mlv_pd and seq id no : 7 for del_pd . translation mixture for rts hy system ( 25 μl ): 6 μl - e . coli lysate ( roche ); 5 μl - reaction mix ( roche ); 6 μl - amino acids ( roche ); 0 . 5 μl - 100 mm met ( roche ); 0 . 5 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 0 . 4 μl - 200 μm assra oligonucleotide ( seq id no : 5 ); 0 . 25 μl - 1 m dtt ; 2 . 5 μl reconstitution buffer ( roche ); 2 . 5 μl nuclease - free water and 1 . 5 μl - 0 . 8 μg / μl mrna mixture 1 : 50 = mlv_pd : del_pd (˜ 1200 ng ). in vitro translation was performed for 20 min at 30 ° c . translation mixture for wakopure system ( 25 μl ): 12 . 5 μl — a solution ( wako ); 5 μl — b solution ( wako ); 0 . 5 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 0 . 4 μl - 200 μm assra oligonucleotide ( seq id no : 6 ); 0 . 25 μl - 1 m dtt ; 5 μl nuclease - free water and 1 . 5 μl - 0 . 8 μg / μl mrna mixture 1 : 50 = mlv_pd : del_pd (˜ 1200 ng ). in vitro translation was performed for 30 min at 37 ° c . both translations (˜ 25 μl ) were stopped by adding 155 μl of ice - cold stopping buffer wbk 500 + dtt + triton ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma )) and centrifuged for 5 min at + 4 ° c . and 25 , 000 g . very carefully 160 μl of centrifuged translation mixture was pippeted on the top of 840 μl 35 % ( w / v ) sucrose solution in wbk 500 + dtt + triton ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma ); 35 % ( w / v )- sucrose ( 84097 - fluka )). in order to purify ternary complexes ( tc ) of mrna - ribosome - protein ( trna ) ultracentrifugation was performed using tl - 100 beckman ultracentrifuge ; tla100 . 2 fixed angle rotor ( beckman ); transparent 1 ml ultracentrifugation tubes ( 343778 - beckman ) for 9 min at + 4 ° c . and 100 , 000 rpm . in order to keep small transparent pellet of tc at the bottom of ultracentrifugation tube intact tubes were handled with care . initially 750 μl of solution was removed from the very top of the centrifugation tube . then ( very carefully ) tube walls were washed with 750 μl of wbk 500 ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ). finally all solution was removed starting from the very top of the centrifugation tube and pellet was dissolved in 30 μl of ice - cold stopping buffer wbk 500 + dtt + triton ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma )). as it was determined using radioactively labeled mrna after ultracentrifugation 5 %- 30 % of input mrna is located in ternary complex pellet . therefore it was expected to have less than 360 ng ( 30 % from 1200 ng mrna used in translation reaction ) of mrna in 30 μl of buffer (˜ 12 ng / μl or 9 * 10 9 molecules / μl of ternary complex ). reverse transcription reaction mixture for selection was prepared on ice : 60 μl - 5 × reaction buffer for reverse transcriptase ( fermentas ); 7 . 5 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 15 μl - 20 μm pd — 42 oligonucleotide ( seq id no : 8 ); 188 μl nuclease - free water - mixture divided into two tubes 2 × 135 μl and 0 . 9 μl of purified (& lt ; 8 * 10 9 molecules ) tc ( translation in roche — rts hy kit ) or 0 . 9 μl of purified (& lt ; 8 * 10 9 molecules ) tc ( translation in wako — wakopure ) were added . each reaction mixture (˜ 135 μl ) again was divided into two tubes 45 μl and 90 μl . to the first part — 45 μl of rt mixture 5 μl of nuclease - free water were added . this sample is considered to be the negative selection control ( without dntp ) and has to prove that there is no dna contamination in the reaction mixture and cdna synthesis is strictly linked to reverse transcriptase functional activity coming from mlv rt in ternary complex . to the second part — 90 μl of rt mixture 10 μl - 10 mm each dntp mix ( fermentas ) were added and reaction mixture again was divided into two tubes — 50 μl for selection control and 50 μl supplemented with 1 μl - 200 u / μl of revertaid h − m - mulv reverse transcriptase ( fermentas ) for positive selection control . according to the protocol each reverse transcription reaction mix contains & lt ; 2 . 7 * 10 9 molecules of ternary complex in 50 μl volume . oil - surfactant mixture for emulsification was prepared by mixing abil em 90 ( goldschmidt ) into mineral oil ( m5904 - sigma ) to final concentration of 4 % ( v / v ) ( ghadessy and holliger , 2004 ; us2005064460 ). emulsions were prepared at + 4 ° c . in 5 ml cryogenic vials ( 430492 - corning ) by mixing 950 μl of oil - surfactant mixture with 50 μl of rt mixture . mixing was performed using ms - 3000 magnetic stirrer with speed control ( biosan ) at ˜ 2100 rpm ; rotilabo ®-( 3 × 8 mm ) magnetic followers with centre ring ( 1489 . 2 - roth ); water phase was added by 10 μl aliquots every 30 sec , continuing mixing for 2 more minutes ( total mixing time — 4 min ). according to optical microscopy data compartments in our emulsions vary from 0 . 5 μm to 10 μm in size with average diameter of ˜ 2 μm . therefore it was expected to have ˜ 1 * 10 10 water in oil compartments after the emulsification of 50 μl reverse transcription reaction mixture , which contains less than 2 . 7 * 10 9 molecules of ternary complex ( about 1 mrna and reverse transcriptase molecules per 3 - 4 compartments ). all six emulsions representing rt reactions with tc , translation in roche — rts hy kit ( negative selection control , selection control and positive selection control ) and with tc , translation in wako — wakopure ( negative selection control , selection control and positive selection control ) were incubated 1 hr at + 42 ° c . to recover the reaction mixtures emulsions were moved to 1 . 5 ml tube , centrifuged for 1 min at room temperature and 25 , 000 g . oil phase was removed leaving concentrated ( but still intact ) emulsion at the bottom of the tube and 250 μl of pb buffer ( qiagen pcr purification kit ) were added . finally emulsions were broken by extraction with 0 . 9 ml water - saturated ether ; 0 . 9 ml water - saturated ethyl - acetate ( in order to remove abil em 90 detergent ) and again 0 . 9 ml water - saturated ether . water phase was dried for 5 min under vacuum at room temperature . synthesized cdna was purified with qiagen pcr purification kit and eluted in 30 μl of eb buffer ( qiagen pcr purification kit ). amplification of cdna was performed by nested pcr . initial pcr mixture was prepared on ice : 16 μl - 10 × taq buffer with kcl ( fermentas ); 16 μl - 2 mm of each dntp ( fermentas ); 9 . 6 μl - 25 mm mgcl 2 ( fermentas ); 3 . 2 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 0 . 8 μl - 100 μm rd_nde primer ( seq id no : 9 ); 0 . 8 μl - 100 μm pd — 55 primer ( seq id no : 10 ); 74 μl water - mixture was divided into 6 samples × 15 μl ( 6 × 15 μl ) and 30 μl . to 6 × 15 μl of pcr master mix were added 5 μl of cdna ( 1 - 6 rt samples ); to 30 μl of pcr master mix were added 9 μl water and mixture again divided into two tubes 2 × 19 . 5 μl for negative pcr control ( plus 0 . 5 μl water ) and positive pcr control ( plus 0 . 5 μl - 1 : 1 mixture (˜ 1 ng ) of pet_his_mlv_pd and pet_his_del_pd plasmids ). the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 25 cycles ( 45 sec at 94 ° c ., 45 sec at 58 ° c ., and 2 min at 72 ° c .) and final elongation 5 min at 72 ° c . expected length of pcr fragments was 2185 bp for mlv_pd and 2014 bp for del_pd . amplification was analyzed on 1 % agarose gel loading 10 μl of pcr mix per well ( fig3 ). nested pcr was performed using two different sets of primers , giving either partial gene amplification ( for better resolution of mlv : del cdna ratio in rt samples ) or full gene amplification ( to prove possibility of full gene recovery ). nested pcr mixture for partial gene amplification was prepared on ice : 28 μl - 10 × taq buffer with kcl ( fermentas ); 28 μl - 2 mm of each dntp ( fermentas ); 16 . 8 μl - 25 mm mgcl 2 ( fermentas ); 5 . 6 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 1 . 4 μl - 100 μm m_f primer ( seq id no : 11 ); 1 . 4 μl - 100 μm m — 2r primer ( seq id no : 12 ); 185 μl water - mixture divided 2 × 19 μl and 6 × 38 μl . to 2 × 19 μl of pcr master mix was added 1 μl of positive or negative controls of first pcr ( primers set rd_nde // pd — 55 )- 30 pcr cycles amplification ; to 6 × 38 μl of pcr master mix were added 2 μl of first pcr ( primers set rd_nde // pd — 55 ) ( 1 - 6 samples )— each sample again was divided into two 2 × 20 μl for 23 or 30 pcr cycles amplification . the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 23 or 30 cycles ( 45 sec at 94 ° c ., 45 sec at 57 ° c ., and 1 min at 72 ° c .) and final elongation 5 min at 72 ° c . expected length of pcr fragments was 907 bp for mlv_pd and 736 bp for del_pd . amplification was analyzed on 1 % agarose gel loading 10 μl of pcr mix per well ( fig4 ). nested pcr mixture for full gene amplification was prepared on ice : 28 μl - 10 × taq buffer with kcl ( fermentas ); 28 μl - 2 mm of each dntp ( fermentas ); 16 . 8 μl - 25 mm mgcl 2 ( fermentas ); 5 . 6 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 1 . 4 μl - 100 μm m_esp primer ( seq id no : 13 ); 1 . 4 μl - 100 μm m_eri primer ( seq id no : 14 ); 185 μl water - mixture divided 2 × 19 μl and 6 × 38 μl . to 2 × 19 μl of pcr master mix was added 1 μl of positive or negative controls of first pcr ( primers set rd_nde // pd — 55 )- 30 pcr cycles amplification ; to 6 × 38 μl of pcr master mix were added 2 μl of first pcr ( primers set rd_nde // pd — 55 ) ( 1 - 6 samples )— each sample again was divided into 2 × 20 μl for 23 or 30 pcr cycles amplification . the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 23 or 30 cycles ( 45 sec at 94 ° c ., 45 sec at 55 ° c ., and 2 min at 72 ° c .) and final elongation 5 min at 72 ° c . expected length of pcr fragments was 2077 bp for mlv_pd and 1906 bp for del_pd . amplification was analyzed on 1 % agarose gel loading 10 μl of pcr mix per well ( fig5 ). to demonstrate proof of principle for compartmentalized ribosome display ( crd ) method selection was performed using starting 1 : 50 = mlv : del mixture of two mrna encoding active ( mlv ) and inactive ( del ) reverse transcriptases fused to protein d spacer ( fig1 ). in vitro translation was performed using two different translation systems roche - rts100 e . coli hy or wako - wakopure in order to understand which translation system is better in our experimental setup . for each translation system three compartmentalized rt reactions were performed : negative selection control without dntp , which has to prove that there is no dna contamination in the reaction mixture ; selection control , which has to demonstrate the enrichment of genes encoding active ( mlv ) reverse transcriptase over genes encoding inactivated enzyme ( del ), because only active enzyme can synthesize cdna ; and positive selection control supplemented with external revertaid h - commercial reverse transcriptase , which has to serve as positive rt control synthesizing cdna from both mlv_pd and del_pd mrna in all compartments , showing real ratio of genes in reaction mixture without selection pressure applied . synthesized cdna was amplified by nested pcr . the picture of agarose gel electrophoresis of initial pcr ( 25 cycles ) ( fig3 ) shows weak pcr fragments bands only in case of both positive selection controls ( translation systems — roche and wako ). this is normal , because these samples contain external rt enzyme , which synthesizes cdna much more efficiently comparing to reactions containing only one in vitro synthesized reverse transcriptase molecule per compartment . the picture of agarose gel electrophoresis of nested pcr ( partial gene amplification ) is shown in fig4 . amplification results after 23 and 30 pcr cycles are consistent : 1 ) there is no amplification ( no dna contamination ) in negative selection controls ( w / o dntp ); 2 ) very efficient amplification of del_pd cdna ( 736 bp dna fragment ) is observed in positive selection controls ( external rt enzyme ) and no mlv_pd cdna amplification is visible , because initial ratio of mlv_pd to del_pd mrna is 1 : 50 ; 3 ) amplification of both cdna mlv_pd ( 907 bp dna fragment ) and del_pd ( 736 bp dna fragment ) are observed in case of selection controls ; 4 ) ratio of mlv_pd : del_pd ˜ 1 : 1 is observed in case of reverse transcriptase synthesized by roche in vitro translation system , what means ˜ 50 times enrichment of mlv_pd genes over del_pd genes starting from initial 1 : 50 ratio ; 5 ) ratio of mlv_pd : del_pd ˜ 1 : 3 is observed in case of reverse transcriptase synthesized by wako in vitro translation system , what means ˜ 16 times enrichment of mlv_pd genes over del_pd genes starting from initial 1 : 50 ratio . the picture of agarose gel electrophoresis of nested pcr ( full gene amplification ) is shown in fig5 . amplification results after 23 and 30 pcr cycles are consistent in between and comparing to results of nested pcr used for partial gene amplification ( fig5 ): 1 ) there is no amplification ( no dna contamination ) in negative selection controls ( w / o dntp ); 2 ) very efficient amplification of del_pd cdna ( 1906 bp dna fragment ) is observed in positive selection controls ( external rt enzyme ) and no mlv_pd cdna amplification is visible , because initial ratio of mlv_pd to del_pd mrna is 1 : 50 ; 3 ) amplification of both cdna mlv_pd ( 2077 bp dna fragment ) and del_pd 1906 bp dna fragment ) are observed in case of selection controls ; 4 ) it is difficult to determine ratio of mlv_pd : del_pd in case of full gene amplification , because relative difference between 2077 bp ( mlv_pd ) and 1906 bp ( del_pd ) dna fragments is not big enough , but in general ratios are similar to results of nested pcr used for partial gene amplification . as a result of this example we can conclude that during reverse transcription reaction performed in crd format we have enriched genes encoding active mlv reverse transcriptase over the genes encoding inactive enzyme by a factor of 50 in case of roche translation system or by a factor of 16 in case of wako translation system used to synthesize enzymes in vitro . crd — selection for reverse transcriptase , which shows improved performance at higher temperatures in order to understand , how efficiently compartmentalized ribosome display ( crd ) selection works , evolution experiment of moloney murine leukemia virus reverse transcriptase ( m - mulv rt ) was performed . general scheme of experiment is shown in fig6 . initial mutants library of reverse transcriptase was constructed by error - prone pcr using nucleotide analogues dptp and 8 - oxo - dgtp . whole gene (˜ 2 kb ) mutagenesis was performed introducing 2 - 3 nucleotides or 1 - 2 amino acids mutations per gene . pcr fragment encoding mutants library of reverse transcriptase ( in fusion with protein d ) mlv_pd was used to synthesize mrna . purified mrna was used for in vitro translation reaction . ternary complexes ( tc ) of mrna - ribosome - mlv_pd ( trna ) were formed in translation mixture and stabilized by low temperature and high concentration of mg 2 + ions . mixture of tc was purified by ultracentrifugation on sucrose cushions . precipitated tc was dissolved in ice - cold buffer ( 50 mm mg 2 + ) and used to prepare reverse transcription reaction mix supplemented with external dntp set and primer for rt reaction . ice - cold rt reaction mixture was emulsified giving ˜ 1 * 10 10 water in oil compartments ˜ 2 μm in size . optimal reaction temperature of mlv rt is ˜ 42 ° c . in order to select for reverse transcriptase variants , which are working better at higher temperatures emulsified rt reaction mixture ( less than one tc ( mrna + mlv rt ) per compartment ) was incubated for 1 hr at 50 ° c . at this temperature successful synthesis of full length cdna was performed better in compartments containing more active or thermostable mlv reverse transcriptase variants . subsequent pcr was used to amplify full length cdna and enrichment for more active and thermostable reverse transcriptase genes was performed . by pcr amplified genes were moved back to crd format restoring intact 5 ′ ( start fragment — t7 polymerase promoter , sd and his - tag coding sequences ) and 3 ′ ( end fragment — gs linker , protein d and second gs linker ) sequences by ligation - pcr . reconstructed pcr fragment , containing enriched library of reverse transcriptase genes , was used for subsequent mrna transcription and next crd selection round . each selection round was performed at higher and higher temperatures of rt reaction : 50 ° c . ( 1 st round ); 52 . 5 ° c . ( 2 nd round ); 55 ° c . ( 3 rd round ); 57 . 5 ° c . ( 4 th round ) and 60 ° c . ( 5 th round ). amplified library of reverse transcriptase genes ( without c terminal pd linker ) after 5 th selection round was cloned into plasmid vector . individual clones were sequenced and analyzed . pool of evolved proteins as well as individual mutants were purified via his - tag using affinity chromatography . mlv reverse transcriptase specific activities at 37 ° c ., 50 ° c . and residual activity at 37 ° c . after 5 min enzyme incubation at 50 ° c . were determined initial plasmid pet_his_mlv_pd ( seq id no : 1 and fig2 ) was used as a starting material for error - prone pcr . mutations were introduced using nucleotide analogues dptp and 8 - oxo - dgtp . pcr mixture for error prone pcr was prepared on ice : 10 μl - 10 × taq buffer with kcl ( fermentas ); 10 μl - 2 mm of each dntp ( fermentas ); 6 μl - 25 mm mgcl 2 ( fermentas ); 2 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 0 . 5 μl - 100 μm m_esp primer ( seq id no : 13 ); 0 . 5 μl - 100 μm m_eri primer ( seq id no : 14 ); 1 μl - 10 μm dptp ( trilink biotechnolgies ); 5 μl - 100 μm 8 - oxo - dgtp ( trilink biotechnolgies ); 3 . 75 μl - 40 ng / μl ( totally 150 ng ) of pet_his_mlv_pd plasmid ; 61 . 25 μl water . the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 30 cycles ( 30 sec at 94 ° c ., 30 sec at 55 ° c ., and 2 min at 72 ° c .) and final elongation 5 min at 72 ° c . amplification was 150 - 300 fold from 150 ng of plasmid ( 7873 bp ) target to ˜ 6 - 12 μg of amplified product ( 2077 bp pcr fragment for pet_his_mlv_pd ). pcr fragment was purified using qiagen pcr purification kit , digested with esp3i ( recognition sequence cgtctc ( ⅕ )) and ecori ( recognition sequence g ↓ aattc ) and finally purified from agarose gel using qiagen gel extraction kit giving dna concentration ˜ 50 ng / μl . mutagenesis efficiency and library quality was checked by sequencing of individual clones subcloned back into original pet_his_mlv_pd plasmid digested with ncoi and ecori . as it was expected mutations were distributed randomly all over the amplified sequence of mlv rt gene ( appendix 1 ). among 10 sequenced genes 23 nucleotide mutations ( 1 transversion , 20 transitions , 2 deletions — labelled red in appendix 1 ) were found giving 15 amino acids exchanges , 6 silent mutations , 1 stop codon and 2 frame shifts of coding frame — on average 1 - 2 amino acids substitutions per gene . mutated library was ligated with start ( 244 bp ) and end ( 398 bp ) fragments in order to get pcr fragment suitable for crd selection ( fig7 ). start fragment ( containing t7 polymerase promoter , sd and his - tag coding sequences ) was constructed by pcr amplification of initial 983 bp start fragment ( target — plasmid pet_his_del_pd ( seq id no : 2 ), primers — pro - pivex ( seq id no : 3 ) and m — 1r ( seq id no : 15 )) and subsequent digestion with ncoi ( recognition sequence c ↓ catgg ) giving 244 bp dna fragment . end fragment ( containing gs linker , protein d and second gs linker sequences ) was constructed by pcr amplification of initial 1039 bp end fragment ( target — plasmid pet_his_del_pd ( seq id no : 2 ), primers — m — 3f ( seq id no : 16 ) and pd - ter ( seq id no : 4 )) and subsequent digestion with ecori ( recognition sequence g ↓ aattc ) giving 398 bp dna fragment . ligation reaction ( 150 μl ) was prepared at room temperature : 15 μl - 10 × ligation buffer for t4 dna ligase ( fermentas ); 15 μl - 1 u / μl t4 dna ligase ( fermentas ); 26 μl - 50 ng / μl mutated mlv rt library digested with esp3i ( ncoi compatible end ) and ecori (˜ 1300 ng or ˜ 5 . 9 * 10 11 molecules ); 9 . 4 μl - 35 ng / μl start fragment digested with ncoi (˜ 329 ng or ˜ 1 . 2 * 10 12 molecules ); 15 . 7 μl - 35 ng / μl end fragment digested with ecori (˜ 548 ng or ˜ 1 . 2 * 10 12 molecules ); 68 . 9 μl - water . ligation was performed overnight at + 4 ° c . reaction mixture was treated once with phenol and twice with chloroform , precipitated and dissolved in 53 μl of water . approximate ligation yield ˜ 20 % was determined comparing amplification efficiency of ligation mixture and known amount of plasmid pet_his_mlv_pd . taking into account 20 % ligation yield diversity of mlv rt mutants library was defined as ˜ 1 . 2 * 10 11 molecules ( 50 μl ). ligated mlv rt library was amplified by pcr ( 1 ml - prepared on ice ): 100 μl - 10 × taq buffer with kcl ( fermentas ); 100 μl - 2 mm of each dntp ( fermentas ); 60 μl - 25 mm mgcl 2 ( fermentas ); 80 μl - dmso ( d8418 - sigma ); 20 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 5 μl - 100 μm pro - pivex primer ( seq id no : 3 ); 5 μl - 100 μm pd - ter - primer ( seq id no : 17 ); 20 μl - ligated mlv rt library (˜ 5 * 10 10 molecules ); 610 μl - water . the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 15 cycles ( 30 sec at 94 ° c ., 30 sec at 53 ° c ., and 3 min at 72 ° c .) and final elongation 5 min at 72 ° c . amplification was ˜ 200 fold from ˜ 5 * 10 10 molecules ( corresponds to ˜ 150 ng ) of final ligation fragment 2702 bp in size target to ˜ 30 μg ( 30 ng / μl ) of amplified product ( 2702 bp pcr fragment ). transcription mixture ( 100 μl ) was prepared : 20 μl - 5 × t7 transcription buffer ( 1 m hepes - koh ph 7 . 6 ; 150 mm mg acetate ; 10 mm spermidin ; 0 . 2 m dtt ); 28 μl - 25 mm of each ntp ( fermentas ); 4 μl - 20 u / μl t7 rna polymerase ( fermentas ); 2 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 30 μl - 30 ng / μl of mutants library ( pro - pivex // pd - ter -) pcr mixture (˜ 900 ng or ˜ 3 * 10 11 molecules ); 16 μl nuclease - free water . transcription was performed 3 hr at 37 ° c . ( library diversity ˜ 5 * 10 10 molecules ). transcription mixture was diluted to 200 μl with ice - cold nuclease - free water and 200 μl of 6 m licl solution were added . mixture incubated 30 min at + 4 ° c . and centrifuged for 30 min at + 4 ° c . in cooling centrifuge at max speed ( 25 , 000 g ). supernatant was discarded and rna pellet washed with 500 μl of ice - cold 75 % ethanol . tube again was centrifuged for 5 min at + 4 ° c . in cooling centrifuge at max speed and supernatant was discarded . rna pellet was dried for 12 min at room temperature and subsequently resuspended in 200 μl of nuclease - free ice - cold water by shaking for 15 min at + 4 ° c . and 1400 rpm . tube again was centrifuged for 5 min at + 4 ° c . in cooling centrifuge at max speed in order to separate not dissolved rna . about 180 μl of supernatant were moved to new tube with 20 μl of 10 × dnase i buffer ( mg 2 + ) ( fermentas ); 1 μl - 1 u / μl dnasei ( rnase - free ) ( fermentas ) and incubated for 30 min at + 37 ° c . in order to degrade dna . to reaction mixture were added 20 μl of 3 m sodium acetate ph 5 . 0 solution and 500 μl of ice - cold 96 % ethanol . finally rna was precipitated by incubation for 30 min at − 20 ° c . and centrifugation for 30 min at + 4 ° c . in cooling centrifuge at max speed ( 25 , 000 g ). supernatant was discarded and rna pellet washed with 500 μl of ice - cold 75 % ethanol . tube again was centrifuged for 5 min at + 4 ° c . in cooling centrifuge at max speed and supernatant was discarded . rna pellet was dried for 12 min at room temperature and subsequently resuspended in 33 μl of nuclease - free ice - cold water by shaking for 10 min at + 4 ° c . and 1400 rpm . rna solution was aliqouted 3 × 10 μl and liquid nitrogen frozen . concentration of mrna was measured spectrophotometrically and double checked on agarose gel using riboruler ™ rna ladder , high range ( fermentas )- mlv rt library mrna ˜ 2 . 1 μg / μl . in vitro translation was performed using rts100 e . coli hy ( 03 186 148 001 - roche ) translation system ( 25 μl ): 6 μl - e . coli lysate ( roche ); 5 μl - reaction mix ( roche ); 6 μl - amino acids ( roche ); 0 . 5 μl - 100 mm met ( roche ); 0 . 5 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 0 . 4 μl - 200 μm assra oligonucleotide ( seq id no : 5 ); 0 . 25 μl - 1 μm dtt ; 3 μl reconstitution buffer ( roche ); 2 . 5 μl nuclease - free water and 0 . 6 μl - 2 . 1 μg / μl mrna (˜ 1200 ng ). reaction mixture was incubated for 20 min at 30 ° c . translation was stopped adding 155 μl of ice - cold stopping buffer wbk 500 + dtt + triton ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma )) and centrifuged for 5 min at + 4 ° c . and 25 , 000 g . very carefully 160 μl of centrifuged translation mixture was pippeted on the top of 840 μl 35 % ( w / v ) sucrose solution in wbk 500 + dtt + triton ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma ); 35 % ( w / v )- sucrose ( 84097 - fluka )). in order to purify ternary complexes ( tc ) of mrna - ribosome - mlv ( trna ) ultracentrifugation was performed using tl - 100 beckman ultracentrifuge ; tla100 . 2 fixed angle rotor ( beckman ); transparent 1 ml ultracentrifugation tubes ( 343778 - beckman ) for 9 min at + 4 ° c . and 100 , 000 rpm . in order to keep small transparent pellet of tc at the bottom of ultracentrifugation tube intact tubes were handled with care . initially 750 μl of solution was removed from the very top of the centrifugation tube . than ( very carefully ) tube walls were washed with 750 μl of wbk 500 ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ). finally all solution was removed starting from the very top of the centrifugation tube and pellet was dissolved in 30 μl of ice - cold stopping buffer wbk 500 + dtt + triton ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma )). as it was determined experimentally , using radioactively labeled mrna , ultracentrifugation yields 5 %- 30 % of input mrna in ternary complex pellet . therefore it was expected to have less than 360 ng ( 30 % from 1200 ng mrna used in translation reaction ) of mrna in 30 μl of buffer (˜ 12 ng / μl or 9 * 10 9 molecules / μl of ternary complex ). reverse transcription reaction mixture for selection was prepared on ice : 60 μl - 5 × reaction buffer for reverse transcriptase ( fermentas ); 7 . 5 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 15 μl - 20 μm pd — 42 oligonucleotide ( seq id no : 8 ); 186 μl nuclease - free water and 1 . 8 μl of purified (& lt ; 1 . 8 * 10 10 molecules ) tc ( translation in roche - rts hy kit ). reaction mixture was divided into two tubes 45 μl and 225 μl . to first part — 45 μl of rt mixture were added 5 μl of nuclease - free water . this sample was considered to be negative selection control ( without dntp ) and has to prove that there is no dna contamination in the reaction mixture and cdna synthesis is strictly linked to reverse transcriptase functional activity coming from mlv rt in ternary complex . to second part — 225 μl of rt mixture were added 25 μl - 10 mm each dntp mix ( fermentas ) and reaction mixture again was divided into two tubes - 200 μl ( 4 × 50 μl ) for selection control (& lt ; 1 . 2 * 10 10 molecules of tc totally ) and 50 μl supplemented with 1 μl - 200 u / μl of revertaid h − m - mulv reverse transcriptase ( fermentas ) for positive selection control . according to protocol each reverse transcription reaction mix contains & lt ; 3 * 10 9 molecules of ternary complex in 50 μl volume . oil - surfactant mixture for emulsification was prepared by mixing abil em 90 ( goldschmidt ) into mineral oil ( m5904 - sigma ) to final concentration of 4 % ( v / v ) ( ghadessy and holliger , 2004 ; us2005064460 ). emulsions were prepared at + 4 ° c . in 5 ml cryogenic vials ( 430492 - corning ) by mixing 950 μl of oil - surfactant mixture with 50 μl of rt mixture . mixing was performed using ms - 3000 magnetic stirrer with speed control ( biosan ) at ˜ 2100 rpm ; rotilabo ®-( 3 × 8 mm ) magnetic followers with centre ring ( 1489 . 2 - roth ); water phase was added by 10 μl aliquots every 30 sec , continuing mixing for 2 more minutes ( total mixing time - 4 min ). according to optical microscopy data compartments in our emulsions vary from 0 . 5 μm to 10 μm in size with average diameter of ˜ 2 μm . therefore it was expected to have ˜ 1 * 10 10 water in oil compartments after the emulsification of 50 μl reverse transcription reaction mixture , which contains less than 3 * 10 9 molecules of ternary complex ( about 1 mrna and reverse transcriptase molecules per 3 - 4 compartments ). all emulsions were incubated 1 hr at + 50 ° c . in order to select for reverse transcriptase variants , which work better at higher temperatures . to recover the reaction mixtures emulsions were moved to 1 . 5 ml tube , centrifuged for 10 min at room temperature and 25 , 000 g . oil phase was removed leaving concentrated ( but still intact ) emulsion at the bottom of the tube . emulsions were broken by extraction with 0 . 9 ml water - saturated ether ; 0 . 9 ml water - saturated ethyl - acetate ( in order to remove abil em 90 detergent ) and again 0 . 9 ml water - saturated ether . water phase was dried for 5 min under vacuum at room temperature and 250 μl of pb buffer ( qiagen pcr purification kit ) were added . four selection samples were merged into two tubes . synthesized cdna was further purified with qiagen pcr purification kit and eluted in 30 μl of eb buffer ( qiagen pcr purification kit ) in case of negative and positive selection controls and 2 × 30 μl in case of selection control . amplification of cdna was performed by nested pcr . first of all small pcr amplification was performed in order to check negative and positive selection controls and determine minimal number of pcr cycles required for efficient amplification of cdna in selection samples . pcr mixture ( 200 μl ) was prepared on ice : 20 μl - 10 × taq buffer with kcl ( fermentas ); 20 μl - 2 mm of each dntp ( fermentas ); 12 μl - 25 mm mgcl 2 ( fermentas ); 2 . 5 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 1 μl - 2 . 5 u / μl pfu dna polymerase ( fermentas ); 1 μl - 100 μm rd_nde primer ( seq id no : 9 ); 1 μl - 100 μm pd — 55 primer ( seq id no : 10 ); 50 μl - purified cdna of selection controls ; 92 μl water . the cycling protocol for 2185 bp pcr fragment was : initial denaturation step 3 min at 94 ° c ., 25 cycles ( 45 sec at 94 ° c ., 45 sec at 58 ° c ., and 2 min at 72 ° c .) and final elongation 5 min at 72 ° c . nested pcr mixture ( 500 μl ) for full gene amplification was prepared on ice : 50 μl - 10 × taq buffer with kcl ( fermentas ); 50 μl - 2 mm of each dntp ( fermentas ); 30 μl - 25 mm mgcl 2 ( fermentas ); 6 . 25 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 2 . 5 μl - 2 . 5 u / μl pfu dna polymerase ( fermentas ); 2 . 5 μl - 100 μm m_esp primer ( seq id no : 13 ); 2 . 5 μl - 100 μm m_eri primer ( seq id no : 14 ); 50 μl of first pcr ( primers set rd_nde // pd — 55 ); 306 μl water . the cycling protocol for 2077 bp pcr fragment was : initial denaturation step 3 min at 94 ° c ., 22 cycles ( 45 sec at 94 ° c ., 45 sec at 55 ° c ., and 2 min at 72 ° c .) and final elongation 5 min at 72 ° c . final pcr fragment of selection sample was agarose - gel purified using qiagen gel extraction kit ( elution in 60 μl ˜ 50 ng / μl ). purified pcr fragment was digested with ecori and esp3i for 1 hr at 37 ° c . and again agarose - gel purified ( elution in 30 μl ˜ 50 ng / μl ). recovered mlv reverse transcriptase library after 1 st selection round was ligated with start and end fragments ( construction described earlier in this example ) in order to get pcr fragment ( fig7 ) suitable for 2 nd round of crd selection ( fig6 ). ligation reaction ( 40 μl ) was prepared at room temperature : 4 μl - 10 × ligation buffer for t4 dna ligase ( fermentas ); 2 μl - 1 u / μl t4 dna ligase ( fermentas ); 4 μl - 50 ng / μl selected library digested with esp3i and ecori (˜ 200 ng or ˜ 0 . 9 * 10 11 molecules ); 1 . 1 μl - 35 ng / μl start fragment digested with ncoi (˜ 35 ng or ˜ 1 . 5 * 10 11 molecules ); 1 . 76 μl - 35 ng / μl end fragment digested with ecori (˜ 61 ng or ˜ 1 . 5 * 10 11 molecules ); 27 . 2 μl - water . ligation was performed 1 hr at room temperature . ligated mlv rt library was amplified by pcr ( 300 μl - prepared on ice ): 30 μl - 10 × taq buffer with kcl ( fermentas ); 30 μl - 2 mm of each dntp ( fermentas ); 18 μl - 25 mm mgcl 2 ( fermentas ); 24 μl - dmso ( d8418 - sigma ); 3 . 7 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 1 . 5 μl - 2 . 5 u / μl pfu dna polymerase ( fermentas ); 1 . 5 μl - 100 μm pro - pivex primer ( seq id no : 3 ); 1 . 5 μl - 100 μm pd - ter - primer ( seq id no : 17 ); 25 . 5 μl - ligated mlv rt library (& lt ; 0 . 6 * 10 10 molecules ); 164 . 3 μl - water . the cycling protocol for 2702 bp pcr fragment was : initial denaturation step 3 min at 94 ° c ., 15 cycles ( 45 sec at 94 ° c ., 45 sec at 53 ° c ., and 3 min at 72 ° c .) and final elongation 5 min at 72 ° c . pcr fragment was agarose - gel purified using qiagen gel extraction kit ( elution in 30 μl ˜ 100 ng / μl ). second selection round was performed following general setup of 1 st selection round of experimental scheme with minor modifications in pcr cycles , emulsified rt reaction temperature and few more details . transcription — 10 μl - 100 ng / μl (˜ 1000 ng ) of agarose - gel purified pcr fragment were taken ; final concentration of mrna used in 2 nd selection round was 1 . 5 μg / μl ; translation — 0 . 8 μl - 1 . 5 μg / μl (˜ 1 . 2 μg ) of mrna were taken ; emulsified rt reaction was performed 1 hr at 52 . 5 ° c . ; 1 st pcr ( rd_nde // pd — 55 )— 24 cycles were performed ; 2 nd ( nested ) pcr ( m_esp // m_eri )— 23 cycles were performed ; final concentration of digested pcr fragment — 80 ng / μl ; ligation — 200 ng (˜ 0 . 9 * 10 11 molecules ) of mlv rt library were taken ; pcr ( on ligation mix )—& lt ; 0 . 6 * 10 10 molecules of selected library were taken and 15 pcr cycles were performed ; concentration of final agarose - gel purified pcr fragment was 200 ng / μl . third selection round was performed following general setup of 1 st selection round of experimental scheme with minor modifications in pcr cycles , emulsified rt reaction temperature and few more details . transcription — 5 μl - 200 ng / μl (˜ 1000 ng ) of agarose - gel purified pcr fragment were taken ; final concentration of mrna used in 3 rd selection round was 1 . 5 μg / μl ; translation — 0 . 8 μl - 1 . 5 μg / μl (˜ 1 . 2 μg ) of mrna were taken ; emulsified rt reaction was performed 1 hr at 55 ° c . ; 1 st pcr ( rd_nde // pd — 55 )— 25 cycles were performed ; 2 nd ( nested ) pcr ( m_esp // m_eri )— 22 cycles were performed ; final concentration of digested pcr fragment — 70 ng / μl ; ligation − 200 ng (˜ 0 . 9 * 10 11 molecules ) of mlv rt library were taken ; pcr ( on ligation mix )—& lt ; 0 . 6 * 10 10 molecules of selected library were taken and 15 pcr cycles were performed ; concentration of final agarose - gel purified pcr fragment was 100 ng / μl . fourth selection round was performed following general setup of 1 st selection round of experimental scheme with minor modifications in pcr cycles , emulsified rt reaction temperature and few more details . transcription — 10 μl - 100 ng / μl (˜ 1000 ng ) of agarose - gel purified pcr fragment were taken ; final concentration of mrna used in 4 th selection round was 1 . 8 μg / μl ; translation — 0 . 67 μl - 1 . 8 μg / μl (˜ 1 . 2 μg ) of mrna were taken ; emulsified rt reaction was performed 1 hr at 57 . 5 ° c . ; 1 st pcr ( rd_nde // pd — 55 )— 25 cycles were performed ; 2 nd ( nested ) pcr ( m_esp // m_eri )— 24 cycles were performed ; final concentration of digested pcr fragment — 50 ng / μl ; ligation — 200 ng (˜ 0 . 9 * 10 11 molecules ) of mlv rt library were taken ; pcr ( on ligation mix )—& lt ; 0 . 6 * 10 10 molecules of selected library were taken and 15 pcr cycles were performed ; concentration of final agarose - gel purified pcr fragment was 100 ng / μl . fifth selection round was performed following general setup of 1 st selection round of experimental scheme with some modifications in pcr cycles , emulsified rt reaction temperature and final stage of analysis . transcription — 10 μl - 100 ng / μl (˜ 1000 ng ) of agarose - gel purified pcr fragment were taken ; final concentration of mrna used in 5 th selection round was 1 . 1 μg / μl ; translation — 1 . 1 μl - 1 . 1 μg / μl (˜ 1 . 2 μg ) of mrna were taken ; emulsified rt reaction was performed 1 hr at 60 ° c . ; 1 st pcr ( rd_nde // pd — 55 )— 25 cycles were performed ; 2 nd ( nested ) pcr ( m_esp // m_eri )— 33 cycles were performed ; nested pcr mixture ( 5000 for full gene amplification was prepared on ice : 50 μl - 10 × taq buffer with kcl ( fermentas ); 50 μl - 2 mm of each dntp ( fermentas ); 30 μl - 25 mm mgcl 2 ( fermentas ); 6 . 25 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 2 . 5 μl - 2 . 5 u / μl pfu dna polymerase ( fermentas ); 2 . 5 μl - 100 μm m_esp primer ( seq id no : 13 ); 2 . 5 μl - 100 μm m_hind3 + primer ( seq id no : 18 ); 50 μl of first pcr ( primers set rd_nde // pd — 55 ); 306 μl water . the cycling protocol for 2077 bp pcr fragment was : initial denaturation step 3 min at 94 ° c ., 22 cycles ( 45 sec at 94 ° c ., 45 sec at 55 ° c ., and 3 min at 72 ° c .) and final elongation 5 min at 72 ° c . final pcr fragment of selection sample was agarose - gel purified using qiagen gel extraction kit ( elution in 60 μl ˜ 60 ng / μl ). purified pcr fragment was digested with hindiii and esp3i for 1 hr at 37 ° c . and again agarose - gel purified ( elution in 40 μl ˜ 50 ng / μl ). recovered mlv reverse transcriptase library after 5 th selection round was ligated into plasmid vector prepared from pet_his_mlv_pd ( seq id no : 1 and fig2 ) digested with ncoi and hindiii , giving new 7474 bp plasmid pet_his_mlv ( seq id no : 19 ) encoding mlv rt with n - terminal his - tag and no pd fusion on c - terminus for fast protein purification using affinity chromatography . ligated mlv rt library after 5 th selection round was electroporated into t7 expression strain er2566 . individual clones were sequenced and analyzed . pool of evolved proteins as well as individual mutants and primary wt m - mulv reverse transcriptase in the same construction were grown in 200 ml of lb to a590 ˜ 0 . 7 and purified via his - tag by affinity chromatography using 2 ml of qiagen - ni - nta superflow resin ( all purification was performed under native conditions according to suppliers recommendations ). elution was performed in 1 ml of eb ( 50 mm — nah 2 po 4 , 300 mm — nacl , 250 mm — imidazol , ph 8 . 0 , 10mm — β - mercaptoethanol and 0 . 1 % of triton x - 100 ). all proteins were dialyzed against 50 times excess of storage buffer ( 50 mm tris - hcl ( ph 8 . 3 at 25 ° c . ), 0 . 1 m nacl , 1 mm edta , 5 mm dtt , 0 . 1 % ( v / v ) triton x - 100 and 50 % ( v / v ) glycerol ). protein purity was checked on sds - page ( usually ˜ 40 - 80 % of target protein ). protein concentration was determined using bredford method based bio - rad protein assay ( 500 - 0006 ). mlv reverse transcriptase specific activities were measured at 37 ° c ., 50 ° c . ( enzyme diluted in special dilution buffer : 30 mm tris - hcl ph 8 . 3 at 25 ° c ., 10 mm dtt , 0 . 5 mg / ml bsa ) and rest of activity at 37 ° c . after 5 min enzyme incubation at 50 ° c . ( enzyme diluted and its stability measured in 1 × rt reaction buffer : 50 mm tris - hcl ph 8 . 3 at 25 ° c ., 4 mm mgcl 2 , 10mm dtt , 50 mm kcl ). enzyme activity in all cases was assayed in the following final mixture : 50 mm tris - hcl ( ph 8 . 3 at 25 ° c . ), 6 mm mgcl 2 , 10 mm dtt , 40 mm kcl , 0 . 5 mm dttp , 0 . 4 mbq / ml [ 3h ]- dttp , 0 . 4 mm polya . oligo ( dt ) 12 - 18 . activity units were determined measuring incorporation of dtmp into a polynucleotide fraction ( adsorbed on de - 81 ) in 10 min at particular reaction temperature and comparing to known amounts of commercial enzyme . during analysis of selection data we have accumulated 104 sequences expressing full length m - mulv reverse transcriptase . the total clustalw alignment of all proteins ( appendix 2 ) is compiled without n terminal his tag in order to have the same numeration of amino acids as is usually used in literature . wild type sequence denoted as mlv is always given as the first sequence . mutations are marked using white font color in black background ( appendix 2 ) amino acids positions , mutations of which somehow improve m - mulv reverse transcriptase properties and are described in different patent applications are marked in the alignment as columns of amino acids ( white font ) highlighted in grey . mutations originating from our selection and located in grey columns serve as proof that our selection procedure precisely targeted the beneficial hot spot or even exact amino acid mutations described elsewhere . out of 104 sequenced clones we found 98 unique sequences and 1 wt ( l5 — 87 ) sequence . five sequences are repeated twice ( l5 — 21 and l5 — 111 ; l5 — 43 and l5 — 112 ; l5 — 49 and l5 — 63 ; l5 — 64 and l593 ; l5 — 85 and l596 ). in total we had randomly expressed 55 proteins . out of 55 expressed proteins 40 enzymatically active mutant variants ( including control wt ) of m - mulv reverse transcriptase were successfully purified to 40 - 80 % homogeneity according to sds - page . total protein concentration in purified rt samples was in range of 0 . 6 - 5 . 5 mg / ml . mutant rt variants were tested for reverse transcriptase activity at 37 ° c ., 50 ° c . and residual activity at 37 ° c . after 5 min incubation at 50 ° c . ( fig8 ). reverse transcriptase activity at 37 ° c . is normalized to be 100 % and is omitted in fig8 . thus only two types of columns ( percents of rt activity at 50 ° c . and residual rt activity at 37 ° c . after 5 min incubation at 50 ° c .) are shown . as a control wt m - mulv reverse transcriptase used for mutants library construction is presented . this primary enzyme was expressed in the same vector and purified in the same way as mutant variants of rt . an average value of wt enzyme rt activity at 50 ° c . is about 45 % comparing to activity at 37 ° c . almost all tested proteins , with few exceptions , have higher than 45 % activity at 50 ° c . an average value of rt activity at 50 ° c . for all tested mutants is about − 92 % and is more than 2 times higher comparing to wt enzyme ( 45 %). some mutants are 100 % or even more active at 50 ° c . as they are at 37 ° c . : 20 , 23 , l5 — 16 , l5 — 24 , l5 — 30 , l5 — 35 , l5 — 37 , l5 — 43 , l5 — 46 , l5 — 47 , l5 — 49 , l5 — 52 , l5 — 55 , l5 — 64 , l5 — 65 , l5 — 68 , l5 — 72 . the best mutants found have rt activity at 50 ° c . about 140 % and more ( 3 times higher than 45 % of wt ): 20 ( 165 %), l5 — 37 ( 162 %), l5 — 43 ( 156 %), l5 — 46 ( 135 %), l5 — 47 ( 179 %), l5 — 52 ( 137 %), l5 — 64 ( 142 %) and l5 — 68 ( 153 %). even though majority of mutants have very high rt activities at 50 ° c ., they are not as thermostable . residual rt activity at 37 ° c . after 5 min incubation at 50 ° c . of wt control is ˜ 11 %. the same average residual activity of selected enzymes is also similar ˜ 12 %. although some tested rt variants are substantially more thermostable and have residual activity 2 - 3 times higher than wt enzyme ( 11 %): l5 — 8 ( 25 %), l5 — 43 ( 32 %), l5 — 46 ( 27 %), l5 — 64 ( 28 %), l5 — 65 ( 25 %), l5 — 68 ( 31 %). specific activity ( u / mg of protein ) for partially purified wt enzyme is ˜ 200 , 000 u / mg ( fig9 ). selected rt variants were expressed and purified in very diverse manner and an average specific activity (˜ 155 , 000 u / mg ) is slightly lower as for wt control ( fig9 ). in some cases specific activity is decreased , in some — increased ( 20 —˜ 274 , 000 u / mg ; l5 — 11 —˜ 273 , 000 u / mg ; l528 —˜ 230 , 000 u / mg ; l5 — 30 —˜ 224 , 000 u / mg ; l5 — 35 —˜ 316 , 000 u / mg ; l5 — 43 —˜ 328 , 000 u / mg ; l5 — 46 —˜ 304 , 000 u / mg ; l5 — 52 —˜ 310 , 000 u / mg ; l5 — 64 —˜ 256 , 000 u / mg ; l5 — 65 —˜ 247 , 000 u / mg ). it is obvious that our selection system works well . using increased temperature of rt reaction as a selection pressure factor , we have managed to evolve faster ( specific rt activity at 50 ° c . is higher ) and more thermostable ( residual rt activity at 37 ° c . after 5 min preincubation at 50 ° c .) reverse transcriptases . the source of valuable information is an alignment of selected protein sequences ( appendix 2 ). sequences of analyzed proteins , whose activity at 50 ° c . was substantially better as compared to primary wt m - mulv ( 70 % and more comparing to 45 % of wt activity ) are highlighted in grey ( appendix 2 ). number of mutagenized amino acids varied in range of 0 ( wt or l5 — 87 ) to 12 ( l5 — 9 ). list of mutations found in all selected rt variants is given in appendix 3 . proteins are sorted by decreasing number of mutations . most mutants ( 53 out of 104 ) had 4 - 6 mutations per sequence . it is obvious that reverse transcriptase sequence has some hot spots , which are very important and beneficial for the rt reaction in general and for the thermostability of enzyme . those hot spots can be easily identified in multiple sequence alignment ( appendix 2 ) as conglomeration of mutations at particular position . especially important are mutations found in better performing variants of m - mulv reverse transcriptase ( sequences are highlighted in grey — appendix 2 ). summarized information about the most frequent mutations ( in decreasing order ) is given in appendix 4 . as in previous appendix mutant proteins with substantially higher activity at 50 ° c . are highlighted in grey . if same mutation repeats for many times and tested reverse transcriptases with this mutation are better performing at 50 ° c ., that means this mutation is somehow beneficial for reverse transcription reaction . according to the frequency with which mutations are found they can be divided into five classes : 21 - 31 repeats ; 14 - 18 repeats ; 4 - 7 repeats ; 2 - 3 repeats and 1 repeat . the first group of the most frequent mutations comprises four amino acids d524 ( 31 repeats ); d200 ( 30 repeats ); d653 ( 23 repeats ) and d583 ( 21 repeats ). two amino acids ( d524 and d583 ) are known to complex magnesium ions in active centre of ribonuclease h domain . mutants d524g , d583n and e562q are used to turn off rnase h activity of m - mulv reverse transcriptase ( gerard et al ., 2002 ), what improves synthesis of cdna . results of our selection are strikingly similar . mutation of aspartate 524 is found in 31 sequences out of 98 . moreover , d524n substitution is found once , d524a — 10 times and finally d524g — 20 times . thus our selection not only precisely targeted important amino acids , but also the same amino acid substitutions , which are known to be the best . exactly the same situation is with mutation of aspartate 583 , which is repeated in 21 selected proteins out of 104 . substitution d583e is found once , d583a — 3 times , d583g — 7 times and finally d583n — 10 times . again , same amino acid and same substitution ( d583n ), which is known to be the best is selected most frequently . commercial enzyme superscript ii from invitrogen has three mutations : d524g , d583n and e562q ( wo2004024749 ). an interesting thing is that mutation of the third amino acid substitution e562 in our selection is found only once ( e562k in l5 — 71 ). this result suggests that most likely glutamate 562 is not as important as aspartates 524 and 583 , or for some reasons exchange of this amino acid can cause some side effects and is not beneficial for rt reactions performed at increased temperatures (& gt ; 50 ° c .). further analysis of selected proteins sequences allowed to identify many more hot amino acids positions , mutations of which are described in other patent applications for improved m - mulv reverse transcriptase : h204r — 7 repeats ( u . s . pat . no . 7 , 078 , 208 ); h638r — 4 repeats ( us20050232934a1 ); t197a — 2 repeats ( u . s . pat . no . 7 , 056 , 716 ); m289v ( l ), t306a ( m )— 2 repeats ( u57078208 ); e302k , n454k — 2 repeats ( wo07022045a2 ); e69g , l435p — 1 sequence ( wo07022045a2 ); y64c , q190r , v223m , f3095 — 1 sequence ( u . s . pat . no . 7 , 056 , 716 ); e562k — 1 sequence ( u . s . pat . no . 7 , 078 , 208 ). there are also two selected sequences of reverse transcriptases which have combination of three amino acids substitutions described in literature ( 30 — d200n , t306m , d524n , d583g ; l5 — 28 — t306a , f3095 , d524a , h594r , f6255 ). in addition to known mutations we have identified many more amino acids positions which are mutated quite often : d200n ( a , g )— 30 repeats ; d653n ( g , a , h , v )— 23 repeats ; l603w ( m )— 18 repeats ; t330p — 15 repeats ; l139p — 14 repeats ; q221r — 6 repeats ; t287a - 6 repeats ; 149v ( t )— 5 repeats ; n479d — 5 repeats ; h594r ( o )— 5 repeats ; f625s ( l )— 5 repeats ; p65s — 4 repeats ; h126s ( r )— 4 repeats ; l333q ( p )— 4 repeats ; a502v — 4 repeats ; e607k ( g , a )— 4 repeats ; k658r ( o )— 4 repeats ; h8p ( r ) 3 repeats ; p130s — 3 repeats ; e233k — 3 repeats ; q237r — 3 repeats ; n249d — 3 repeats ; a283d ( t )— 3 repeats ; a307v — 3 repeats ; y344h — 3 repeats ; p407s ( l )— 3 repeats ; m428l — 3 repeats ; q430r — 3 repeats ; d449g ( a )— 3 repeats ; a644v ( t )— 3 repeats ; n649s — 3 repeats ; l671p — 3 repeats ; e673g ( k )— 3 repeats ; n678i — 3 repeats ( appendix 4 ). best performing variants of rt usually have mutations of amino acids , which are modified most frequently : 20 ( 50 ° c .- 123 %)— d200n ( 30 repeats ), l603w ( 18 repeats ) and slightly modified c terminus — n678i , s679p , r680a ; l5 — 35 ( 50 ° c .- 125 %)— d200n ( 30 repeats ), t330p ( 15 repeats ), n479d ( 5 repeats ); l5 — 37 ( 50 ° c .- 162 %)— h123s ( 4 repeats ), l149f ( 1 sequence ), d200n ( 30 repeats ), n454k ( 2 repeats ), d583n ( 21 repeats ); l5 — 43 ( 50 ° c .- 160 %)— d200n ( 30 repeats ), q237r ( 3 repeats ), t330p ( 15 repeats ), d524g ( 31 repeats ), f625s ( 5 repeats ), d653n ( 23 repeats ); l5 — 46 ( 50 ° c .- 135 %)— d200n ( 30 repeats ), t330p ( 15 repeats ), d583n ( 21 repeats ), t644t ( 3 repeats ); l5 — 47 ( 50 ° c .- 179 %)— n107s ( 1 repeat ), h126r ( 4 repeats ), t128a ( 1 repeat ), 1179v ( 2 repeats ), d200n ( 30 repeats ), h642y ( 2 repeats ), d653n ( 23 repeats ); l5 — 52 ( 50 ° c .- 137 %)— d200n ( 30 repeats ), t330p ( 15 repeats ), q374r ( 2 repeats ), ( d583n ( 21 repeats ); l5 — 64 ( 50 ° c .- 142 %)— d200n ( 30 repeats ), d216g ( 2 repeats ), d524g ( 31 repeats ), e545g ( 2 repeats ); l5 — 65 ( 50 ° c .- 127 %)— d200n ( 30 repeats ), q238h ( 1 repeat ), l570i ( 1 repeat ), l603w ( 18 repeats ); l5 — 68 ( 50 ° c .- 153 %)— m39v ( 2 repeats ), 149v ( 2 repeats ), q91r ( 2 repeats ), h204r ( 7 repeats ), t287a ( 6 repeats ), n454k ( 2 repeats ), f625l ( 5 repeats ), d653h ( 23 repeats ). the combined data set of measured rt activities and sequence alignment analysis of mutant proteins allowed us to determine many beneficial mutations ( and combinations thereof ) in the sequence of m - mulv reverse transcriptase sequence . in vitro evolution experiment described in 2 nd example was very efficient . gradually increased temperature of reverse transcription reaction was used as a selection pressure and generated a lot of different mutant variants of m - mulv rt . most of them were able to perform better at elevated temperatures comparing to primary enzyme . sequence analysis of evolved reverse transcriptases indicates hot spots and most important amino acids positions ( replacements ), responsible for complex improvement of enzyme properties . in order to elucidate individual impact of different mutations single and multiple mutants of m - mulv rt were constructed , partially purified and analyzed . starting point for mutants construction was 7474 bp plasmid pet_his_mlv ( seq id no : 19 ) encoding m - mulv rt with n - terminal his - tag for fast protein purification using affinity chromatography . m - mulv reverse transcriptase specific activity at 37 ° c ., relative activity at 50 ° c . and relative residual activity at 37 ° c . after 5 min enzyme incubation at 50 ° c . were determined in some cases rnase h activity was checked and cdna synthesis reaction at different temperatures on 1 kb or 4 . 5 kb rna was performed . initial plasmid pet_his_mlv ( seq id no : 19 ) was used as a starting material for mutagenic pcr . mutations were introduced using mutagenic primers . individual clones were sequenced and analyzed . m - mulv rt mutants were expressed in t7 expression strain er2566 . individual proteins and primary wt m - mulv reverse transcriptase in the same construction were grown in 200 ml of lb to a590 ˜ 0 . 7 and purified via his - tag by affinity chromatography using 2 ml of qiagen - ni - nta superflow resin ( all purification was performed under native conditions according to suppliers recommendations ). elution was performed in 1 ml of eb ( 50 mm — nah 2 po 4 , 300 mm — nacl , 250 mm — imidazol , ph 8 . 0 , 10 mm — β - mercaptoethanol and 0 . 1 % of triton x - 100 ). all proteins were dialyzed against 50 times excess of storage buffer ( 50 mm tris - hcl ( ph 8 . 3 at 25 ° c . ), 0 . 1 m nacl , 1 mm edta , 5 mm dtt , 0 . 1 % ( v / v ) triton x - 100 and 50 % ( v / v ) glycerol ). protein purity was checked on sds - page ( usually ˜ 40 - 80 % of target protein ). protein concentration was determined using bradford reagent ( fermentas # r1271 ). mlv reverse transcriptase activities were measured at 37 ° c ., 50 ° c . ( enzyme diluted in special dilution buffer : 30 mm tris - hcl ph 8 . 3 at 25 ° c ., 10 mm dtt , 0 . 5 mg / ml bsa ) and rest of activity at 37 ° c . after 5 min enzyme incubation at 50 ° c . ( enzyme diluted and its stability measured in 1 × rt reaction buffer : 50 mm tris - hcl ph 8 . 3 at 25 ° c ., 4 mm mgcl 2 , 10 mm dtt , 50 mm kcl ). enzyme activity in all cases was assayed in the following final mixture : 50 mm tris - hcl ( ph 8 . 3 at 25 ° c . ), 6 mm mgcl 2 , 10 mm dtt , 40 mm kcl , 0 . 5 mm dttp , 0 . 4 mbq / m1 [ 3h ]- dttp , 0 . 4 mm polya . oligo ( dt ) 12 - 18 . activity units were determined measuring incorporation of dtmp into a polynucleotide fraction ( adsorbed on de - 81 ) in 10 min at particular reaction temperature and comparing to known amounts of commercial enzyme . rnase h activity of m - mulv reverse transcriptase variants was measured according to u . s . pat . no . 5 , 405 , 776 . rnase h activity of purified enzymes was assayed in reaction mixtures ( 50 μl ) containing 50 mm tris - hcl ph 8 . 3 , 2 mm mncl 2 , 1 mm dtt and [ 3h ]( a ) n *( dt ) n ( 5 μm [ 3h ]( a ) n , 35 cpm / μmol ; 20 μm ( dt ) n ). reactions were incubated at 37 ° c . for 10 min and were stopped by adding 10 μl of trna ( 1 mg / ml ) and 20 μl of cold 50 % tca . after 10 minutes on ice , the mixture was centrifuged for 10 minutes in an eppendorf centrifuge ( at 25000 g ). forty μl of the supernatant was counted in a lsc - universal cocktail ( roth — rotiszint eco plus ). one unit of rnase h activity is the amount of enzyme required to solubilize one mole of [ 3h ]( a ) n in [ 3h ]( a ) n *( dt ) n in 10 min at 37 ° c . “ revertaid ™ first strand cdna synthesis kit ” (# k1622 - fermentas ) and its control 1 . 1 kb rna with a 3 ′- poly ( a ) tail in combination with oligo ( dt ) 18 primer was used to check purified reverse transcriptases for their ability to synthesize cdna at different temperatures . alternatively 4 . 5 kb rna ( synthesized from eco31i linearized ptz19r plasmid , which additionally contains piece of phage lambda dna 5505 - 8469 bp ) was used to test reverse transcription reaction . cdna was synthesized 1 hr in 20 μl reaction volume using kit &# 39 ; s components 1 μg of synthetic rna , following provided protocol with only some minor modifications ( without 5 min preincubation at 37 ° c .). reverse transcription reactions were performed in 96 well pcr plate in eppendorf mastercycler gradient pcr machine applying corresponding temperature gradient . synthesized cdna was analyzed by alkaline agarose gel electrophoresis ( staining with ethidium bromide ). samples of cdna synthesis analysis on alkaline agarose gels are given in fig1 . according to the frequency , with which mutations are found during the evolution of m - mulv reverse transcriptase , they can be divided into five classes : 21 - 31 repeats ; 14 - 18 repeats ; 4 - 7 repeats ; 2 - 3 repeats and 1 repeat . construction of individual reverse transcriptase mutants in general was performed according to this information . most frequently found mutations were tested first . reverse transcriptase specific activity at 37 ° c ., relative activity at 50 ° c . and relative residual activity at 37 ° c . after 5 min enzyme incubation at 50 ° c . were determined . in some cases rnase h activity was checked and cdna synthesis reaction at different temperatures on 1 kb or 4 . 5 kb rna was performed . all experimental data on individual mutants are presented in appendix 5 . second column (“ selection frequency ”) indicates the number of sequenced mutants , which had exact mutation and the number in the parentheses indicates total number of particular amino acid mutations found in selection . for example d200n — 25 ( 30 ) means , that aspartate 200 replacement to asparagine was found 25 times out of 30 total d200 mutations . reverse transcriptase specific activity measured at 37 ° c . is given in units per mg of protein . relative enzyme activity at 50 ° c . and relative residual rt activity at 37 ° c . after 5 min incubation at 50 ° c . are given in percents normalized to specific activity ( 100 %) of the same enzyme measured at 37 ° c . as a control ( first line ) is given wt m - mulv reverse transcriptase used for mutants library construction . this primary enzyme is expressed in the same vector and purified in the same way as mutant variants of rt . specific activity of wt enzyme is about 200 , 000 u / mg at 37 ° c ., relative activity at 50 ° c . ( comparing to activity at 37 ° c . )— 45 - 50 % ( 90 , 000 - 100 , 000 u / mg ) and relative residual rt activity at 37 ° c . after 5 min incubation at 50 ° c . ( comparing to activity at 37 ° c .) is about 11 % (˜ 22 , 000 u / mg ). wild type enzyme has about 160 - 200 u / mol of rnase h activity and can synthesize full length 1 kb cdna at 48 ° c . it is known , that m - mulv reverse transcriptase is protected from thermal inactivation by the binding to template - primer substrate and contrary , enzyme is less thermostable in solution alone ( gerard et al ., 2002 ). relative residual rt activity at 37 ° c . after 5 min incubation at 50 ° c . directly indicates enzyme thermostability in solution without substrate . meanwhile relative activity at 50 ° c . represents enzyme thermostability in complex with rna / dna substrate and speed of cdna synthesis . fast mutant variant of reverse transcriptase will give increased numbers of polymerase units at 50 ° c ., even if it &# 39 ; s thermostability will be the same as wild type enzyme . highest temperature of cdna synthesis ( in our case 1 kb or 4 , 5 kb ) is the most comprehensive parameter , which represents general ability of enzyme to synthesize cdna at increased temperatures . reverse transcriptase mutants , which have at least 10 % increased : specific activity at 37 ° (≧ 220 , 000 u / mg , 200 , 000 u / mg - wt ), relative activity at 50 ° c . comparing to mutant activity at 37 ° c . (≧ 54 %, 45 - 50 %- wt ) or relative residual activity at 37 ° c . after 5 min incubation at 50 ° c . comparing to mutant activity at 37 ° c . (≧ 13 %, 11 %- wt ),— are shadowed in gray and considered as significantly improved enzymes . mutants able to synthesize full length 1 kb cdna at temperatures higher than 48 ° c . are also shadowed in gray . reverse transcriptase mutants with increased specific activity (≧ 220 , 000 u / mg ) at 37 ° c . are ( appendix 5 ): d200 ( d200n — 254 , 000 u / mg ; d200g — 276 , 000 u / mg ; d200h — 234 , 000 u / mg ), t330 ( t330n — 223 , 000 u / mg ; t330d — 240 , 000 u / mg ), q221 ( q221r — 268 , 000 u / mg ), h594 ( h594k — 270 , 000 u / mg ; h594q — 231 , 000 u / mg ), d449 ( d449e — 224 , 000 u / mg ; d449n — 221 , 000 u / mg ), m39 ( m39n — 349 , 000 u / mg ), m66 ( m66l — 237 , 000 u / mg ; m66v — 227 , 000 u / mg ; m66i — 240 , 000 u / mg ), h126 ( h126r — 227 , 000 u / mg ), w388 ( w388r — 266 , 000 u / mg ), 1179 ( 1179v — 251 , 000 u / mg ). reverse transcriptase mutants with increased relative activity ( 54 %) at 50 ° c . ( comparing to activity at 37 ° c .) are ( appendix 5 ): d200 ( d200n — 84 %; d200a — 87 %; d200q — 103 %; d200e — 79 %; d200v — 131 %; d200w — 103 %; d200g — 88 %; d200k — 102 %; d200r — 68 %; d200h — 54 %), l603 ( l603w — 105 %; l603f — 104 %; l603y — 95 %; l603m — 77 %), d653 ( d653n — 93 %; d653k — 106 %; d653a — 99 %; d653v — 98 %; d653q — 93 %; d653l - 83 %; d653h — 116 %; d653g — 90 %; d653w — 93 %; d653e — 80 %), t330 ( t330p — 80 %; t330n — 69 %; t330d — 55 %; t330v — 65 %; t330s — 67 %), q221 ( q221r — 94 %; q221k — 77 %; q221e — 64 %; q221m — 58 %; q221y — 77 %), e607 ( e607k — 84 %; e607a — 98 %; e607g — 72 %; e607d — 69 %), l139 ( l139p — 59 %), t287 ( t287s — 68 %), n479 ( n479d — 81 %), h594 ( h594r — 69 %; h594k — 80 %; h594q — 75 %; h594n — 61 %), d449 ( d449g — 79 %; d449e — 77 %; d449n — 75 %; d449a — 99 %; d449v — 83 %), m39 ( m39v — 54 %; m39n — 71 %), m66 ( m66l — 79 %; m66v — 73 %; m66i — 80 %), l333 ( l333q — 54 %), h126 ( h126r — 58 %), p130 ( p130s — 70 %), q91 ( q91r — 56 %), w388 ( w388r — 72 %), r390 ( r390w — 64 %), q374 ( q374r — 56 %), e5 ( e5k — 67 %). reverse transcriptase mutants with increased relative residual activity (≧ 13 %) at 37 ° c . after 5 min incubation at 50 ° c . ( comparing to activity at 37 ° c .) are ( appendix 5 ): d200 ( d200n — 15 %; d200a — 18 %; d200q — 23 %; d200r — 27 %; d200h — 27 %), l603 ( l603w — 23 %; l603y — 13 %; l603p — 15 %), d653 ( d653n — 21 %; d653k — 15 %; d653a — 18 %; d653v — 16 %; d653q — 18 %; d653h — 13 %; d653g — 13 %; d653w — 13 %; d653e — 19 %), t330 ( t330p — 21 %; t330n — 13 %; t330d — 16 %; t330s — 15 %), t287 ( t287a — 13 %; t287f — 13 %), h594 ( h594r — 14 %; h594q — 13 %), d449 ( d449g — 13 %), m39 ( m39v — 13 %), m66 ( m66l — 13 %), y344 ( y344h — 13 %), q91 ( q91r — 13 %), n649 ( n649s — 16 %), w388 ( w388r — 14 %). mutants able to synthesize full length 1 kb cdna at temperatures higher than 48 ° c . are ( appendix 5 ): d200 ( d200n — 50 . 4 ° c . ; d200h — 50 . 4 ° c . ), l603 ( l603w — 53 . 1 ° c . ; l603f — 50 . 4 ° c . ; l603y — 47 . 8 - 50 . 4 ° c . ), d653 ( d653n — 50 . 4 - 53 . 1 ° c . ; d653k — 50 . 4 - 53 . 1 ° c . ; d653a — 50 . 4 ° c . ; d653v — 50 . 4 ° c . ; d653q — 50 . 4 ° c . ; d653l — 50 . 4 ° c . ; d653h — 50 . 4 - 53 . 1 ° c . ; d653g — 50 . 4 ° c . ; d653w — 50 . 4 ° c . ), q221 ( q221r — 50 . 4 ° c . ), e607 ( e607k — 47 . 8 - 50 . 4 ° c . ), h594 ( h594k — 47 . 8 - 50 . 4 ° c . ; h594q — 47 . 8 - 50 . 4 ° c .). samples of 1 kb cdna synthesis analysis on alkaline agarose gels are given in fig1 a - d . according to collected biochemical data most important positions in m - mulv reverse transcriptase sequence , which can impact cdna synthesis at increased temperatures , are : d200 , l603 , d653 , t330 , q221 , e607 , l139 , t287 , n479 , h594 , d449 , m39 , m66 , l333 , h126 , y344 , p130 , q91 , n649 , w388 , r390 , i179 , q374 , e5 . in general mutations of interest can be combined in between and m - mulv reverse transcriptase thermostability with and without substrate , velocity , processivity and overall ability to synthesize cdna at increased temperatures can be further improved . some data , which illustrates enzyme improvement by combinatorial approach are presented in appendix 6 . single mutants d200n and l603w have relative activity at 50 ° c . 84 % and 105 %. highest temperatures of 1 kb cdna synthesis are 50 . 4 ° c . and 53 . 1 ° c . double mutant d200n ; l603w has relative activity 131 % at 50 ° c . and can synthesize 1 kb cdna at 56 ° c . triple mutant d200n ; l603w ; t330p ( 80 % at 50 ° c . ; 1 kb cdna at 47 . 8 ° c .) is improved further and has relative activity 175 % at 50 ° c . and can synthesize 1 kb cdna at 56 - 58 ° c . quadruple mutant d200n ; l603w ; t330p ; e607k ( 84 % at 50 ° c . ; 1 kb cdna at 47 . 8 - 50 . 1 ° c .) has relative activity 174 % at 50 ° c . and can synthesize 1 kb cdna at 60 - 62 ° c . quintuple mutant d200n ; l603w ; t330p ; e607k ; l139p ( 59 % at 50 ° c . ; 1 kb cdna at 47 . 8 ° c .) has relative activity 176 % at 50 ° c . and can synthesize 1 kb cdna at 62 ° c . and that is about 14 ° c . higher temperature comparing to wild type m - mulv reverse transcriptase ( 1 kb cdna at 47 . 8 ° c .). additive character of thermostability is also observed in case of : n479d , h594r mutants ( d200n ; l603w — 131 % at 50 ° c ., 1 kb cdna at 56 ° c . versus d200n ; l603w ; n479d ; h594r — 182 % at 50 ° c ., 1 kb cdna at 56 - 58 ° c ., 4 . 5 kb cdna at 56 - 58 ° c . ), t330p mutant ( d200n ; l603w ; d653n ; d524g — 155 % at 50 ° c ., 1 kb cdna at 58 - 60 ° c . versus d200n ; l603w ; d653n ; d524g ; t330p — 180 % at 50 ° c ., 1 kb cdna at 60 - 62 ° c .). samples of 4 . 5 kb cdna synthesis analysis on alkaline agarose gels are given in fig1 e - g . modification of crd — selection for dna dependent dna polymerase activity using biotin - dutp ( proof of principle ) this example illustrates activity - based selection strategy of reverse transcriptase as dna dependent dna polymerase , which is able to incorporate modified nucleotides into dna - dna substrate . the principle scheme of selection is schematically illustrated in fig1 . two plasmids pet_his_mlv_d583n_pd ( encoding rnase h minus moloney murine leukemia virus ( m - mlv ) reverse transcriptase fused to protein d spacer ) and its derivative pet_his_del_pd ( encoding inactivated reverse transcriptase ; 57 amino acids deletion in pol domain and mutation d583n in rnase h domain , example 1 , seq id no : 2 ) were used as a starting material in this example . initial dna fragments encoding active and inactive reverse transcriptases were synthesized in two separate polymerase chain reactions using plasmids pet_his_mlv_d583n_pd and pet_his_del_pd as a target . synthesized pcr fragments were used in transcription reaction for synthesis of mrna , which lacks stop codon at the 3 ′ end . purified mrnas were mixed to a ratio of 1 : 20 = mlv ( active rt ): del ( inactive rt ). double stranded dna adapter ( required for selection of dna dependent dna polymerase activity ) was ligated to 3 ′ mrna by t4 dna ligase . this mrna - dsdna complex is used for in vitro translation reaction . the ribosome moving along the mrna stops translation at the site of rna - dna hybridization ( tabuchi et al ., 2001 ). ribosome - mrna / dsdna - protein complexes were stabilized ( as in conventional ribosome display ) by dilution of translation mixture with ice - cold buffer containing 50 mm mg 2 + . mixture of ternary complexes ( tc ) was purified by ultracentrifugation on sucrose cushions . purified ternary complexes (& lt ; 3 * 10 9 molecules taken ) containing mrna - dsdna linked to in vitro translated m - mulv ( rnase h −) reverse transcriptases were used to prepare reaction mixture additionally supplemented with biotin - dutp and reaction buffer . ice - cold reaction mixture was emulsified yielding ˜ 1 * 10 10 water in oil compartments ˜ 2 μm in size . emulsified reaction mixture ( less than one tc , ribosome - mrna / dsdna - protein per compartment ) was incubated for 30 min at 37 ° c . after the temperature of compartmentalized reaction mixture is raised most of tcs dissociate releasing mrna / dsdna and reverse transcriptase . successful incorporation reaction can occur only in compartments containing active m - mulv ( rnase h −) reverse transcriptase resulting in biotinylation of mrna / dsdna complex . biotinylated complex can be selectively immobilized on streptavidin coated magnetic beads and specifically amplified by rt - pcr . as a result of successful experiment genes encoding active enzyme ( in our case rt - pcr fragment of mlv_d583n_pd reverse transcriptase ) should be enriched over genes encoding inactive enzyme ( del_pd ). ( 1 ) determination of ligation efficiency . efficiency of ligation reaction was determined by ligation of mlv_pd mrna ( synthesized from pet_his_mlv_pd plasmid , example 1 ) with primer long + tb ( seq id no : 23 ) using ddc - long2 primer ( seq id no : 22 ) as a splint . the 5 ′ end of long + tb was previously phosphorylated using t4 polynucleotide kinase ( fermentas ). annealing mix of 36 μl was prepared by mixing of 8 . 5 μmol (˜ 10 μg ) of purified mlv_pd mrna with 4 times of molar excess of long + tb ( seq id no : 23 ) and 4 . 2 times of molar excess of ddc - long2 ( seq id no : 22 ) in nuclease free water . the mixture was incubated at 70 ° c . for 5 min and then cooled to room temperature for 20 min . before adding ligation reaction components mixture was moved to cooling stand for 2 min . 4 . 5 μl of 10 × ligation buffer and 4 . 5 μl of t4 dna ligase ( 5v / μl ) ( fermentas ) were added to 36 μl of annealing mixture . ligation reaction was performed for 30 min at 37 ° c . and followed extraction once with equal volume of roti ® phenol / chloroform ( roth ) and twice with equal volume of chloroform . assuming that mrna amount was close to initial amount taken for ligation reaction ˜ 5 μg of ligation products mix was diluted to 43 μl by nuclease free water . aliquot of 2 μl of resultant mixture was left for analysis on agarose gel before immobilization on dynabeads m - 280 streptavidin beads ( dynabeads ® kilobase binder ™ kit ( dynal biotech )). 10 μl of resuspended dynabeads was transferred to a 1 . 5 ml microcentrifuge tube , washed with 50 μl of binding solution provided in the kit . the tube was placed on the magnet for 1 - 2 min until beads settled at the tube and solution was removed . dynabeads were gently resuspended by pipetting in 38 μl of binding solution supplemented with 2 μl of aqueous trna ( trna from yeast ( roche )) solution ( 1 μg / μl ) to minimize non - specific mrna binding . 40 μl of dynabeads in binding solution was added to solution (˜ 40 μl ) containing ligation products mix . the tube was incubated shaking at the + 22 ° c . in termomixer ( eppendorf ) for 60 min . after the binding of ligated mrna / dsdna , supernatant was removed and the beads were washed three times with 50 μl of washing solution ( supplied in the kit ). collected dynabeads with immobilizes ligated mrna / dsdna complex was resuspended in 26 μl of nuclease free water and followed extraction once with 40 μl of roti ® phenol / chloroform ( roth ) and twice with 40 μl volume of chloroform to release mrna / dsdna complex from magnetic beads . 1 ; 2 and 5 μl of final mix were analysed on agarose gel along with sample ( 2 μl ) left before immobilization and mass ruler ™ high range dna ladder . amount of mrna in mrna / dna complex purified on streptavidin beads was determined comparing mrna amounts before and after the immobilization . the yield of recovered mrna was ˜ 60 %, what means that at least 60 % of mrna was successfully ligated with dna duplex , resulting in mrna / dsdna complex . ( 2 ) determination of biotin - dutp incorporation efficiencies into mrna / dsdna complex and into self primed mrna . as it was shown in first mrna to dsdna ligation experiment ligation reaction efficiency is ˜ 60 % and more . free mrna left in ligation mixture can self prime and participate in extension reaction using m - mulv reverse transcriptase and biotin - dutp . this experiment was performed in order to demonstrate that mrna / dsdna complex ( ligation product ) is better substrate than free self primed mrna . mlv_d583n_pd mrna was ligated with long + oligonucleotide ( seq id no : 21 ) according procedure described above ( construction of initial plasmids pet_his_mlv_d583n_pd and pet_his_del_pd in details is described in example 1 ). ˜ 12 . 5 ng of prepared ( mlv_d583n_pd mrna / long +) substrate was combined with ˜ 12 . 5 ng of del_pd mrna previously incubated at 70 ° c . for 5 min , then cooled to room temperature for 20 min in nuclease free water , in total volume of 12 . 5 μl . the second mix for dttp or biotin - dutp incorporation by reverse transcriptase was prepared : 8 μl of 5 × reaction buffer for reverse transcriptase ; 1 μl - 40 u / μl ribolock ™ rnase inhibitor ( fermentas ); 18 . 6 μl nuclease - free water ; 0 . 4 μl - 200 u / μl revertaid ™ minus m - mulv reverse transcriptase ( fermentas ). prepared mix was divided to two tubes 2 × 15 μl and 1 μl of 1 mm dttp ( fermentas ) or 1 μl of 1 mm biotin - dutp ( fermentas ) was added . subsequentially 5 μl of substrate ( from the first mix ) was added to dttp and biotin - dutp containing mixtures . reactions were carried out at 37 ° c . for 60 min . after that 1 μl of 0 . 5m edta ( ph 8 . 0 ) was added to both samples and reaction mixtures were extracted once with equal volume of roti ® phenol / chloroform ( roth ) and once with equal volume of chloroform followed by purification on g - 50 microcolumns ( ge healthcare ). 2 μl of resultant reaction products were left for direct rt reaction ( without streptavidin beads purification ) and the remaining part of solutions were used for biotinylated mrna - dsdna complex immobilization on dynabeads m - 280 streptavidin beads ( dynabeads ® kilobase binder ™ kit ( dynal biotech )). 10 μl of resuspended dynabeads was transferred to a 1 . 5 ml microcentrifuge tube , washed with 25 μl of provided binding solution . the tube was placed on the magnet for 1 - 2 min until beads settled at the bottom of the tube and solution was removed . dynabeads were gently resuspended by pipetting in 90 μl of binding solution and 2 μl of aqueous trna ( trna from yeast ( roche )) solution ( 1 μg / μl ) was added to minimize non - specific mrna binding . 40 μl of dynabeads in binding solution was added to solution (˜ 40 μl ) containing the elongated by dttp and biotin - dutp rna - dna fragments . the tubes were incubated at the + 22 ° c . in termomixer ( eppendorf ) for 40 min by shaking ( 1400 rpm ). after the binding step , supernatant was removed and the beads were washed three times with 50 μl of washing solution ( provided in the kit ) shaking ( 1400 rpm ) for 5 min at + 22 ° c . collected dynabeads with immobilizes elongated mrna - dsdna complex was resuspended in reverse transcription reaction mixture . reverse transcription reaction mixture was prepared on ice : 20 μl - 5 × reaction buffer for reverse transcriptase ( fermentas ); 10 μl - 10 mm dntp ( fermentas ); 2 . 5 μl - 40 ribolock rnase inhibitor ( fermentas ); 5 - 20 μm pd — 42 oligonucleotide ( seq id no : 8 ); 2 . 5 μl revertaid ™ minus m - mulv reverse transcriptase ( 200 u / μl )( fermentas ); 55 μl nuclease - free water . prepared mix was divided to five 19 μl ( 5 × 19 μl ) aliquots : two were used to resuspend dynabeads with immobilized elongated mrna / dsdna complex or mrna , the other two were transferred to the tubes with samples left without streptavidin beads purification and to the rest of 19 μl aliquot was added 1 μl of nuclease free water - negative reaction control to prove , that reaction mixture is not contaminated by dna . all reaction mixtures were incubated by shaking ( 1000 rpm ) at the + 42 ° c . in termomixer ( eppendorf ) for 1 hour until cdna synthesis reaction was finished . amplification of cdna was performed by nested pcr . initial pcr mixture was prepared on ice : 14 μl - 10 × taq buffer with kcl ( fermentas ); 14 μl - 2 mm of each dntp ( fermentas ); 8 . 4 μl - 25 mm mgcl 2 ( fermentas ); 2 . 8 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 0 . 7 μl - 100 μm m_f primer ( seq id no : 11 ); 0 . 7 μl - 100 μm m — 2r primer ( seq id no : 12 ); 92 . 4 μl water - mixture was divided into 7 samples for 19 μl ( 7 × 19 μl ). to 5 × 19 μl of pcr master mix were added 1 μl of cdna ( 1 - 5 rt samples ); to the 6 th tube of pcr master mix 1 μl of water was added - negative pcr control ; to the 7 th tube ( positive pcr control )- 1 μl of pet_his_mlv_d583n_pd plasmid (˜ 1 ng ) was added . the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 25 cycles ( 45 sec at 94 ° c ., 45 sec at 57 ° c ., and 1 min at 72 ° c .) and final elongation 3 min at 72 ° c . predicted amplicons size 907 bp for mlv_d583n_pd and 736 bp for del_pd cdna . pcr products were analyzed on 1 % agarose gel loading 10 μl of pcr mix per well ( fig1 ). as it was expected efficient cdna amplification is observed only in elongation reaction with biotin - dutp . very faint bands of amplified cdna can be detected in elongation reaction with dttp and can be explained by weak nonspecific binding of mrna to streptavidin beads . after purification on streptavidin beads dna encoding mlv_d583n_pd gene ( 907 bp ) is enriched over dna of del_pd ( 736 bp ). that means mrna / dsdna complex is elongated by biotin - dutp much more efficiently than self primed del_pd mrna . ( 3 ) preparation of mrna mixture ( mlv_d583n_pd : del_pd = 1 : 20 ) and rna / dsdna complex . preparation of pcr fragments for in vitro transcription . pcr mixture was prepared on ice : 20 μl - 10 × taq buffer with kcl ( fermentas ); 20 μl - 2 mm of each dntp ( fermentas ); 12 μl - 25 mm mgcl 2 ( fermentas ); 16 μl - dmso ( d8418 - sigma ); 4 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 1 - 100 μm pro - pivex primer ( seq id no : 3 ); 1 - 100 μm pd - ter - primer ( seq id no : 20 ); 122 μl water - mixture divided into two tubes 2 × 98 μl . to 2 × 98 μl of pcr master mix were added either 2 μl of pet_his_mlv_d583n_pd ( diluted to ˜ 1 ng / μl ) or 2 μl of pet_his_del_pd ( diluted to ˜ 1 ng / μl ) ( construction of initial plasmids pet_his_mlv_d583n_pd and pet_his_del_pd in details is described in example 1 ). the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 30 cycles ( 45 sec at 94 ° c ., 45 sec at 53 ° c ., and 2 min at 72 ° c .) and final elongation 5 min at 72 ° c . amplification efficiency was 7000 fold from 2 ng of plasmid ( 7873 bp ) target to ˜ 5 μg ( 50 ng / μl ) of amplified product ( 2702 bp pcr fragment mlv_d583n_pd from pet_his_mlv_d583n_pd ; 2531 bp pcr fragment del_pd from pet_his_del_pd ). transcription mixture was prepared : 80 μl - 5 × t7 transcription buffer ( 1 m hepes - koh ph 7 . 6 ; 150 mm mg acetate ; 10 mm spermidin ; 0 . 2 m dtt ); 56 μl - 112 mm of each ntp ( fermentas ); 16 μl - 20 u / μl t7 rna polymerase ( fermentas ); 8 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 114 μl nuclease - free water - mixture divided into two tubes 2 × 165 μl and add 35 μl - 20 ng / μl of pcr fragment mlv_d583n_pd ( unpurified pcr mixture ) or 35 μl - 20 ng / μl of pcr fragment del_pd pcr ( unpurified pcr mixture ). transcription was performed 2 hr at 37 ° c . both transcription mixtures were diluted to 200 μl with ice - cold nuclease - free water and 200 μl of 6 m licl solution were added . mixtures incubated 25 min at + 4 ° c . and centrifuged for 25 min at + 4 ° c . in cooling centrifuge at max speed ( 25 , 000 g ). supernatant was discarded and rna pellet washed with 500 μl of ice - cold 75 % ethanol . tubes were centrifuged again for 5 min at + 4 ° c . at max speed and supernatant was discarded . rna pellet was dried for 5 min at room temperature and subsequently resuspended in 400 μl of nuclease - free ice - cold water by shaking for 15 min at + 4 ° c . and 1400 rpm . tubes were centrifuged again for 5 min at + 4 ° c . at max speed to separate not dissolved rna . about 380 μl of supernatant were transferred to new tube with 42 μl of 10 × dnase i buffer ( mg 2 + ) ( fermentas ); 3 μl - 1 u / μl dnasei ( rnase - free ) ( fermentas ) and incubated for 20 min at + 37 ° c . to degrade dna . reaction mixtures were extracted once with equal volume of roti ® phenol / chloroform ( roth ) and twice with equal volume of chloroform to remove dnasei . 43 μl of 3 m sodium acetate ph 5 . 0 solution and 1075 μl of ice - cold 96 % ethanol were added to each tube . finally rna was precipitated by incubation for 30 min at − 20 ° c . and centrifugation for 25 min at + 4 ° c . at max speed ( 25 , 000 g ). supernatant was discarded and rna pellet washed with 500 μl of ice - cold 75 % ethanol . tubes were centrifuged again for 4 min at + 4 ° c . at max speed and supernatant was discarded . rna pellet was dried for 5 min at room temperature and subsequently resuspended in 150 μl of nuclease - free ice - cold water by shaking ( 1400 rpm ) for 15 min at + 4 ° c . rna solution was aliquot for 10 μl and liquid nitrogen frozen . concentration of mrna was measured spectrophotometrically and double - checked on agarose gel along with riboruler ™ rna ladder , high range ( fermentas ). mrna / dsdna complex was produced by ligation of long + oligodeoxynucleotide to mrna using ddc - long2 oligodeoxynucleotide as a splint . the 5 ′ end of long + was previously phosphorylated using t4 polynucleotide kinase ( fermentas ). oligodeoxynucleotide ddc - long2 has 3 ′ end modification ( ddc ) in order to prevent possibility of 3 ′ end extension by reverse transcriptase on its natural rna - dna substrate . annealing mix of 50 μl was prepared by mixing of 17 μmol of purified mrna mixture at ratio of 1 : 20 = mlv_d583n_pd ( active rt ): del_pd ( inactive rt ) with 4 . 3 time of molar excess of long + and 4 . 1 time of molar excess of ddc - long2 in nuclease free water . the mixture was incubated at 70 ° c . for 5 min and then cooled to room temperature for 20 min . before adding ligation reaction components mixture was moved to cooling stand for 2 min . 5 μl of 10 × ligation buffer and 5 μl of t4 dna ligase ( 5v / μl ) ( fermentas ) were added to 40 μl of annealing mixture . negative ligation reaction was carried out using the same annealing mixture in 1 × ligation buffer without t4 dna ligase . prepared ligation reaction mixtures were incubated at 37 ° c . for 30 min . to stop ligation 1 μl of 0 . 5m edta ( ph 8 . 0 ) was added to both tubes and reaction mixtures were extracted once with equal volume of roti ® phenol / chloroform ( roth ) and twice with equal volume of chloroform followed by concentration of reaction products at 30 ° c . for 10 min in vacuum concentrator 5301 ( eppendorf ). desalting was performed using illiustra probequant g - 50 microcolumns ( ge healthcare ). concentrations of ligation products were determined on agarose gel along with riboruler ™ rna ladder , high range ( fermentas )— mrna mixture ( mlv_d583n_pd : del_pd = 1 : 20 ) ligated to long +/ ddc - long2 oligodeoxynucleotides ˜ 0 . 24 μg / μl ( sample with t4 dna ligase ) and simple mrna mixture with long +/ ddc - long2 oligodeoxynucleotides ˜ 0 . 06 μg / μl ( sample without t4 dna ligase ). ( 4 ) general control of mrna / dsdna complex by incorporation of [ α - p 33 ] datp . prepared mrna / dsdna complex ( substrate ) was tested for dttp ( or biotin - dutp ) and subsequent [ α - p 33 ] datp incorporation by reverse transcriptase . reaction mixture : 16 μl of 5 × reaction buffer for reverse transcriptase ; 4 μl - 40 u / μl ribolock ™ rnase inhibitor ( fermentas ); 2 μl [ α - p 33 ] datp ( 10mci )/ ml , srf - 203 ( hartmann analytic )); 35 μl nuclease free water ; 1 μl - 200 u / μl revertaid ™ minus m - mulv reverse transcriptase ( fermentas ). prepared mix was divided to two tubes 2 × 28 μl and 2 μl of 1 mm dttp ( fermentas ) or 2 μl of 1 mm biotin - dutp ( fermentas ) was added . resultant mixtures were divided to two tubes 2 × 15 μl . to first tube 1 . 25 μl (˜ 0 . 3 μg ) of ligation products plus 3 . 75 μl of nuclease free water were added . to second tube 5 μl (˜ 0 . 3 μg ) of negative ligation reaction products were added . reaction mixtures were incubated at 37 ° c . for 30 min . after that , 1 μl of 0 . 5m edta ( ph 8 . 0 ) was added to all tubes and reaction mixtures were extracted once with equal volume of roti ® phenol / chloroform ( roth ) and twice with equal volume of chloroform followed by purification on illiustra probequant g - 50 microcolumns ( ge healthcare ). reactions products were analyzed on agarose gel along with riboruler ™ rna ladder , high range ( fermentas ) fig1 a . in all samples ( with and without ligase ) we can see discreet band (˜ 2500b ) of del_pd mrna ( 20 times smaller amount of mlv_d583n_pd mrna ˜ 2700b , which was present in mrna mixture can not be distinguished ). subsequently agarose gel was dried on filter paper and radiolabeled mrna / dsdna complex ( at the same position as mrna ) was detected only in case of positive ligation samples ( with ligase ) and not in case of negative ligation samples ( without ligase ) fig1 b . previously prepared mrna / dsdna complex ( mrna mix mlv_d583n_pd ( active rt ): del_pd ( inactive rt )= 1 : 20 ) was used for in vitro translation employing synthetic wakopure system ( 295 - 59503 - wako ). translation mixture for wakopure system ( 25 μl ): 12 . 5 μl — a solution ( wako ); 5 μl — b solution ( wako ); 0 . 5 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 0 . 25 μl - 1 m dtt ; 1 . 75 μl nuclease - free water and 5 μl - 0 . 24 μg / μl mrna / dsdna substrate (˜ 1200 ng ). in vitro translation was performed for 120 min at 37 ° c . translations (˜ 25 μl ) was stopped by adding 155 μl of ice - cold stop buffer wbk 500 + dtt + triton ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma )) and centrifuged for 5 min at + 4 ° c . and 25 , 000 g . very carefully 160 μl of centrifuged translation mixture was transferred on the top of 840 μl 35 % ( w / v ) sucrose solution in wbk 500 + dtt + triton x - 100 ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma ); 35 % ( w / v )- sucrose ( 84097 - fluka )) to transparent 1 ml ultracentrifugation tubes ( 343778 - beckman ). ternary complexes ( tc ) consisted of mrna / dsdna - ribosome - protein ( trna ) were purified by ultracentrifugation at tl - 100 beckman ultracentrifuge in tla100 . 2 fixed angle rotor ( beckman ) at 100 , 000 rpm for 9 min at + 4 ° c . initially 750 μl of solution was removed from the very top of the centrifugation tube . then ( very carefully to keep small transparent pellet of tc at the bottom of ultracentrifugation tube intact ) tube walls were washed with 750 μl of wbk 500 ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ). finally all solution was removed starting from the very top of the centrifugation tube and pellet was dissolved in 30 μl of ice - cold stop buffer wbk 500 + dtt + triton ( 50 mm tris - acetate ph 7 . 5 at 25 ° c . ; 50 mm nacl ; 50 mm mg - acetate ; 500 mm kcl ; 10 mm dtt ; 0 . 1 % ( v / v )- triton x - 100 ( t8787 - sigma )). as it was determined using radioactively labeled mrna after ultracentrifugation 5 %- 30 % of input mrna is located in ternary complex pellet . therefore it was predicted to have less than 360 ng ( 30 % from 1200 ng mrna used in translation reaction ) of mrna in 30 μl of buffer (˜ 12 ng / μl or 9 * 10 9 molecules / μl of ternary complex ). modified nucleotide incorporation reaction mix was prepared on ice by mixing : 5 μl - 5 × reaction buffer for reverse transcriptase ( fermentas ); 1 . 25 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 2 . 5 μl - 1 mm biotin - dutp ( fermentas ); 40 . 95 μl nuclease - free water and 0 . 3 μl of purified (& lt ; 2 . 7 * 10 9 molecules ) tc . according to the protocol 50 μl of nucleotide incorporation reaction mix contains & lt ; 2 . 7 * 10 9 molecules of ternary complex . oil - surfactant mixture was prepared by mixing abil em 90 ( goldschmidt ) into mineral oil ( m5904 - sigma ) to final concentration of 4 % ( v / v ) ( ghadessy and holliger , 2004 ; us2005064460 ). emulsions were prepared at + 4 ° c . in 5 ml cryogenic vials ( 430492 - corning ) by mixing 950 μl of oil - surfactant mixture with 50 μl of rt mixture . mixing was performed using ms - 3000 magnetic stirrer with speed control ( biosan ) at ˜ 2100 rpm ; rotilabo ®-( 3 × 8 mm ) magnetic bar with centre ring ( 1489 . 2 - roth ); water phase was added in 10 μl aliquots every 30 sec , continuing mixing for 2 more minutes ( total mixing time - 4 min ) according to optical microscopy data compartments in prepared emulsions vary from 0 . 5 μm to 10 μm in size with average diameter of − 2 μm . therefore it was expected to have 1 * 10 10 water in oil compartments after the emulsification of 50 μl reverse transcription reaction mixture , which contains less than 2 . 7 * 10 9 molecules of ternary complex ( about one mrna - dsdna complex and reverse transcriptase molecules per 3 - 4 compartments ). to recover the reaction mixture from emulsion 20 μl of 0 . 1m edta was added to emulsion , stirred for 10 sec , then 50 μl phenol / chloroform mix was added and stirred for additional 10 sec . after that , emulsion was transferred to 1 . 5 ml microcentrifuge tube , 0 . 5 ml of water - saturated ether was added mixed by vortexing and centrifuged for 10 min at room temperature for 16 , 000 g . oil - ether phase was removed leaving concentrated ( but still intact ) emulsion at the bottom of the tube . finally emulsions were broken by extraction with 0 . 9 ml water - saturated ether ; 0 . 9 ml water - saturated ethyl - acetate ( in order to remove abil em 90 detergent ) and twice 0 . 9 ml water - saturated ether . water phase was dried for 12 min under vacuum at room temperature followed removal of incorporated nucleotides on illiustra probequant g - 50 microcolumns ( ge healthcare ). 2 μl aliquot of resultant mixture was left for direct rt reaction ( without streptavidin beads purification ) and the remaining part of solution was used for biotinylated mrna / dsdna complex immobilization on dynabeads m - 280 streptavidin beads ( dynal biotech ). dynabeads ® kilobase binder ™ kit ( dynal biotech ) was used for isolation of biotinylated mrna - dsdna complex according provided product description . 5 μl of resuspended dynabeads was transferred to a 1 . 5 ml microcentrifuge tube , washed with 20 μl of provided binding solution . the tube was placed on the magnet for 1 - 2 min until beads settled at the tube and solution was removed . dynabeads were gently resuspended by pipetting in 50 μl of binding solution and 1 μl of aqueous trna ( trna from yeast ( roche )) solution ( 1 μg / μl ) was added to minimize non - specific mrna binding . 50 μl of dynabeads in binding solution were added to solution (˜ 50 μl ) containing the biotinylated rna - dna fragments . the tube was incubated shaking at the + 22 ° c . in termomixer ( eppendorf ) for 1 hour . after the binding of mrna / dsdna , supernatant was removed from the beads , and the beads were washed two times with 50 μl of washing solution ( provided in the kit ) shaking ( 1400 rpm ) for 5 min at + 22 ° c . and once with 50 μl of washing solution shaking ( 1400 rpm ) for 12 min at + 22 ° c . collected dynabeads with immobilized biotinylated mrna / dsdna complex were resuspended in reverse transcription reaction mixture . reverse transcription reaction mixture for selected mrna / dsdna complex was prepared on ice : 12 μl - 5 × reaction buffer for reverse transcriptase ( fermentas ); 6 μl - 10 mm dntp ( fermentas ); 1 . 5 μl - 40 u / μl ribolock rnase inhibitor ( fermentas ); 0 . 3 μl - 20 μm pd — 42 oligonucleotide ( seq id no : 8 ); 1 . 5 μl revertaid ™ minus m - mulv reverse transcriptase ( 200 u / μl )( fermentas ); 35 . 7 μl nuclease - free water . prepared mix was divided to three 19 μl aliquots : one was used to resuspend dynabeads with immobilized biotinylated mrna / dsdna complex , the other aliquot of 19 μl was transferred to the tube with sample of elongated mrna / dsdna complex left without streptavidin beads purification and to the rest of 19 μl aliquot was added 1 μl of nuclease free water ( negative reaction control — to prove that reaction mixture is not contaminated ). all reaction mixtures were incubated by shaking ( 1000 rpm ) at the + 42 ° c . in termomixer ( eppendorf ) for 1 hour . amplification of cdna was performed by nested pcr . initial pcr mixture was prepared on ice : 10 μl - 10 × taq buffer with kcl ( fermentas ); 10 μl - 2 mm of each dntp ( fermentas ); 6 μl - 25 mm mgcl 2 ( fermentas ); 2 μl - 1 u / μl lc ( recombinant ) taq dna polymerase ( fermentas ); 1 μl - 2 . 5 u / μl pfu dna polymerase ( fermentas ); 0 . 5 μl - 100 μm rd_nde primer ( seq id no : 9 ); 0 . 5 μl - 100 μm pd — 55 primer ( seq id no : 10 ); 65 μl water - mixture was divided into 5 samples for 19 μl ( 5 × 19 μl ). to 3 × 19 μl of pcr master mix were added 1 μl of cdna ( 1 - 3 rt samples ); to one tube with 19 μl of pcr master mix 1 μl water was added - negative pcr control . for positive pcr control 1 μl of pet_his_mlv_pd plasmid (˜ 1 ng ) was added . the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 30 cycles ( 45 sec at 94 ° c ., 45 sec at 58 ° c ., and 3 min at 72 ° c .) and final elongation 5 min at 72 ° c . nested pcr mixture for partial gene amplification ( for better resolution of mlv_d583n_pd : del_pd cdna ratio in rt samples ) was prepared on ice : 20 μl - 10 × taq buffer with kcl ( fermentas ); 20 μl - 2 mm of each dntp ( fermentas ); 12 μl - 25 mm mgcl 2 ( fermentas ); 0 . 9 μl - 5 u / μl taq dna polymerase ( fermentas ); 1 . 0 μl - 100 μm m_f primer ( seq id no : 11 ); 1 . 0 μl - 100 μm m — 2r primer ( seq id no : 12 ); 135 . 1 μl water - mixture was divided 5 × 38 μa 2 μl of first pcr ( primers set rd_nde // pd — 55 ) product were added to prepared nested pcr mixture . master mix was mixed again and divided into two tubes ( 2 × 20 μl ) for 30 or 35 pcr cycles amplification . the cycling protocol was : initial denaturation step 3 min at 94 ° c ., 30 or 35 cycles ( 45 sec at 94 ° c ., 45 sec at 57 ° c ., and 1 min at 72 ° c .) and final elongation 3 min at 72 ° c . expected length of pcr fragments was 907 bp for mlv_d583n_pd and 736 bp for del_pd . amplification was analyzed on 1 % agarose gel loading 10 μl of pcr mix per well ( fig1 ). 1 . double stranded ( dsdna ) adaptor was successfully ligated to mrna using t4 dna ligase . ligation efficiency is about 60 % as it was determined by ligation of dsdna - biotin adapter . mrna / dsdna complex can be specifically purified on streptavidin beads , providing an opportunity to discriminate between biotin labelled and unlabelled substrates . free mrna is much worse substrate for dna dependent dna polymerase comparing to mrna / dsdna . as a consequence 60 % ligation efficiency of dsdna to mrna is good enough and such a substrate can be successfully used in evolution scheme . 2 . general selection experiment using mrna / dsdna complex ( mrna mixture mlv_d583n_pd : del_pd = 1 : 20 ) was performed . in vitro translation was performed using wakopure protein translation system and compartmentalized biotin - dutp incorporation reaction in to dsdna was carried out to demonstrate the enrichment of genes encoding active ( mlv_d583n_pd ) reverse transcriptase over genes encoding inactivated enzyme ( del_pd ). according to selection scheme ( fig1 ) incorporation reaction of biotin - dutp should occur only in aqueous compartments containing active ( mlv_d583n_pd ) reverse transcriptase resulting in biotinylation of mrna / dsdna complex . dna dependent dna polymerase is selected by binding of the biotinylated complex to streptavidin immobilized on magnetic beads , and then the selected gene is amplified by rt - pcr . genes encoding active enzyme ( in our case rt - pcr fragment for mlv_d583n_pd reverse transcriptase ) were enriched over genes encoding inactive enzyme ( fig1 ). initial ratio of genes mlv_d583n_pd : del_pd was 1 : 20 and final ratio ( after enrichment ) was ˜ 1 : 1 . respectively an enrichment factor in this particular experiment was ˜ 20 folds . enrichment factors calculated from different experiments varied in range from 5 to 200 . and it was confirmed that dna dependent dna polymerase could be selected for modified nucleotide incorporation applying compartmentalized ribosome display ( crd ) method . selection of conventional dna dependent dna polymerases can be performed straight away . biotin - dutp can be exchanged to different nucleotide analogues of interest including nucleotide analogues having 3 ′ modifications . after the incorporation of such nucleotide analogues into dna strand 3 ′ end is blocked , can &# 39 ; t be extended and elongation reaction will be terminated . this approach is used in sequencing by synthesis ( sbs ) scheme and dna polymerase suitable for sbs can be easily evolved using compartmentalized ribosome display ( crd ) technique . applicants incorporate by reference the material contained in the accompanying computer readable sequence listing identified as sequence_listing . txt , having a file creation date of oct . 1 , 2009 1 : 41 : 10 p . m . and file size of 652 kb . l5_9 12 mutations , 83d -& gt ; n , 135l -& gt ; p , 166p -& gt ; s , 214h -& gt ; r , 222y -& gt ; c , 293t -& gt ; a , 344y -& gt ; h , 407p -& gt ; l , 415a -& gt ; v , 436a -& gt ; t , 444v -& gt ; a , 447p -& gt ; l 18 11 mutations , 83d -& gt ; n , 139l -& gt ; p , 200d -& gt ; n , 330t -& gt ; p , 479n -& gt ; d , 577k -& gt ; q , 583d -& gt ; g , 618l -& gt ; v , 678n -& gt ; i , 679s -& gt ; p , 680r -& gt ; a l5_79 10 mutations , 74i -& gt ; t , 104p -& gt ; r , 325y -& gt ; h , 333l -& gt ; q , 430q -& gt ; r , 597i -& gt ; t , 616e -& gt ; k , 649n -& gt ; s , 658k -& gt ; r , 673e -& gt ; g l5_82 10 mutations , 26l -& gt ; p , 130p -& gt ; s , 137s -& gt ; g , 343a -& gt ; t , 356a -& gt ; g , 524d -& gt ; g , 539a -& gt ; t , 603l -& gt ; w , 653d -& gt ; n , 676s -& gt ; p l5_117 10 mutations , 14s -& gt ; p , 95n -& gt ; s , 139l -& gt ; p , 190q -& gt ; r , 333l -& gt ; q , 339d -& gt ; g , 380v -& gt ; a , 383q -& gt ; p , 524d -& gt ; g , 532g -& gt ; r 21 9 mutations , 131n -& gt ; s , 179i -& gt ; t , 204h -& gt ; r , 323p -& gt ; l , 353t -& gt ; a , 583d -& gt ; a , 678n -& gt ; i , 679s -& gt ; p , 680r -& gt ; a l5_3 9 mutations , 125i -& gt ; v , 138g -& gt ; r , 143h -& gt ; r , 380v -& gt ; a , 552l -& gt ; p , 603l -& gt ; w , 622k -& gt ; r , 658k -& gt ; q , 668s -& gt ; p l5_20 9 mutations , 12e -& gt ; a , 87l -& gt ; p , 95n -& gt ; s , 200d -& gt ; a , 221q -& gt ; r , 242a -& gt ; t , 331g -& gt ; e , 428m -& gt ; l , 603l -& gt ; w l5_75 9 mutations , 11h -& gt ; y , 325y -& gt ; h , 333l -& gt ; q , 430q -& gt ; r , 597i -& gt ; t , 616e -& gt ; k , 649n -& gt ; s , 658k -& gt ; r , 673e -& gt ; g l5_76 9 mutations , 66m -& gt ; l , 105g -& gt ; e , 200d -& gt ; n , 289m -& gt ; v , 314i -& gt ; t , 436a -& gt ; t , 491l -& gt ; p , 573a -& gt ; t , 583d -& gt ; n 8 8 mutations , 89p -& gt ; s , 139l -& gt ; p , 287t -& gt ; a , 330t -& gt ; p , 514l -& gt ; f , 607e -& gt ; k , 635c -& gt ; s , 663e - & gt ; d l5_13 8 mutations , 130p -& gt ; s , 159r -& gt ; k , 200d -& gt ; a , 221q -& gt ; r , 330t -& gt ; p , 449d -& gt ; g , 524d -& gt ; g , 583d -& gt ; e l5_41 8 mutations , 60s -& gt ; a , 139l -& gt ; p , 168f -& gt ; s , 199f -& gt ; y , 346e -& gt ; d , 450r -& gt ; h , 524d -& gt ; g , 583d -& gt ; g l5_68 8 mutations , 39m -& gt ; v , 49i -& gt ; v , 91q -& gt ; r , 204h -& gt ; r , 287t -& gt ; a , 454n -& gt ; k , 625f -& gt ; l , 653d - & gt ; h l5_72 8 mutations , 139l -& gt ; p , 200d -& gt ; n , 330t -& gt ; p , 393a -& gt ; t , 572m -& gt ; l , 594h -& gt ; q , 671l -& gt ; p , 672i -& gt ; t l5_114 8 mutations , 17p -& gt ; s , 108d -& gt ; e , 249n -& gt ; d , 307a -& gt ; v , 344y -& gt ; h , 524d -& gt ; g , 653d -& gt ; g , 673e -& gt ; k l5_115 8 mutations , 8h -& gt ; p , 139l -& gt ; p , 197t -& gt ; a , 200d -& gt ; g , 358g -& gt ; w , 524d -& gt ; g , 623a -& gt ; v , 653d -& gt ; a 17 7 mutations , 49i -& gt ; v , 65p -& gt ; s , 200d -& gt ; n , 409c -& gt ; r , 470v -& gt ; a , 502a -& gt ; v , 583d -& gt ; n l5_15 7 mutations , 17p -& gt ; s , 108d -& gt ; e , 249n -& gt ; d , 307a -& gt ; v , 344y -& gt ; h , 524d -& gt ; g , 653d -& gt ; g l5_21 , l5_111 7 mutations , 65p -& gt ; s , 233e -& gt ; k , 407p -& gt ; s , 478l -& gt ; p , 603l -& gt ; w , 638h -& gt ; r , 653d -& gt ; n l5_40 7 mutations , 24t -& gt ; a , 41l -& gt ; r , 127p -& gt ; s , 151l -& gt ; f , 330t -& gt ; p , 503h -& gt ; r , 653d -& gt ; g l5_47 7 mutations , 107n -& gt ; s , 126h -& gt ; r , 128t -& gt ; a , 179i -& gt ; v , 200d -& gt ; n , 642h -& gt ; y , 653d -& gt ; n l5_71 7 mutations , 97p -& gt ; s , 184t -& gt ; a , 250l -& gt ; p , 289m -& gt ; l , 497d -& gt ; g , 551a -& gt ; t , 562e -& gt ; k l5_78 7 mutations , 11h -& gt ; r , 148v -& gt ; m , 330t -& gt ; p , 459h -& gt ; r , 502a -& gt ; v , 653d -& gt ; g , 667t -& gt ; a l5_81 7 mutations , 166p -& gt ; s , 199f -& gt ; l , 446q -& gt ; r , 468d -& gt ; a , 501e -& gt ; k , 530q -& gt ; h , 558a -& gt ; v l5_118 7 mutations , 221q -& gt ; r , 283a -& gt ; t , 287t -& gt ; a , 369f -& gt ; i , 376y -& gt ; c , 434i -& gt ; t , 603l -& gt ; w 13 6 mutations , 51p -& gt ; s , 136l -& gt ; w , 207l -& gt ; p , 428m -& gt ; l , 560r -& gt ; w , 603l -& gt ; w l5_30 6 mutations , 200d -& gt ; a , 330t -& gt ; p , 449d -& gt ; g , 479n -& gt ; d , 583d -& gt ; n , 671l -& gt ; p l5_32 6 mutations , 69e -& gt ; g , 135l -& gt ; p , 139l -& gt ; p , 431p -& gt ; q , 583d -& gt ; g , 679s -& gt ; f l5_43 , l5_112 6 mutations , 200d -& gt ; n , 237q -& gt ; r , 330t -& gt ; p , 524d -& gt ; g , 625f -& gt ; s , 653d -& gt ; n l5_53 6 mutations , 130p -& gt ; s , 139l -& gt ; p , 417a -& gt ; v , 524d -& gt ; g , 583d -& gt ; n , 634h -& gt ; y l5_56 6 mutations , 8h -& gt ; p , 211r -& gt ; w , 292p -& gt ; l , 486l -& gt ; p , 524d -& gt ; a , 594h -& gt ; r l5_57 6 mutations , 49i -& gt ; v , 173r -& gt ; k , 302e -& gt ; k , 392v -& gt ; a , 603l -& gt ; w , 669t -& gt ; s l5_60 6 mutations , 112v -& gt ; a , 200d -& gt ; n , 280l -& gt ; p , 322a -& gt ; t , 379g -& gt ; s , 653d -& gt ; n l5_62 6 mutations , 118v -& gt ; a , 204h -& gt ; r , 282e -& gt ; g , 346e -& gt ; d , 524d -& gt ; a , 528l -& gt ; i l5_84 6 mutations , 126h -& gt ; r , 139l -& gt ; p , 417a -& gt ; v , 491l -& gt ; p , 524d -& gt ; g , 653d -& gt ; a l5_95 6 mutations , 124d -& gt ; g , 187r -& gt ; g , 263q -& gt ; r , 494n -& gt ; k , 583d -& gt ; g , 618l -& gt ; v l5_107 6 mutations , 139l -& gt ; p , 233e -& gt ; k , 295k -& gt ; e , 633i -& gt ; t , 642h -& gt ; r , 643s -& gt ; g 16 5 mutations , 241r -& gt ; q , 259a -& gt ; t , 311r -& gt ; h , 544t -& gt ; i , 656a -& gt ; t 20 5 mutations , 200d -& gt ; n , 603l -& gt ; w , 678n -& gt ; i , 679s -& gt ; p , 680r -& gt ; a 23 5 mutations , 221q -& gt ; r , 332t -& gt ; i , 524d -& gt ; a , 644a -& gt ; v , 661i -& gt ; v l5_2 5 mutations , 5e -& gt ; k , 43v -& gt ; i , 184t -& gt ; a , 391p -& gt ; l , 543e -& gt ; k l5_14 5 mutations , 37g -& gt ; w , 197t -& gt ; a , 200d -& gt ; g , 433v -& gt ; a , 603l -& gt ; w l5_18 5 mutations , 457m -& gt ; t , 462a -& gt ; t , 504g -& gt ; r , 559q -& gt ; r , 655a -& gt ; v l5_23 5 mutations , 30p -& gt ; l , 223v -& gt ; m , 388w -& gt ; r , 390r -& gt ; w , 435l -& gt ; p l5_24 5 mutations , 139l -& gt ; p , 221q -& gt ; r , 603l -& gt ; w , 649n -& gt ; s , 658k -& gt ; r l5_28 5 mutations , 306t -& gt ; a , 309f -& gt ; s , 524d -& gt ; a , 594h -& gt ; r , 625f -& gt ; s l5_37 5 mutations , 126h -& gt ; s , 149l -& gt ; f , 200d -& gt ; n , 454n -& gt ; k , 583d -& gt ; n l5_49 , l5_63 5 mutations , 50i -& gt ; v , 194n -& gt ; s , 204h -& gt ; r , 287t -& gt ; a , 524d -& gt ; a l5_55 5 mutations , 5e -& gt ; k , 200d -& gt ; n , 240t -& gt ; a , 653d -& gt ; h , 671l -& gt ; p l5_69 5 mutations , 14s -& gt ; t , 49i -& gt ; t , 538a -& gt ; t , 603l -& gt ; w , 653d -& gt ; n l5_88 5 mutations , 70a -& gt ; v , 139l -& gt ; p , 479n -& gt ; d , 524d -& gt ; g , 625f -& gt ; l l5_101 5 mutations , 65p -& gt ; s , 204h -& gt ; r , 283a -& gt ; d , 391p -& gt ; s , 583d -& gt ; n l5_104 5 mutations , 132p -& gt ; s , 164s -& gt ; g , 388w -& gt ; r , 524d -& gt ; g , 533q -& gt ; k 3 4 mutations , 221q -& gt ; r , 428m -& gt ; l , 602g -& gt ; r , 603l -& gt ; w 5 4 mutations , 139l -& gt ; p , 283a -& gt ; d , 358g -& gt ; v , 653d -& gt ; n 7 4 mutations , 204h -& gt ; r , 433v -& gt ; a , 524d -& gt ; g , 572m -& gt ; i 11 4 mutations , 29f -& gt ; l , 148v -& gt ; m , 390r -& gt ; w , 653d -& gt ; a 12 4 mutations , 23s -& gt ; p , 88v -& gt ; a , 237q -& gt ; r , 623a -& gt ; v 30 4 mutations , 200d -& gt ; n , 306t -& gt ; m , 524d -& gt ; n , 583d -& gt ; g l5_1 4 mutations , 249n -& gt ; d , 409c -& gt ; r , 470v -& gt ; a , 502a -& gt ; v l5_6 4 mutations , 287t -& gt ; a , 524d -& gt ; a , 594h -& gt ; r , 680r -& gt ; p l5_8 4 mutations , 475v -& gt ; g , 524d -& gt ; g , 679s -& gt ; p , 680r -& gt ; a l5_11 4 mutations , 15k -& gt ; t , 200d -& gt ; n , 576k -& gt ; r , 607e -& gt ; a l5_16 4 mutations , 91q -& gt ; l , 583d -& gt ; g , 600r -& gt ; k , 603l -& gt ; m l5_29 4 mutations , 174d -& gt ; g , 312l -& gt ; p , 502a -& gt ; v , 524d -& gt ; g l5_39 4 mutations , 12e -& gt ; v , 86i -& gt ; v , 200d -& gt ; n , 646a -& gt ; v l5_42 4 mutations , 192f -& gt ; l , 333l -& gt ; p , 556t -& gt ; a , 603l -& gt ; w l5_44 4 mutations , 92s -& gt ; p , 430q -& gt ; r , 479n -& gt ; d , 583d -& gt ; n l5_46 4 mutations , 200d -& gt ; n , 330t -& gt ; p , 583d -& gt ; n , 644a -& gt ; t l5_52 4 mutations , 200d -& gt ; n , 330t -& gt ; p , 374q -& gt ; r , 583d -& gt ; n l5_61 4 mutations , 64y -& gt ; c , 351l -& gt ; v , 449d -& gt ; a , 530q -& gt ; h l5_64 , l5_93 4 mutations , 200d -& gt ; n , 216d -& gt ; g , 524d -& gt ; a , 545e -& gt ; g l5_65 4 mutations , 200d -& gt ; n , 238q -& gt ; h , 570l -& gt ; i , 603l -& gt ; w l5_85 , l5_96 4 mutations , 200d -& gt ; n , 330t -& gt ; p , 583d -& gt ; a , 638h -& gt ; r l5_90 4 mutations , 200d -& gt ; n , 298r -& gt ; g , 330t -& gt ; p , 374q -& gt ; r l5_103 4 mutations , 96t -& gt ; m , 200d -& gt ; n , 559q -& gt ; p , 607e -& gt ; g l5_106 4 mutations , 524d -& gt ; g , 583d -& gt ; n , 635c -& gt ; r , 670l -& gt ; f l5_120 4 mutations , 252y -& gt ; h , 308g -& gt ; s , 441e -& gt ; g , 603l -& gt ; w 1 3 mutations , 49i -& gt ; v , 524d -& gt ; a , 594h -& gt ; r 28 3 mutations , 8h -& gt ; r , 632i -& gt ; t , 644a -& gt ; v l5_4 3 mutations , 208a -& gt ; v , 225d -& gt ; g , 680r -& gt ; a l5_25 3 mutations , 39m -& gt ; l , 302e -& gt ; k , 628k -& gt ; e l5_35 3 mutations , 200d -& gt ; n , 330t -& gt ; p , 479n -& gt ; d l5_51 3 mutations , 110r -& gt ; g , 431p -& gt ; q , 653d -& gt ; n l5_58 3 mutations , 66m -& gt ; l , 90c -& gt ; y , 653d -& gt ; v l5_66 3 mutations , 93p -& gt ; l , 457m -& gt ; r , 603l -& gt ; w l5_73 3 mutations , 67s -& gt ; p , 139l -& gt ; p , 307a -& gt ; v l5_80 3 mutations , 326p -& gt ; s , 583d -& gt ; g , 676s -& gt ; p l5_92 3 mutations , 126h -& gt ; s , 200d -& gt ; n , 653d -& gt ; g l5_94 3 mutations , 494n -& gt ; d , 524d -& gt ; g , 607e -& gt ; k l5_97 3 mutations , 484l -& gt ; p , 498i -& gt ; v , 653d -& gt ; a 10 2 mutations , 481a -& gt ; t , 524d -& gt ; g l5_59 2 mutations , 450r -& gt ; h , 503h -& gt ; r l5_99 2 mutations , 36t -& gt ; i , 524d -& gt ; g l5_116 2 mutations , 653d -& gt ; h , 662t -& gt ; a l5_48 1 mutations , 77h -& gt ; r wt_mlv , l5_87 0 mutations d -& gt ; g , 20 sequences ( 7 , 10 , l5 — 8 , l5 — 13 , l5 — 15 , l5 — 29 , l5 — 41 , l5 — 43 , l5 — 53 , l5 — 82 , l5 — 84 , l5 — 88 , l5 — 94 , l5 — 99 , l5 — 104 , l5 — 106 , l5 — 112 , l5 — 114 , l5 — 115 , l5 — 117 ) d -& gt ; a , 10 sequences ( 1 , 23 , l5 — 6 , l5 — 28 , l5 — 49 , l5 — 56 , l5 — 62 , l5 — 63 , l5 — 64 , l5 — 93 ) d -& gt ; n , 1 sequences ( 30 ) d -& gt ; g , 2 sequences ( l5 — 14 , l5 — 115 ) d -& gt ; a , 3 sequences ( l5 — 13 , l5 — 20 , l5 — 30 ) d -& gt ; n , 25 sequences ( 17 , 18 , 30 , l5 — 11 , l5 — 35 , l5 — 37 , l5 — 39 , l5 — 43 , l5 — 46 , l5 — 47 , l5 — 52 , l5 — 55 , l5 — 60 , l5 — 64 , l5 — 65 , l5 — 72 , l5 — 76 , l5 — 85 , l5 — 90 , l5 — 92 , l5 — 93 , l5 — 96 , l5 — 103 , l5 — 112 ) d -& gt ; v , 1 sequences ( l5 — 58 ) d -& gt ; g , 5 sequences ( l5 — 15 , l5 — 40 , l5 — 78 , l5 — 92 , l5 — 114 ) d -& gt ; a , 4 sequences ( 11 , l5 — 84 , l5 — 97 , l5 — 15 ) d -& gt ; n , 10 sequences ( 5 , l5 — 21 , l5 — 43 , l5 — 47 , l5 — 51 , l5 — 60 , l5 — 69 , l5 — 82 , l5 — 111 , l5 — 112 ) d -& gt ; h , 3 sequences ( l5 — 55 , l5 — 116 ) d -& gt ; e , 1 sequences ( l5 — 13 ) d -& gt ; g , 7 sequences ( 18 , 30 , l5 — 16 , l5 — 32 , l5 — 41 , l5 — 80 , l5 — 95 ) d -& gt ; a , 3 sequences ( 21 , l5 — 85 , l5 — 96 ) d -& gt ; n , 10 sequences ( 17 , l5 — 30 , l5 — 37 , l5 — 44 , l5 — 46 , l5 — 52 , l5 — 53 , l5 — 76 , l5 — 101 , l5 — 106 ) l -& gt ; w , 17 sequences ( 3 , 13 , 20 , l5 — 3 , l5 — 14 , l5 — 20 , l5 — 21 , l5 — 24 , l5 — 42 , l5 — 57 , l5 — 57 , l5 — 66 , l5 — 69 , l5 — 82 , l5 — 111 , l5 — 118 , l5 — 120 ) l -& gt ; m , 1 sequences ( l5 — 16 ) t -& gt ; p , 15 sequences ( 8 , 18 , l5 — 13 , l5 — 30 , l5 — 40 , l5 — 43 , l5 — 46 , l5 — 52 , l5 — 72 , l5 — 78 , l5 — 85 , l5 — 90 , l5 — 96 , l5 — 112 ) l -& gt ; p , 14 sequences ( 5 , 8 , 18 , l5 — 24 , l5 — 32 , l5 — 41 , l5 — 53 , l5 — 72 , l5 — 73 , l5 — 84 , l5 — 88 , l5 — 107 , l5 — 115 , l5 — 117 ) h -& gt ; r , 7 sequences ( 7 , 21 , l5 — 49 , l5 — 62 , l5 — 63 , l5 — 68 : l5 — 101 ) q -& gt ; r , 6 sequences ( 3 , 23 , l5 — 13 , l5 — 20 , l5 — 24 , l5 — 118 ) t -& gt ; a , 6 sequences ( 8 , l5 — 6 , l5 — 49 , l5 — 63 , l5 — 68 , l5 — 118 ) r -& gt ; p , 1 sequences ( l5 — 6 ) r -& gt ; a , 5 sequences ( 18 , 20 , 21 , l5 — 4 , l5 — 8 ) i -& gt ; t , 1 sequences ( l5 — 69 ) i -& gt ; v , 4 sequences ( 1 , 17 , l5 — 57 , l5 — 68 ) h -& gt ; q , 1 sequences ( l5 — 72 ) h -& gt ; r , 4 sequences ( 1 , l5 — 6 , l5 — 28 , l5 — 56 ) f -& gt ; s , 3 sequences ( l5 — 28 , l5 — 43 , l5 — 112 ) f -& gt ; l , 2 sequences ( l5 — 68 , l5 — 88 ) s -& gt ; f , 1 sequences ( l5 — 32 ) s -& gt ; p , 4 sequences ( 18 , 20 , 21 , l5 — 8 ) h -& gt ; s , 2 sequences ( l5 — 37 , l5 — 92 ) h -& gt ; r , 2 sequences ( l5 — 47 , l5 — 84 ) l -& gt ; q , 3 sequences ( l5 — 75 , l5 — 79 , l5 — 117 ) l -& gt ; p , 1 sequences ( l5 — 42 ) e -& gt ; g , 1 sequences ( l5 — 103 ) e -& gt ; a , 1 sequences ( l5 — 11 ) e -& gt ; k , 2 sequences ( 8 , l5 — 94 ) k -& gt ; q , 1 sequences ( l5 — 3 ) k -& gt ; r , 3 sequences ( l5 — 24 , l5 — 75 , l5 — 79 ) h -& gt ; p , 2 sequences ( l5 — 56 , l5 — 115 ) h -& gt ; r , 1 sequences ( 28 ) a -& gt ; d , 2 sequences ( 5 , l5 — 101 ) a -& gt ; t , 1 sequences ( l5 — 118 ) p -& gt ; s , 2 sequences ( l5 — 21 , l5 — 111 ) p -& gt ; l , 1 sequences ( l5 — 9 ) d -& gt ; g , 2 sequences ( l5 — 13 , l5 — 30 ) d -& gt ; a , 1 sequences ( l5 — 61 ) a -& gt ; t , 1 sequences ( l5 — 46 ) a -& gt ; v , 2 sequences ( 23 , 28 ) e -& gt ; g , 2 sequences ( l5 — 75 , l5 — 79 ) e -& gt ; k , 1 sequences ( l5 — 114 ) highest temperature conc ., specific activity of cdna synthesis mg / ml 37 ° c . ( u / mg ) 50 ° c . 1kb 4 . 5kb m - mulv ( wt ) 3 . 4 ~ 200 000 45 - 50 % 47 . 8 ° c . d200n , l603w ( 20 mut .) 0 . 46 260 000 131 % 56 ° c . d200n , l603w , t330p 4 . 17 321 194 175 % 56 - 58 ° c . 53 ° c . ( m0_1 ) d200n , l603w , t330p , 4 . 27 311 576 174 % 60 - 62 ° c . 56 ° c . e607k ( m1 ) d200n , l603w , t330p , 2 . 91 348 694 176 % 62 ° c . 61 ° c . e607k , l139p ( m2 ) d200n , l603w , n479d , 5 . 37 371 510 182 % 56 - 58 ° c . 56 ° c . h594r ( m3 ) d200n , l603w , d653n , 4 . 84 358 112 155 % 58 - 60 ° c . 58 ° c . d524g ( m4 ) d200n , l603w , d653n , 5 . 23 272 031 180 % 60 - 62 ° c . 58 ° c . d524g , t330p ( m6 ) seq id no : 1 locus pet - his - mlv - pd 7873 bp dna circular 5 - jun - 2007 source organism comment this file is created by vector nti http :// www . invitrogen . com / comment origdb | genbank comment vntdate | 448383004 | comment vntdbdate | 448383004 | comment lsowner | comment vntname | pet - his - mlv - pd | comment vntauthorname | remigijus skirgaila | comment vntauthortel |+ 370 - 5 - 2394224 | comment vntauthoreml | skirgaila @ fermentas . lt | comment vntauthorwww | www . fermentas . com | features location / qualifiers cds 3534 . . . 4391 / vntifkey = “ 4 ” / label = ap / note = “ orf : frame # 2 start : atg stop : taa ” cds complement ( 6586 . . . 7674 ) / vntifkey = “ 4 ” / label = laci / note = “ orf : frame # 3 start : gtg stop : tga ” terminator 2864 . . . 2910 / vntifkey = “ 43 ” / label = t7 \ terminator rep_origin 2947 . . . 3402 / vntifkey = “ 33 ” / label = f1 \ origin rep_origin complement ( 2936 . . . 2936 ) / vntifkey = “ 33 ” / label = ori promoter 174 . . . 190 / vntifkey = “ 30 ” / label = pt7 misc_feature 2394 . . . 2669 / vntifkey = “ 21 ” / label = pd misc_feature 2364 . . . 2393 / vntifkey = “ 21 ” / label = gs \ linker misc_feature 2670 . . . 2759 / vntifkey = “ 21 ” / label = gs \ linker misc_feature 306 . . . 2363 / vntifkey = “ 21 ” / label = mlv \ h + misc_feature 258 . . . 305 / vntifkey = “ 21 ” / label = his base count 1873 a 2155 c 2084 g 1761 t origin 1 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc 61 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc 121 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctatacga aattaatacg 181 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 241 aagaggagaa attacatatg agaggatcgc atcaccatca ccatcacgga tctggttcca 301 tgggcatgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 361 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 421 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 481 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 541 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 601 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 661 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 721 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 781 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 841 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 901 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 961 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 1021 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 1081 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 1141 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 1201 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 1261 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 1321 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 1381 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 1441 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 1501 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 1561 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 1621 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 1681 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 1741 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 1801 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 1861 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 1921 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 1981 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 2041 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 2101 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 2161 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 2221 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 2281 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccca 2341 attcccgctt aattaatgaa ttcggatccg gtggcggttc cggcggtgga tctatgggta 2401 ccgcaaccgc gcccggcgga ttgagtgcga aagcgcctgc aatgaccccg ctgatgctgg 2461 acacctccag ccgtaagctg gttgcgtggg atggcaccac cgacggtgct gccgttggca 2521 ttcttgcggt tgctgctgac cagaccagca ccacgctgac gttctacaag tccggcacgt 2581 tccgttatga ggatgtgctc tggccggagg ctgccagcga cgagacgaaa aaacggaccg 2641 cgtttgccgg aacggcaatc agcatcgttg gatctggtgg cggttccggc ggtggatctg 2701 gtggcggttc cggcggtgga tctggtggcg gttccggcgg tggatcgtgt cttctttaag 2761 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc 2821 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 2881 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg 2941 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 3001 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 3061 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 3121 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 3181 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 3241 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 3301 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 3361 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg 3421 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 3481 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 3541 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 3601 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 3661 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 3721 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 3781 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 3841 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 3901 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 3961 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 4021 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag 4081 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 4141 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 4201 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 4261 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 4321 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 4381 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 4441 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 4501 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 4561 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 4621 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 4681 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 4741 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 4801 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 4861 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4921 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 4981 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 5041 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 5101 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 5161 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 5221 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 5281 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 5341 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac 5401 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 5461 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 5521 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 5581 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca 5641 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt 5701 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt 5761 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata 5821 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta 5881 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc 5941 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag 6001 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc 6061 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt 6121 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta 6181 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt 6241 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca 6301 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc 6361 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc 6421 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc 6481 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc 6541 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag 6601 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 6661 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt 6721 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag 6781 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt 6841 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc 6901 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc 6961 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg 7021 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag 7081 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc 7141 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat 7201 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac 7261 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc 7321 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac 7381 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg 7441 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag 7501 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc 7561 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga 7621 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa 7681 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt 7741 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta 7801 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca 7861 acagtccccc ggc seq id no : 2 locus pet_his_del_pd 7702 bp dna circular 6 - jun - 2007 source organism comment this file is created by vector nti http :// www . invitrogen . com / comment vntdate | 448455242 | comment vntdbdate | 448455855 | comment lsowner | comment vntname | pet_his_del_pd | comment vntauthorname | remigijus skirgaila | comment vntauthortel |+ 370 - 5 - 2394224 | comment vntauthoreml | skirgaila @ fermentas . lt | comment vntauthorwww | www . fermentas . com | features location / qualifiers misc_feature 2499 . . . 2588 / vntifkey = “ 21 ” / label = gs \ linker misc_feature 2193 . . . 2222 / vntifkey = “ 21 ” / label = gs \ linker misc_feature 2223 . . . 2498 / vntifkey = “ 21 ” / label = pd promoter 174 . . . 190 / vntifkey = “ 30 ” / label = pt7 rep_origin complement ( 2765 . . . 2765 ) / vntifkey = “ 33 ” / label = ori rep_origin 2776 . . . 3231 / vntifkey = “ 33 ” / label = f1 \ origin terminator 2693 . . . 2739 / vntifkey = “ 43 ” / label = t7 \ terminator cds complement ( 6415 . . . 7503 ) / vntifkey = “ 4 ” / label = laci / note = “ orf : frame # 3 start : gtg stop : tga ” cds 3363 . . . 4220 / vntifkey = “ 4 ” / label = ap / note = “ orf : frame # 2 start : atg stop : taa ” misc_feature 258 . . . 305 / vntifkey = “ 21 ” / label = his misc_feature 306 . . . 2192 / vntifkey = “ 21 ” / label = del base count 1823 a 2115 c 2033 g 1731 t origin 1 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc 61 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc 121 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctatacga aattaatacg 181 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 241 aagaggagaa attacatatg agaggatcgc atcaccatca ccatcacgga tctggttcca 301 tgggcatgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 361 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 421 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 481 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 541 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 601 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 661 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 721 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 781 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 841 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 901 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 961 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 1021 aaggtactcg ggccctgtta caaaccctag ggacggcagg cttctgtcgc ctctggatcc 1081 ctgggtttgc agaaatggca gcccccttgt accctctcac caaaacgggg actctgttta 1141 attggggccc agaccaacaa aaggcctatc aagaaatcaa gcaagctctt ctaactgccc 1201 cagccctggg gttgccagat ttgactaagc cctttgaact ctttgtcgac gagaagcagg 1261 gctacgccaa aggtgtccta acgcaaaaac tgggaccttg gcgtcggccg gtggcctacc 1321 tgtccaaaaa gctagaccca gtagcagctg ggtggccccc ttgcctacgg atggtagcag 1381 ccattgccgt actgacaaag gatgcaggca agctaaccat gggacagcca ctagtcattc 1441 tggcccccca tgcagtagag gcactagtca aacaaccccc cgaccgctgg ctttccaacg 1501 cccggatgac tcactatcag gccttgcttt tggacacgga ccgggtccag ttcggaccgg 1561 tggtagccct gaacccggct acgctgctcc cactgcctga ggaagggctg caacacaact 1621 gccttgatat cctggccgaa gcccacggaa cccgacccga cctaacggac cagccgctcc 1681 cagacgccga ccacacctgg tacacggatg gaagcagtct cttacaagag ggacagcgta 1741 aggcgggagc tgcggtgacc accgagaccg aggtaatctg ggctaaagcc ctgccagccg 1801 ggacatccgc tcagcgggct gaactgatag cactcaccca ggccctaaag atggcagaag 1861 gtaagaagct aaatgtttat actaatagcc gttatgcttt tgctactgcc catatccatg 1921 gagaaatata cagaaggcgt gggttgctca catcagaagg caaagagatc aaaaataaag 1981 acgagatctt ggccctacta aaagccctct ttctgcccaa aagacttagc ataatccatt 2041 gtccaggaca tcaaaaggga cacagcgccg aggctagagg caaccggatg gctgaccaag 2101 cggcccgaaa ggcagccatc acagagactc cagacacctc taccctcctc atagaaaatt 2161 catcacccaa ttcccgctta attaatgaat tcggatccgg tggcggttcc ggcggtggat 2221 ctatgggtac cgcaaccgcg cccggcggat tgagtgcgaa agcgcctgca atgaccccgc 2281 tgatgctgga cacctccagc cgtaagctgg ttgcgtggga tggcaccacc gacggtgctg 2341 ccgttggcat tcttgcggtt gctgctgacc agaccagcac cacgctgacg ttctacaagt 2401 ccggcacgtt ccgttatgag gatgtgctct ggccggaggc tgccagcgac gagacgaaaa 2461 aacggaccgc gtttgccgga acggcaatca gcatcgttgg atctggtggc ggttccggcg 2521 gtggatctgg tggcggttcc ggcggtggat ctggtggcgg ttccggcggt ggatcgtgtc 2581 ttctttaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct 2641 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa 2701 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc 2761 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 2821 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 2881 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 2941 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 3001 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 3061 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 3121 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 3181 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca 3241 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 3301 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3361 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 3421 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 3481 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 3541 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 3601 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 3661 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 3721 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 3781 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 3841 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 3901 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 3961 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 4021 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4081 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4141 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4201 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 4261 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 4321 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 4381 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 4441 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 4501 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 4561 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 4621 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 4681 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 4741 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 4801 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 4861 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 4921 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 4981 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 5041 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 5101 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 5161 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 5221 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg 5281 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg 5341 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 5401 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa 5461 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc 5521 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg 5581 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg 5641 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat 5701 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag 5761 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag 5821 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc 5881 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc 5941 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc 6001 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg 6061 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg 6121 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac 6181 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct 6241 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata 6301 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt 6361 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc 6421 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 6481 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa 6541 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt 6601 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct 6661 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc 6721 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg 6781 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc 6841 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc 6901 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg 6961 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat 7021 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc 7081 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac 7141 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac 7201 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat 7261 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt 7321 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc 7381 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt 7441 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac 7501 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca 7561 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc 7621 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga 7681 tggcgcccaa cagtcccccg gc seq id no : 3 oligonucleotide ( 22 b ) pro - pivex 5 ′- gcgagcccgatcttccccatcg - 3 ′ seq id no : 4 oligonucleotide ( 21 b ) pd - ter 5 ′- aagaagacacgatccaccgcc - 3 ′ seq id no : 5 oligonucleotide ( 38 b ) assra 5 ′- ttaagctgctaaagcgtagttttcgtcgtttgcgacta - 3 ′ seq id no : 6 protein ( 833 aa ) mlv_pd mrgshhhhhhgsgsmgmtlniedehrlhetskepdvslgstwlsdfpqawaetggmglavrqapliiplkatstp vsikqypmsqearlgikphiqrlldqgilvpcqspwntpllpvkkpgtndyrpvqdlrevnkrvedihptvpnpy nllsglppshqwytvldlkdaffclrlhptsqplfafewrdpemgisgqltwtrlpqgfknsptlfdealhrdla dfriqhpdlillqyvddlllaatseldcqqgtrallqtlgnlgyrasakkaqicqkqvkylgyllkegqrwltea rketvmgqptpktprqlreflgtagfcrlwipgfaemaaplypltktgtlfnwgpdqqkayqeikqalltapalg lpdltkpfelfvdekqgyakgvltqklgpwrrpvaylskkldpvaagwppclrmvaaiavltkdagkltmgqplv ilaphavealvkqppdrwlsnarmthyqallldtdrvqfgpvvalnpatllplpeeglqhncldilaeahgtrpd ltdqplpdadhtwytdgssllqegqrkagaavtteteviwakalpagtsaqraelialtqalkmaegkklnvytd sryafatahihgeiyrrrglltsegkeiknkdeilallkalflpkrlsiihcpghqkghsaeargnrmadqaark aaitetpdtstlliensspnsrlinefgsgggsgggsmgtatapgglsakapamtplmldtssrklvawdgttdg aavgilavaadqtsttltfyksgtfryedvlwpeaasdetkkrtafagtaisivgsgggsgggsgggsgggsggg sgggscll seq id no : 7 protein ( 766 aa ) del_pd mrgshhhhhhgsgsmgmtlniedehrlhetskepdvslgstwlsdfpqawaetggmglavrqapliiplkatstp vsikqypmsgearlgikphigrlldqgilvpcqspwntpllpvkkpgtndyrpvqdlrevnkrvedihptvpnpy nllsglppshqwytvldlkdaffclrlhptsqplfafewrdpemgisgqltwtrlpqgfknsptlfdealhrdla dfriqhpdlillqyvddlllaatseldcqqgtrallqtlgtagfcrlwipgfaemaaplypltktgtlfnwgpdq qkayqeikqalltapalglpdltkpfelfvdekqgyakgvltqklgpwrrpvaylskkldpvaagwppclrmvaa iavltkdagkltmgqplvilaphavealvkqppdrwlsnarmthyqallldtdrvqfgpvvalnpatllplpeeg lqhncldilaeahgtrpdltdqplpdadhtwytdgssllqegqrkagaavtteteviwakalpagtsaqraelia ltqalkmaegkklnvytdsryafatahihgeiyrrrglltsegkeiknkdeilallkalflpkrlsiihcpghqk ghsaeargnrmadqaarkaaitetpdtstlliensspnsrlinefgsgggsgggsmgtatapgglsakapamtpl mldtssrklvawdgttdgaavgilavaadqtsttltfyksgtfryedvlwpeaasdetkkrtafagtaisivgsg ggsgggsgggsgggsgggsgggscll seq id no : 8 oligonucleotide ( 15 b ) pd_42 5 ′- ttacggctggaggtg - 3 ′ seq id no : 9 oligonucleotide ( 28 b ) rd_nde 5 ′- ctttaagaaagaggagaaattacatatg - 3 ′ seq id no : 10 oligonucleotide ( 14 b ) pd_55 5 ′- gccgggcgcggttg - 3 ′ seq id no : 11 oligonucleotide ( 23 b ) m_f 5 ′- gatcaagccccacatacagagac - 3 ′ seq id no : 12 oligonucleotide ( 19 b ) m_2r 5 ′- gccctgcttctcgtcgaca - 3 ′ seq id no : 13 oligonucleotide ( 40 b ) m_esp 5 ′- atcgtctcccatgggcatgaccctaaatatagaagatgag - 3 ′ seq id no : 14 oligonucleotide ( 32 b ) m_eri 5 ′- aatgaattcattaattaagcgggaattgggtg - 3 ′ seq id no : 15 oligonucleotide ( 18 b ) m_1r 5 ′- cagggcccgagtaccttg - 3 ′ seq id no : 16 oligonucleotide ( 17 b ) m_3f 5 ′- ccagttcggaccggtgg - 3 ′ seq id no : 17 oligonucleotide ( 19 b ) pd_ter - 5 ′- aagaagacacgatccaccg - 3 ′ seq id no : 18 oligonucleotide ( 36 b ) m_hind3 + 5 ′- cggatcaagcttaattaattaagcgggaattgggtg - 3 ′ seq id no : 19 locus pet_his_mlv 7474 bp dna circular 4 - jun - 2007 source organism comment this file is created by vector nti http :// www . invitrogen . com / comment origdb | genbank comment vntdate | 446486240 | comment vntdbdate | 448293778 | comment lsowner | comment vntname | pet_his_mlv | comment vntauthorname | remigijus skirgaila | comment vntauthortel |+ 370 - 5 - 2394224 | comment vntauthoreml | skirgaila @ fermentas . lt | comment vntauthorwww | www . fermentas . com | features location / qualifiers cds 3135 . . . 3992 / vntifkey = “ 4 ” / label = ap / note = “ orf : frame # 2 start : atg stop : taa ” cds complement ( 6187 . . . 7275 ) / vntifkey = “ 4 ” / label = laci / note = “ orf : frame # 3 start : gtg stop : tga ” terminator 2465 . . . 2511 / vntifkey = “ 43 ” / label = t7 \ terminator rep_origin 2548 . . . 3003 / vntifkey = “ 33 ” / label = f1 \ origin rep_origin complement ( 2537 . . . 2537 ) / vntifkey = “ 33 ” / label = ori promoter 174 . . . 190 / vntifkey = “ 30 ” / label = pt7 cds 306 . . . 2360 / vntifkey = “ 4 ” / label = mlv cds 258 . . . 305 / vntifkey = “ 4 ” / label = his base count 1810 a 2046 c 1942 g 1676 t origin 1 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc 61 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc 121 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctatacga aattaatacg 181 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 241 aagaggagaa attacatatg agaggatcgc atcaccatca ccatcacgga tctggttcca 301 tgggcatgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 361 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 421 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 481 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 541 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 601 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 661 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 721 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 781 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 841 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 901 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 961 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 1021 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 1081 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 1141 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 1201 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 1261 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 1321 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 1381 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 1441 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 1501 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 1561 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 1621 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 1681 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 1741 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 1801 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 1861 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 1921 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 1981 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 2041 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 2101 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 2161 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 2221 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 2281 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccca 2341 attcccgctt aattaattaa gcttgcggcc gcactcgagc accaccacca ccaccactga 2401 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa 2461 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga 2521 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg 2581 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2641 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2701 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2761 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2821 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2881 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 2941 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 3001 tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 3061 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 3121 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 3181 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 3241 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 3301 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 3361 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 3421 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 3481 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 3541 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 3601 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 3661 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac 3721 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 3781 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 3841 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 3901 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 3961 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 4021 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 4081 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 4141 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 4201 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 4261 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 4321 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 4381 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 4441 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 4501 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 4561 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 4621 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 4681 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 4741 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 4801 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 4861 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 4921 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 4981 cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 5041 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 5101 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 5161 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 5221 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca 5281 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg 5341 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt 5401 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact 5461 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg 5521 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta 5581 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag 5641 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt 5701 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt 5761 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg 5821 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg 5881 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat 5941 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg 6001 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt 6061 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc 6121 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg 6181 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 6241 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag 6301 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg 6361 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat 6421 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg 6481 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag 6541 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat 6601 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt 6661 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc 6721 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg 6781 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac 6841 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat 6901 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc 6961 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat 7021 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag 7081 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc 7141 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac 7201 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg 7261 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa 7321 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca 7381 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg 7441 catgcaagga gatggcgccc aacagtcccc cggc seq id no : 20 oligonucleotide ( 19 b ) pd - ter - 5 ′- aagaagacacgatccaccg - 3 ′ seq id no : 21 oligonucleotide ( 35 b ) long + 5 ′- cgaacgtggcgagaaaggaagggaagaaagaagtc - 3 ′ seq id no : 22 oligonucleotide ( 71 b + 3 ′ modification ddc ) ddc - long2 5 ′- tttttttagacttctttcttcccttcctttctcgccacgttcgaagaagacacg atccaccgccggttccg - ddc - 3 ′ seq id no : 23 oligonucleotide ( 35 b + 3 ′ biotin ( teg ) long + tb 5 ′- cgaacgtggcgagaaaggaagggaagaaagaagtc - bio - 3 ′ mattheakis lc , bhatt rr , dower wj . an in vitro polysome display system for identifying ligands from very large peptide libraries . proc natl acad sci usa . 1994 september 13 ; 91 ( 19 ): 9022 - 6 . matsuura t , pluckthun a . selection based on the folding properties of proteins with ribosome display . febs lett . 2003 march 27 ; 539 ( 1 - 3 ): 24 - 8 . hanes j , pluckthun a . in vitro selection and evolution of functional proteins by using ribosome display . proc natl acad sci usa . 1997 may 13 ; 94 ( 10 ): 4937 - 42 . he m , taussig mj . antibody - ribosome - mrna ( arm ) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites . nucleic acids res . 1997 december 15 ; 25 ( 24 ): 5132 - 4 . irving ra , coia g , roberts a , nuttall sd , hudson pj . ribosome display and affinity maturation : from antibodies to 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tawfik ds , griffiths a d . altering the sequence specificity of haeiii methyltransferase by directed evolution using in vitro compartmentalization . protein eng des sel . 2004 january ; 17 ( 1 ): 3 - 11 . ghadessy fj , ong jl , holliger p . directed evolution of polymerase function by compartmentalized self - replication . proc natl acad sci usa . 2001 april 10 ; 98 ( 8 ): 4552 - 7 . epub 2001 mar . 27 . ghadessy fj , ramsay n , boudsocq f , loakes d , brown a , iwai s , vaisman a , woodgate r , holliger p . generic expansion of the substrate spectrum of a dna polymerase by directed evolution . nat . biotechnol . 2004 june ; 22 ( 6 ): 755 - 9 . epub 2004 may 23 . ong jl , loakes d , jaroslawski s , too k , holliger p . directed evolution of dna polymerase , rna polymerase and reverse transcriptase activity in a single polypeptide . j mol . biol . 2006 aug . 18 ; 361 ( 3 ): 537 - 50 . epub 2006 jul . 5 . bernath k , hai m , mastrobattista e , griffiths ad , magdassi s , tawfik ds . in vitro compartmentalization by double emulsions : sorting and gene enrichment by fluorescence activated cell sorting . anal biochem . 2004 february 1 ; 325 ( 1 ): 151 - 7 . mastrobattista e , taly v , chanudet e , treacy p , kelly bt , griffiths ad . high - throughput screening of enzyme libraries : in vitro evolution of a beta - galactosidase by fluorescence - activated sorting of double emulsions . chem . biol . 2005 december ; 12 ( 12 ): 1291 - 300 . bertschinger j , neri d . covalent dna display as a novel tool for directed evolution of proteins in vitro . protein eng des sel . 2004 september ; 17 ( 9 ): 699 - 707 . epub 2004 nov . 2 . doi n , yanagawa h . stable : protein - dna fusion system for screening of combinatorial protein libraries in vitro . febs lett . 1999 august 27 ; 457 ( 2 ): 227 - 30 . yonezawa m , doi n , kawahashi y , higashinakagawa t , yanagawa h . dna display for in vitro selection of diverse peptide libraries . nucleic acids res . 2003 october 1 ; 31 ( 19 ): e118 . sepp a , choo y . cell - free selection of zinc finger dna - binding proteins using in vitro compartmentalization . j mol . biol . 2005 november 25 ; 354 ( 2 ): 212 - 9 . epub 2005 oct . 3 . sepp a , tawfik ds , griffiths ad . microbead display by in vitro compartmentalisation : selection for binding using flow cytometry . febs lett . 2002 december 18 ; 532 ( 3 ): 455 - 8 . griffiths ad , tawfik ds . directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization . embo j . 2003 january 2 ; 22 ( 1 ): 24 - 35 . bernath k , magdassi s , tawfik ds . directed evolution of protein inhibitors of dna - nucleases by in vitro compartmentalization ( ivc ) and nano - droplet delivery . j mol . biol . 2005 february 4 ; 345 ( 5 ): 1015 - 26 . epub 2004 dec . 7 . thorsen t , roberts rw , arnold fh , quake sr . dynamic pattern formation in a vesicle - generating microfluidic device . phys rev lett . 2001 april 30 ; 86 ( 18 ): 4163 - 6 . okushima s , nisisako t , torii t , higuchi t . controlled production of monodisperse double emulsions by two - step droplet breakup in microfluidic devices . langmuir . 2004 november 9 ; 20 ( 23 ): 9905 - 8 . song h , tice jd , ismagilov rf . a microfluidic system for controlling reaction networks in time . angew chem int ed engl . 2003 february 17 ; 42 ( 7 ): 768 - 72 . link dr , grasland - mongrain e , duni a , sarrazin f , cheng z , cristobal g , marquez m , weitz da . electric control of droplets in microfluidic devices . angew chem int ed engl . 2006 april 10 ; 45 ( 16 ): 2556 - 60 . matsuura t , yanagida h , ushioda j , urabe i , yomo t . nascent chain , mrna , and ribosome complexes generated by a pure translation system . biochem biophys res commun 2007 january 12 ; 352 ( 2 ): 372 - 7 . epub 2006 nov . 17 . gerard gf , d &# 39 ; alessio jm , kotewicz ml , noon mc . influence on stability in escherichia coli of the carboxy - terminal structure of cloned moloney murine leukemia virus reverse transcriptase . dna . 1986 august ; 5 ( 4 ): 271 - 9 . forrer p , jaussi r . high - level expression of soluble heterologous proteins in the cytoplasm of escherichia coli by fusion to the bacteriophage lambda head protein d . gene . 1998 december 11 ; 224 ( 1 - 2 ): 45 - 52 . gerard gf , potter rj , smith md , rosenthal k , dhariwal g , lee j , chatterjee dk . the role of template - primer in protection of reverse transcriptase from thermal inactivation . nucleic acids res . 2002 july 15 ; 30 ( 14 ): 3118 - 29 . vichier - guerre s , ferris s , auberger n , mahiddine k , jestin jl . a population of thermostable reverse transcriptases evolved from thermus aquaticus dna polymerase i by phage display . angew chem int ed engl . 2006 september 18 ; 45 ( 37 ): 6133 - 7 . ghadessy fj , holliger p . a novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system . protein eng des sel . 2004 march ; 17 ( 3 ): 201 - 4 . epub 2004 feb . 27 . roberts rw , szostak jw . rna - peptide fusions for the in vitro selection of peptides and proteins . proc natl acad sci usa . 1997 november 11 ; 94 ( 23 ): 12297 - 302 . odegrip r , coomber d , eldridge b , hederer r , kuhlman pa , ullman c , fitzgerald k , mcgregor d . cis display : in vitro selection of peptides from libraries of protein - dna complexes . proc natl acad sci usa . 2004 march 2 ; 101 ( 9 ): 2806 - 10 . epub 2004 feb . 23 . reiersen h , løbersli i , løset ga , hvattum e , simonsen b , stacy je , mcgregor d , fitzgerald k , welschof m , brekke oh , marvik oj . covalent antibody display — an in vitro antibody - dna library selection system . nucleic acids res . 2005 january 14 ; 33 ( 1 ): e10 . bertschinger j , neri d . covalent dna display as a novel tool for directed evolution of proteins in vitro . protein eng des sel . 2004 sep . ; 17 ( 9 ): 699 - 707 . epub 2004 nov . 2 . stein v , sielaff i , johnsson k , hollfelder f . a covalent chemical genotype - phenotype linkage for in vitro protein evolution . chembiochem . 2007 december 17 ; 8 ( 18 ): 2191 - 4 . tabuchi i , soramoto s , nemoto n , husimi y . an in vitro dna virus for in vitro protein evolution . febs lett . 2001 november 23 ; 508 ( 3 ): 309 - 12 .