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T7 RNA ポリメラーゼを用いた新規な DNA シークエンス 解析法としての

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1. 0000 00000000000 T7 RNP0 00 elongation phase ugmgmumumuaagdRapnpnpiuiBiBBgtutl processive elongati on phase lU RNAQOOCOOOCOOOOCOOOOOOCOOOCOOCOOOOOODI 000 T promte i 00000000 00 0000000000001 00000 initiation phaseg 0 0 B 0000 Fig 6 4 pBS pGEMI O0 O0 H pPGEMI DNA 5 pGEM D 5 pGEM D D T7 promoter 7 5 pGEMI D
2. HEEL ESTEE HEBEL 0 DNA Hc Bop Bob DERE D DEBES PEDI Bop AE 0 dATP 3 4 ddCTP 3 DNAD 4 DESEE EGET BE BRE EE DE E E DNAD 1
3. SEPT DEBE Deu poor 000 O ODIIEIAIzIIIAIIAIIAII I 2II DEL TE OG ES BLUE EE EE DEBT p DE opp DESEE OE E ERE TEE EE EE BE REO DECRE BEES T HE ABE 11 DE EDDIE 00 0 j DE T emisionPCROOO0O 0CO0O 0C0 000000000 PCR UD DEBES DES DE e RE P De b DEBE dd ERE SEALS T E HT EE EE BE BIET BE ZR HESS TE DAT J Ju 7 5 DD
4. 7 3 3 DAU IH HUD iU H 7 31 82 DAJ II Ur UU BESTE ES EE pp 11 0000 q 2 20 q250 q24 2 L UU D 4o00 0 0 0 00 0000 100 q12 2 0 0 0 qS UH 0000 2 DNA FAD dependent oxi dor educt ase donai n cont ni ng 1 8gp 900 ODOLI DNA DNA U Uu utulIl EL EL uggagagammuguguuuuuuuuudcTeable7 11 00 DAU DU UUUwU cSII EIE EE E ED Eo EDGE ORDE
5. 22 0 0 Dye AJ O0 O0 Dye co 0 p 0COOCOOOCOOCOCOCOOCOOCOCOOOLDI 7 RNAP EIE TE DI RE BE D 1 HE BTE 5 T7 3 dNTP j T7 RNAP 6 RX 3 4 RG J J TMR 3 dCTP R6G 3 dCTP R110 3 dCTP 0 0O H O T7 RNAP O O RNA ugmumuuuaaaaagaggsuguiubluuguuuuuuuagBgB ulsuiul HE BE TECRE BE REORUESEL EE S BE HEBES BE EE RE DP EE BECHER REED Ts d App DEBES e DE DEESSET BIBT bb DI E EE Dye A JD Dye Cn p Tp m BT gud uu gd guum gu 3 dNIP Huet p BEBE BEP DERE SE EE DRE TE P HDT RAPI JU UU LES DERI FE ELE BEBE b E EPA E EE DESDE DC D DE T7 RNAP 0 umgaumgumuuuumuuuuuigtitiestuurusettrLi
6. 77 RGCT GR6GR66T 1661 636066 Go GRCRT CCAAGTTGGAT RGCGTGT 6T GT 6T GT6T GT6T 6T GT 6T GT 6T6666T GT6666 TGCHRRIHE NC CCHTWETC GC 180 190 288 18 228 238 249 258 268 27 n M Jd PCT Gf P GERA GRRRGRRRT GRCRTCGR e s asnata GT6GTGT GTGTGTGTGT GTGTGTGGGG 67666GT GGRCRGGGTT G GGGRGGGT 181 20 21 228 38 24 258 68 21 280 i 1 Lu m f i i WIMA Fig 7 3 Conpari son of sequence anal yses by TS and CS methods results for DNA sequence of GT di nucl eoti de repetiti ve regi on One GT di nucl eoti de repeti ti ve regi on pi cked up f romhurman genom c cl ones was sel ect ed and sequenced by TS and CS methods The upper and ower panel s show el ectropherograms with CS and TS respectively Peaks suddenl y di sappear at the poi nts i ndi cated by arrows in CS To the contrary peaks conti nue beyond the term nation points in TS 0000Fig 7 400070700000 poiyco oo enun p pgmnnnaugnuanagnagagunpnaunpnnaganpnanangcsnnt P PpoyG
7. 4 3 dUTP 3 aCTP 3aQ damPa daPguugagmgungugauaugummamguunHWJuti3bye U Dye Qj Dye A Dye GI HD HU B HU 0 EET EUER SEE ER ELO E BE SEP DEL E EE C DE D ELO ESTE ET DE CE D Dyie U 0 0 0 0 0 0 Dye A0D 0 O0 6 I7RNPII JI IHH DI BEC Eb BE RP E BE EE P E p s awPe g gaggggaggumagugnamugmgn unll 54 6 6 11111151 Db Bb BE BER DE Dg 1 3 dGTP Dye 0 R6G CH 4 3 dATP Dye A TMR CH 4 3 dUTP Dye U ROX CH 3 dCTP Dye c 0 UU UU UD UU DDD TSs0O0O000C0C00C0000
8. PCR products MDA products L nue cec eic xj ML 2344 5 4T Fig 7 5 Comparison of DNA anplificati on patterns by PCR and MDA met hods Tenpl ate was DNA extracted E coli cl ones harbori ng human chromosome 21 and 11 DNA fragment Agarose gel electrophoresis of PCR products upper panel and MDA products lower panel anplified fromseven human genomi c GC rich cl ones Mrepresents the mol ecul ar wei ght marker The results showthat all fragments can be obtained with a high yield more than 1 u g DNA in 10 u I of MA reaction ni xture 80 DAJ Fig 7 6 7 6 DNA 82 441 7 6 5 7 3 1 1 CS 1 5 DESDE HD Lrrrr rt r 0 DNAD I jH uuu B
9. 5 DAU T HL DAU DD UU I DbNA I He BE DEOR SEE EISE ELE DNAD H DNI O DNA 000 pwapnguanmnmuuuguarsuuununnbuueccnnri 80 DNA m gu ududg bg gdugdgugtug g 5 DN 1 J M Keith D A E Cochran G H Lala P Adans D Bryant K R Mtchelson Nucleic Acids Res 32 e35 2004 2 J Kieleczawa J Bionol Tech 17 207 217 2006 3 W Zhang G Hu A B Deisseroth Nucleic Aci ds Res 19 6649 1991 87 4 T M Scheidl Y Mura H A Yee T Tamaoki Biotechniques 19 691 693 1995 5 M Mtz S Paabo C Kilger Biotechniques 29 268 70 2000 6 S H Cross V H Clark
10. fornanide di methyl sul phoxi de DvsO j Cao ug U 1 g e 0 c dGrP dl TP DEZ TD m Dm Wm uu es pm m gos m mg oNAeq T p opp E d d s E EE DE GE UD D BEEHBESEE BE BECHER GCI 0 0 0 HABES 00000 E DEBERE 341 single nucleotide pol ynor phi p OPD i e AE DAU JI U H
11. DNA BE SES DEI DH uggagaggagauauBgu T70 0 O T7 RMA pol T7 0000 i vwvtrenguugagguaguguaauucmguuuuauunulIl 5 RNAP 3 deoxynucl eosi de tri phosphate0p 0 0 0000 0000000000000000000000 0000 00 00 00 5 DAJ O DNAD OOOD 8 HE DE EE DE EPCS ET hO Oo DAI Inn DESEE E BE EPHEESEEEEDSEE ICH 5 EE EAE ETE DE E SEE SEES E DT Bo DE EE EE EE EE ET DESEE 0000 02000 0 0 O DNA p
12. 0 5 DNA J 1 E EJ J ES 76 FO GGRGOGGGGCROCGGG CGGCT OD GGG T 8 28 POTI TOHEA THOA HHT 48 25 T DHI 158 16 170 184 9 21 228 23 198 Fig 7 2 Conparison of sequence analyses by TS and CS methodsf or DNA sequence of GC rich regi on One GC rich regi on picked up from human genomic clones was selected and sequenced by TS and CS methods The upper and lower panels show el ectro pherograms w th CS and TS respecti vel y Peaks suddenl y di sappear at the poi nts i ndi cated by arrows in CS To the contrary peaks conti nue beyond t he term nation points in TS D unrisg 73 5 lU UU CSI DDD
13. 1 1 35 4 5 eo ET LE 4 x T A do ar t Fig 5 8 Effects of pol yam ne addi ti on on the transcription by T7 RNAP Each ane i ndi cates RNA products obtai ned in the absence of additi ve ane 1 Agmati ne lane 4 in the presence of sperm dine lane 2 cadaverine lane 3 and 1 8 di ani noocctane lane 5 57 Ps N X H H N x S N SZ NH Agmatine H N c NH 1 8 diaminooctane 2 S x NS P Jd 2 2 z H HN NZ aNs an J NH Spermidine Fig 5 9 Structures of linear polyamnes as enhancers for T7 RNAP activity 5 10 0 DNAD TSO O0 0 O DNA BEL ORE Tp 0 15000000 0DO0 000 0000 3000 Hi Bd gy gg oH m 0 0 O 1 8 di ami nooctane p 00000000 40000000000000 O DNAOOCOCOCOCOCOOOCCOCOOCOCOCOOOO0O0OD 1 8 diam m nooctanep O0 0 00 00 000 00000 Ts0 0000 uggaguiesoengguuuauauanmmuauudgtiongnnrntH Dag Dap I I 1 T7 RNAP O uu ui U U
14. 5 tP iB rS DE HE i Ud HH TP DB DERE CHEN eecnarnayacnererer 210 220 400 110 i LM AAA Fig 5 3 Eliminati on of peak conpressi on by use of c rGTP in place of r GTP The arrows i ndi cate two conpressi on sites i n human TSH cDNA usi ng r GTP as the substrate a Conpressions in the corresponding sequence were el i ri nated by the use of 7 deaza rGTP for the substrate b 51 ATP CTP UTP GTP ATP CTP UTP ITP Fig 5 4 Elim nation of peak conpressi on by use of rITP in the place of rGTP The arrows or underlines i ndi cate conpressi on sites in p53 exon8 A and ambda phage genom c DNA B usi ng rGTP as the substrate upper panel Conpressi ons i n the correspondi ng sequence were el i m nated by use of rITP for the substrate lower panel 5 3 3 Dpa agagpnpnagugunag 5 5 5 PPaser rn PPasef RNAPO O0 O Dpa uggannggaunanaggg
15. uuBaungaurReaPnpunmuuuuu Transcriptional Sequencing 5 0000 Fig 3 10 0 00 00 00 000TSs000 10 Fig 1100000001 00000 ddNiPO O00000 0 DNAOOCODOCOOCOCODCOODI T7 RNAP O O0 O 0T7 RNAPO O 0000000 I ddNrPo I HP UU U 2 0 H OHII 3 dNTP 3 ed NT EE EE Epp pO ED CET TEE uuuuuullu 000000000000000 T7RNP0 00000000000 0 0 DNA BBm i BT poBBE EE E 19 DESEE DIC LEE T promoter Template DN 3 3 5 J pe of T RNA polymerase with T promoter on template DNA 37 C Repet it ion Fig 3 1 Procedure of Transcriptional Sequenci ng Fig 3 20 00 0 000000000000000000 0 0 0 bDye uibye G Dye AQ Dye c0 O IDD ID IDD DBDU DU DU IBU BL O 0 DNA 536 559 586 nm 615 nmi D D D 488nmp l 5 4 R110 R6G TW 3 uu
16. DNA DNA 5 DNA O 29 DNAPO O DNAD 79 UU UD HDD DTU UD U DAJ IU IU HU HBD DU g g iH DNA C mwa DNAD D D D U rig 7 500 Fig 75 7 8 51 4 6 7 0 0 O Lanes 20 3 5
17. B B thi Devel oprent of Transcriptional Sequenci Method as a Novel DNA Sequenci ng Method Usi ng T7 RNA Pol ymerase 20070 70 ud U 10 Th Sa bua a aw T Lu mcos 1 1 2 a 2 8 14 aE a kuya Ngu Sa 9 uultu 0 20 0000 2 1 DD ierre 13 2 1 1 BB mmm em em e e 0 amp 13 2 2 I KK 6I 13 2 2 1 D0000 cmm e e A e KK 13 2 2 2 Gee m KI 14 2 2 3 DNA jejej e mmI 14 2 3 DAD mmmmmmmmmmmemmemmemmm i emeMtM AItK 14 2 3 1 0g0 0HB80 0H Bc 6 MI 14 2 3 2 Ethanol EtOH 0O D ccc nn M n e n X 15 2 3 3 PCR sss n herr 15 2 3 4 udgagaaddHgpwAamDnu u s i zeetbetzsenee It amp t amp t amp amp cccee 15 2 34 5
18. ugggamugumuguumggauaugu5 5sumgauuuuu TSO O0 DNA 000 85 5 5 5 DNAD O0 0O 0 O DNA uggmgumuuaaaaagaggggbesapnguguggumumumuaaadaadgaggul 1 99A 0000001 5 pGEMI I H O 89 5 U 91 0 preti T7 promter 5 uj 5 uut 68 Sequencing time Sequence accuracy 6 Fig 6 4 Conpari son of sequence accuracy w th pTS1 and comercial vectors Comparison of sequence accuracy within 400 base long regi ons of TS and CS by various plasm ds pTS pGEM and pBS 200 ng of each plasnid were i ncubated for 1 5 10 15 and 30 nin usi ng both methods The i ndi vi dual quality of the sequences was assessed by calcul ati ng the percentage of correct basecalls wth in
19. 78 eT CE OR OY RIY sag T Da 188 118 Im 158 168 178 MM TOCIT TARC CTTRGTORC CT TO GOCOGGGODO T TRRTORCRCCTOG TRETCODRG CRCT TTG GAGO GG03666GG6606GG6663660GG3 6GGUYOT6rG6GTCrGyGrIICRG DA GCCTG GCOR 18 20 38 49 59 68 78 00 98 198 118 128 130 TUR My yl Abd h li i Fig 7 4 Conparison of sequence anal yses by TS and CS methods for DNA sequence of poly G regi on One pol y G regi on pecked up from human genomic cl one was selected and sequenced by TS and CS methods The upper and lower panels show el ectropherograns with CS and TS respectively Peaks suddenl y di sappear at the poi nts i ndi cated by arrows in CS To the contrary peaks conti nue beyond the termination points in TS 7 32 TSHU BU DAU II HUU UD DpDngagggunaggguapnbwpnanmnaunnpnpuursuunananunpn 5 5
20. Gefitiniv 0 O I ressa LE BE ELE ELE HE EH L r J J L DNA O0 5 L L BE TED d ou uut L 0 Fig 6 2 00 190000000 PCR O D O tt J J jJ J Jj Jj m m m m J Jj L prender 111 ij Tm T IU jl hil CA 16 G CTCTGAACCTCAGGCCO CC TT 1CTCA TGT CT G GOAGCT GTCTCCCT T AGT GHG GGTTAXTTNS CC CCOCHCCNT AT C ACONIST OX 5 M0 150 160 170 180 190 200 210 22 210 Fig 6 2 Rapid TS for PCR product Detecti on of deletion by direct TS method The T7 and T3 phage promoter sequences can be appended to both of the PCR pri mers and incorporated i nto the EGFR PCR product 281 bp Usi ng T7 RNAP the
21. si RNA 5 000000 000 GCCETTEPPH BE EC BEBE 00000 51 D00000 000000 siRagguggggg RNA 5 Kr ppel like factor KLF15 0 000000000 DNA Ts OB EPO MEINEM PD EEUU DNAD P 0000 Loop nedi ated i sot her mal SDA U C OD00COOCOOCOCOOOOCOCOOO0COOO0OO0 anpl i fi cati on LAMP 0 C Strand di spl acenent anplifi cati on 5
22. PCRO 0 0 O0 00 cso0o00000000O00 0O 0O OdNiPsO O0OCOOCOOCCOCOCOCOOCOO0OCOCOOO PCRO O DNA pol ynerase 0 O0 O0 U PCROOOOCOOCOOCOCOCOCOOCOO0O0O 000 dNP I PcROOOOCOOOLD I 5 E EE 1 1 BEOEESE SHE BERE BD BE HE ELSE RESI EGET E E EE EE BRL BE TBI ERCBE GE EE DE OOO PRI EP DEGERE EE ED pL SEE DNAP 0 Fig 15 DNAD T E BE Gb I Bo DE DE EE EP BD B pon ES 1 HER HE EE ELA SEDI EET ELTE E ZB EE EE DES EE PE BEGLEITET DNAT D lJ PERI BDOESEE UE DERE EIE HZ PCROO HEB alkaline phosphatase
23. 37 Table 4 2 U U JU J 3 OdAT P O0 0000000000001 00000000 0 RNAP F644Y0 O0 T7 O O O 500 1 3 4 T7 3 T7 644 3 dAT I O0 Fe44Y 00 U lU DU 500 u M 250 u M 100 u M II UD B U D H 1 1 1 1 1 RN JI j HET HE HE 4 ugagaggguauagaudg I7ZRNAPT O0 O0 66 T7 O O F644 ug 667 3 HA HBBIBBHEHHBBU dgutuSydmgiuiu uBmwHBggWdsygdWtBgg g Eb coli DNA pol yrerase 72g DNA pol ymerase T7 DNA pol ymerase 000000000000001 38 WT F644Y 12345678910 Fig 4 3 Chain terni nati on study f or the presence of the incorporated of 3 dNTPs and rNTPs in F644Y and wild type T7 RNAP Tenpl ate DNA and el ectrophoresi s conditi ons are gi ven in Fig 4 2
24. RNAQ ul 4 2 7 T7 F644Y0 F667Y0 0 O0 O T7 O 3 dNTP l T7 RNAP 0 Feezrn D 0 B T7 3 D D 4 2 5 6 3 3 T7 RNAP 0 F644Y dATP 3 dCTP 3 500 p MH 00O 250 p M l ugagagugRaggagauuuuuuRNgugugagaguadgued udgrabor 0000000 T7 RNAPO 00O 3 4 2 8 RNAP 0 F644Y0 O0 0 O I7RNP F644Y O T7 RNAP ug 40 nM Tris HCI pH 8 0 8 mM MgCl 5 mM DIT 2 mM sperm di ne HCl 250 u 500 H MGTP 200 H MCTP 500 u MUTP 0 1 u MR6G CH 3 0 1 H M R110 CH 3 dGTP j 1 u MTMR CH 4 3 dUTP 5 u M ROX CH 4 3 dCTP 000000 DAJ O0 O0 D D Th
25. 7 4 DD 5 6 0 DAU JU U 1 BEES T9 DIOE OoO00O0OOCOCOOCOOOOOOOOOOOOCOOCOOO O gO DNAOONOD DOOO0OCOCOCOOCOCOOCOOOO0OO0OOCOOOCO0O0OC0O0OOOTsOOOOO0OODI 16 1 00000000 DESEADO p 5 T7 RAPO O00000 00000020 0 bpNAg 10 0 D000 200 0000 DNA RNA hybrid 700000 C 000 00 200 0000 0 0 DNA RNA hybrrid 0 O00000 0 0000 7000 2 HH D
26. LUE 1 DESDE EE RE DESEE EE Ho BE ESAE 1 1 ULL SELBE Eo d DESEE S ET 1 UU DD Eb EP EE RE EI DIET ADT 10 0 DAJ HT D HB H 3 DNA DNA I D 1 D 10 lD S BEBE
27. 3 deoxynucl eosi de triphosphate H LH H HU T7 RNAP T7 RNA l 11 ugumnmmBmnir uultu 1 A M Maxam W Gilbert Proc Natl Acad Sci USA 74 560 564 1977 2 F Sanger S Nicklen A R Coul son Proc Natl Acad Sci USA 74 5463 5467 1977 3 T Hunkapiller R J Kaiser B F Kopp L Hood Science 254 59 67 1991 4 International Human Genome Sequencing Consortium Nature 409 860 921 2001 5 International Human Genome Sequencing Consortium Nature 431 931 945 2008 6 S Tabor C C Richardson Proc Natl Acad Sci USA 84 4767 4771 1988 7 J M Prober G L Trainor R J Dam F W Hobbs C W Robertson R J Zagursky A J Cocuzza M A Jensen K Baumeister Science 238 336 341 1987 8 L M Snith J Z Sander R J Kaiser P Hughes C Dodd C R Connell C Heiner S B H Kent L E Hood Nature 321 674 679 1986 9 E V rle C Schneider M Renner M V l ker W Fi ehn Nucl ei c Aci ds Res 22 4354 4355 1994 10 D Seto J Seto P Deshpande L Hood DNA Seq 5 131 140 1995 11 M Mtz S Paabo C Kilger Biotechniques 29 268 270 2000 12 I P loshi khes M
28. 3 dNTP RNAP 3 OH 7 DEJES BE E 4 2 T7 RAPO OOC O 0000 0 00 000 00 T7I 000 T7000 Nati onal titute of Genetics M shi m Japan 0000000 O0 eeoogp p I U L u ugagagggagagggggaggguug 05MNCI O 10 glycerol 9 5 nMTris HCI pH7 5 mM MgCl 50 mM NaCl 0 01 wv NaCl 0 01 wv gelatin 000000 554 OCOOO0OOOOCOOOCOOOO0OCOCO TTOOOCOOOOOCOCOOOOLOI TED 10 nM Tri s HCl pH 7 5 1 27 HA HD BHD DNI JUD E BEBE PD HDD 00 DNO O0 0000000 T70 0 0070 0 0 O0 DNAD TE H BLBH B gdgHgdug eeocm gH gpdu T7Rpol N 5 ATA TTT
29. DNAQ 1000001 74 000000 10 0 DNA Fig 7 OCOOCOOCOOCOCOOOOCOCOO0OOCOCO0O0O0OOO DNP OODCOCOOOOCOCODI RNAP Fig 1 DN Eb DE CHE ED PCS DE TE Db JEDE GRE DES EU ET 0 00 T7 RNAP O O d GC DNA J 00 ZU DU DD B DA JJ ID D U D RNAPU OD DNA J J NAQD D D 5 B00000000 D 0 CA 5
30. D E S Rr 1000 00 00 000 5 2 DDD BUH RNA 73 00000 DNA O Centri Sep coetum 0 00000000000000 00 000000 000 RAJ JU DU ABI Pri sm310 7 3 D D 7 3 1 000000 rsuuunc 5 HT EL BEBE EE TE ELSE EDU EE IST Hp p FB DEBE DEAE EDS BI DE HD BI B E BEES E cso o o o DA Tb D CS BE EP CBE EET p 3 BEES EE n RR EE E C DEG EE HH E BED DNA HE 0 10 0
31. T7 RNAP O0 ET EE EE EE D GE e ET IEEE PBEOHCHE BB TE RNAJU U HU UH Fig 51 tson v tsoncrickpg 0o 0 O Hoogsteen pep pep pp dep ER BRE ES EE SEES EO DB IEEE SET EJ BIET D DO EE BERE DICE Eb DRE Hoogsteen B BED p EET BE BZTIEM B TS Ho WI B pr OE BE Bo Do ORE BEBE BE uggaaguagaagaugausuuguuuuuuu riTP O c rGrP c rATP HET DE ETC DE CELERE EPCDE SEE EE 5 ELEC BE EE HE GT 1 48 Watson Crick type pr di Oytidine 5 ZS SE TD o s E KX 4r EO de KA H s i gt e X gt kon 0 pos 2 H T inbss denine Guanine nosine B Hoogsteen type Cytidine d Cytidine H 901 zd Guanine H KA H i s H seg i xui 7T Deaza Guanine Fig 5 1 Hydrogen bond formati on among the ri bonucl eoti des Fig 5 20 7 T7 RNAP
32. T7 RNAP 7 DE SDESET TEE BESTE REGES EE DEBE ETE IZ DUAE JEEP EE BET RT TR d Ero E DE EL DERE gs 9 2 EGRE pros n BESTE 000000000000000 ne E LB E TEE ET D ELE 7 UuUUUugpUugDudnul Fig 3 30 0 000000000 Pvu lI D BD 0 O pBluescript SKO D DNA 0 H Dye An J D O T7 RNAP 0000 33 nz 100 u MO O 5 u MJ I n0 0 0 O0 0 0 100 u MO po 1410p MOOO UU n Dy es ATE BETIS BE HEBES HDD DB E ETE 0 00 100puMpnpya5uMinuumuguauguauguauguagaguggBiu
33. mmm e 15 2 3 6 DNADJBOUBDHBBBHB c sneKKKKH MHeIII 16 2 3 7 MM amp amp t KK IIR 16 2 3 8 DNgHBgHUBgBBggBHlg ceeeeeA6HKe IIIII 16 239 DNA t M 16 2 3 10 17 0 30 7 3 1 DD ccs mmm ehh 18 3 2 BD UD crm m 18 3 2 1 Wee c R RRKKHKHKII IK 18 3 2 2 T7 RAPI eem e 19 3 3 19 3 31 19 3 3 2 T7 RAPI IHH E SD BE DIC E DIET Bo B ees 21 3 4 DB creer Ihr 24 3 5 DD ccrte 25 0000 U 40 0000000000000000 000 r I U 4 1 creer IHememhhh herr 27 4 2 crrrrrrrrrreeeetmmmmm mmm mRAMM A ARMHIeHn IRA 27 4 2 1 ess 27 4 2 2 T7 RAPI I I U DD DDD 7 28 4 2 3 T RAPO 0O00 0 0 0 T7 RNAP polymerase 0 000e 30 4 2 4 T7 RNAPO 000000 T7 RAPO O 000000 5m ro ne 30 4 2 5 T7 T7 RNAP O0 3 4 ITI 30 4 2 6 T7 Fea4Yt 0 0
34. T RAPO 000000 T7 RAPO 0000000000 Bonner n n D UD DUH Db JE Bk 1 E E E EE D E PE 4000200000 UBBBH reeler O pBluescript DNA pg 0000000000 5uitsT7RNA J JU UU 0 0 RAPI J UO D U 0 00 37 C0 1 s0 000000 RNA00O0000O 6m0 0000 Ssv wv l 00 I mage Anal yzer Fuji Film Tokyo 1 4 2 5 T RAPO J J J J T7 RNAP O O 3 T T7 3 2 100 53 4 3 30 4 0 T7 7 2 6 T7 6 44 T7 RAPO 0 O 3 dATP O0 O RNA uuu T7 F644Y0O0 0 0 0 T7 RAPO O0 O 3 dATP 1 O O0 RNA D 4 2 5000 0 0 0 T7 RNAPO O O0 F644Y0 O0 0 O0 T7 RNA 000000000 500 p M 100 p M 50 u M 2 5 1 3 4
35. 000 bpwannmnuuueanetut TsniI 5 99 502 0 2 000000 400 5 5 8 HE E E 0o00 1 J G Paez P A Janne J C Lee S Tracy H Greulich S Gabriel P Herman F J Kaye N Li ndeman T Boggon K Naoki H Sasaki Y Tujii M J Eck W R Sellers B E Johnson M Meyerson Science 304 1497 1500 2004 2 D L Lyaknov B He X Zhang F W Studier J J Dunn W T McAllister J Mol Biol 2800 201 213 1998 3 D L Lyaknov B He X Zhang F W Studier J J Dunn W T McAllister J Mol Biol 2690 28 40 1997 4 J A Luckey H Drossman A J Kostichka D A Mead J D Cunha T B Norris L M Smth Nucleic Aci ds Research 18 4417 4421 5 M L Metzker J Lu R A Gibbs Science 271 1420 1422 1996 6 MA Innis K B Mambo D H Gelfand M A Brow Proc Natl Acad Sci USA 85 9436 9440 1988 7 O V Makarova E M Makarova R Sousa M Dreyfus Proc Natl Acad Sci USA 92 12250 12254 1995 8 D A Schafer J Gelles M P Sheetz R Landick Nature 352 444
36. 00000 Ecol DNA polymerase I 0000020200 dP H UI U D 3 F76000000000000 0 ddNTP 0 100 27000000 7 3 Fe44g F6670 D D DNAP 000 p GER DE EG BE BP sb 00000000 0 0 pNAP RNAP j Reverse transcriptase 0 B u U I uguaguagaagaHumuuagadmuuulbulI 5 3 ERES D ES B o BE S D DI EE E LC D E RE ET Do p RE E EE E PP p De TET HET EL EE BE BREOM DECRE E SHE HE EE BERE EE RD BD RE ET BEBE P n DEED ES DESI DAJ I TER ABE BEBE PEZ DECEDERE d d 4 5 DDD 0000 F644Y0 F667Y0 000 3 dAT P U HD U n vitro 00 0000 00 000 0 0 0 TrTRRPH GDLOUHDOGOUDUHUDULUI 3 dNTPs 0o 0000000000000000 00000Ts000000 T RNAP 0 F644Y0 667 IE ET SEPA DIESEL BOUE BP EE E BEAT EE DRE ESSE IEEE
37. uupnagganaugganagugpnsuauuprzRePenmnupnsuuupnminuul E HEEL HUU EECOHE EE ES DI BE CIPUE EE DIESE 3 T7 5 DESEE CEDE CEDE E DESEE EE 40 t amp Gtaca amp ta amp tttGtettGatGGetteat tarGcatt vrps o 0 sl s lt v lt r gt y h h Fig 4 4 Electrophoresis of Transcriptional Sequencing using F644Y mut ant The reaction was carried out with PCR product of human thyrotropi n B CDNA and F644Y or WId type W T7 RNAP Thi s fi gure shows the G si gnal s Arrowheads show the hi gh background signals of wWlId type T7 RNAP 4 4 RNAP O O 0O O F6440 0 0 Feeznn uu tl 00000 NPO 3 00 00C00COOCOOOCOCOOCOO0OCOOOD0DO 0000000000 0 T7 RNAPO O00000 00 0 Fea4 T7 RNAP J pol ymerase Helix Y 624 aa 634 aa Helix Z 648 aa 658 aa 0 0 00 loop D HBD 2 O EEG EE BESTE ED EE E DE EE COE 00 EE DE DES EH
38. 5 5 ET ET DAU EE DESEE BE DEL FEDT RNA I UU HU Fig 5 20 lane 3000 4H D 59 RNA EE B BI RNA ll 1 8 di am nooctane 3 per ni di ne I cadaveri n 0 DAU HL D D B LI U sperni di ne 1 0o20 00000000 Dd DNA CSI T7 RNAP 0 O0 g0 000
39. 0000 5 1 u ugaggmmagaguuaguumuauuunltr7ZRNAP 3 OH BW T7 RNAP O 000000000000 0 T7 polymerase I D polyrerase Helix Y 625 634 aa loop 635 648 aa Helix Z 649 658 aa loop 658 684 aa 00000000000001 0 0 O0 K631 Y639 G640 D812 D5370 Osuni Davis C p 0 0 T7 RNAP 812 537 pol ynerase UD BE SG ET GE HET EE ED DEDI EE E I DUDI cr o O K631 639 G640 polynmeraset 0 U D metif BEI E J 5 2 eH BP DE p Sr SEE 2p pp Dp E opp BE C20 Motif BO Motif polyrerasep 0000000001 3 OH 0000 D DDD DUHH ET B n b OE BI EE unn DR E ED ELE E ORE CER ERE EP ED EP HERE E coli DNA pol ymerasel F762 F766 1 Tag DNA pol ymerase F667 V Reverse transcriptase 0 0 F155 0 9r Mycobacterium DNA pol ynmerase 0 0
40. 5 I 55 DORATA E EJ E3 E E3 6CI1ITT1686GI1GCGTGITT616G6C T6 TC 160 170 1680 Fig 5 7 Detection of heterozygotes by TS The top chromatogram shows sequence of p53 exon 8 PCR product by amplification of w DNA The lower chromatogram shows the heterozygous poi nt mutati on f rom274R CGT to 274H CAT i n human col on car ci noma cell li ne HT29 5 3 4 5 EE BEBE BU HE DUST BEBE EE EE EE EE DI HDD 0 polynerase p I RNA LECT T po e e 5 RNAP OOO T7 0 0 RNA S j r n 856 DNAD 856 7 sperm dine T7
41. l 1 100 DNAQ I ID RNA D D U UU I I DNA DNADDDI PCR LI D TS SI DNA ODI DNA PCR T TSO 0 0 0 Ep EE EE S uuunungnnnn S BEER BECEE GE EDBL 60 guanosi ne tri phosphate 2 0000000 inosine triphosphate rITP O 7 deaza guanosi ne tri phosphate c raGrPH O MT290 p530 PRHUBDBUUHOUBUUHUH R2Z728 80n0 g8Bg0HB808U UI
42. Etero BHTBS EB ROBEBE B O BB BE BE Wumggmgl m m DNA 000 1 5 Gene Pure ki t Ni ppon Gene Tokyo Japan 0000000 DNA T D 20 15 ES E rua o 2 3 6 DNA JJ HUU U DbNAT T4 pol ynucl eoti de ki nase Ni ppon Gene Tokyo apa n n au au ul lU Ep DE ET EE 30000000 ES EMEN UE O Phenol Chl oroformlsoanyl alcoho Nippon Gene Tokyo Japan 0 O0 0000000000000 CF9RX 000 0 18 700Xg0 5 l 5 chloroform 8 700 9 500000000000000 1 55 EO j O H O EtOH l 000000 L pm DNA O O O Centrifuge Concentrator VC 96 TAI TEC Saitama
43. D00000 amno JU U DDDUUDDUDUDUDUDUHDUDHDUHDHDUH DA D O Tosoh Tokyo 4 85 Dye ug Dye C Dye A Dye G 3 2 2 TZBNAPH E Bo dpoW BOB WB RW WT T7 40nmMTris HCl pH 8 0 8 mMMgCl 5 mMDTT 2 mM sper ni di ne HCl 250 u MATP 500 u MGTP 200 H MCTP 500 u MUTP DNA 0000000 Pw lt 0000 O0 pBluescript SK Stratagene CA U S A 200 n0 00000000000 25 untsi l T7 RNAP I nvitrogen Tokyo Japan 0 Dye A0D J I 0 0 O 37 cC0o 1000 Dye AQ O0 O O 3100h M 50 u MI 25 p M 10 u MI5A MIT D I I Sephadex G50 col um Amersham Bi osci ences Tokyo Japan 0000000000000 Centrifuge Concentrator VC 96 TAI TEC Saitama Japan 000000000000000 RMAO OOOO ABI Prism377 DNA Sequencer Appl i ed Bi osystens Tokyo Japaa j 0000000000000 loading buffer 6 2 uI O 90 cC0o 300000000 ABI Prism377 DNA Sequencer o O0 0000 00 0000000 Rang 4 20C lJ uut 3 3 DD U 3 3 1
44. CS 1 DH Hg g DEBE BEES Epp DE Bie EE ugggamgbspapmnagmgasaugguauagguuuauuaguauuuuuuudtdl 50 HEIDE EE HH H HEB HOH HHHMHSHIIdLHgSHddHBHHBM Hg ugmgmumumuaaaaagssBBB DE EL pa l Ho HP EE EE p S598 ED EV DIL DEBE uggaggmumuguuagumgs panmgummuumumuuguuuuuug 20050 rm 1 p 5 5 DAU H DD 00000000 1000 0 0 cso 0 5 4000 50000001
45. 0 0 DNAO RNA hybrida DNA 7 O0 elongationcomplex DNA BEBE BEBE esp EE 00000 00 DNAOQO0OO0OO0COOCOO0COO0OCO0O0 0 OTSOCOOCOOCOOCOOOODI I Tso 0000 0 DNAQ uggagmggauauauaugsiRA AngumagagaugbpwgumauuBgubl uIl 21 mer 0000 20 RNA BEBE REOR HEEL DERE BH HOHER HERE ET E pL BI Bp BED 5 Br p EE E DIE 2A nn 200000000 85 5 5
46. 0 TSOO00000000 0000 s0000 0000000000000281 47 uunnunmnunnannnnanpnnnnnnnnaBpnunnnnnnnunn diTPO c dGIP O0 O 4 3 rITPI c rGrP c rATPn HEEL DESEE SET CER ELSE BT RES BH 0 0 0 RNA O DNA OOOO Tmp U 0 0 0 DNA J I JU U UU U H EET 1551 IH 1 1 BEBE BE HEB 1411 RNA DEC Ep RNA DDBOELELULUI ugumauguamusuusugauggauguaguogougguagaguguamsdululul 1 00 C000 00 000000 0Ts00000000000000000001 T7
47. 20 TMR 3 dUTP n on Fig 3 2 Structure of four rhodani ne l abel ed 3 dNTPs Cn i ndi cates the number of CH chain in the linker between rhodani ne dye and base of 3 dNTP 3 3 2 21 uguganagggugggsugguuuur RePmImuiu U L 7 RAJU I 3 ED DE 0 0000 0 De U bye A Dye G 0 0 RNA I I BU l uupnpnauupnpRApgnngpbpanmuuusuuggnagugmnuuubyeurn 6
48. DNAD DNAP Uuuuuun lt ni CSI 5 RIT oO 5 DNA EE OE HE 1 0 uu Is ESSE J OA Template DN A 3 GTICAGTGAC T Primer 55 CA a 2p dNTPs DNA polymerase N b CA dd CA A GT ddC CA A ddG CA A GddT CAAGTCddA CAAGTCA ddC CAAGTCACTddG CAAGTCACddT A C G 3 sam Poly acrylamide gel electrophoresis PAGE Fig 1 1 Procedure of di deoxy sequenci ng reacti on 1 2 Fig
49. m oH I K v jo p wv Fig 5 2 Incorporation of the nucleotide anal og in RNA synthesi s To exam ne whether several types of structure destabilizi ng nucl eoti de analogs are incorporated into T7 transcripts c rGTP was substituted f or rGTP or both c rATP and c rGTP were substituted f or r GTP and rATP The tenplate gene is 10 ng of human TSH PCR product The concentrati ons of substrates were C standard transcri pti on w th 200 u Meach of the four rNTPs 200 u Mof rATP rCTP rUTPwWth 1 200 u MrI TP 2 400 u MrITP 3 800 H MrITP 4 1600 H MrITP 5 200 u MC rGTP 6 400 u Mc rGTP 7 800 u Mc rGTP 8 1600 u Mc rGTP 9 200 u M c rGTP of rCTP and rUTP with 1600 u M c rGTP and c rATP 5 3 2 5 Fig 5 3000 4 TS0 000 DNA f UDDDBDBDBD riTPUUEH c rorrpooigooo go pPNABDBBODBDBDBBOBBDI 5 30 000 2 00 0 O Tyrotoropin cDNA 0 repo 0000 Ts0000 c rorrpo o
50. T7 uuauagaumuusuugaumuususuaummuuuauuuuuBluduHHHlilbiIl EE PE s DC PES E Bo S E E DES HABE B E HB SS BB d Ep Bo p CEP REED BE OP ET E BE ERE 2D HET HEAD DE DESEE HER ELSE EP DESEE DE BED E T7RNAPQ F6440 646 667 733 782 F882 0 0 0 0 O0 T7 F644YD 46 667 F733Y F782Y F882Y 1 33 Motif B 630 670 640 650 660 Helix Z Helix Y Loop 639 644 646 607 660 690 730 740 PICS WORSE T LA GFPVWQE Helix AA 733 Motif C 780 790 10 880 PES QDG dpa SFGTIP m 82 582 Fig 4 1 Mitation sites of T7 RNAP mutant The mutat ed resi dues and hi ghl y conser ved moti f s among vari ous DNA and RNAP are indi catedinthisfi gure The moti f s and mitated sites arelocated above and bel owthe ani no aci d sequence respecti vel y Motif B Motif C Hel i xY Hel i xZ and Hel i are located at 625 652 805 818 625 634 649 658 and 684 699 respecti vel y The seri es of T7 RNAP mutants desi gnedinthi s study consi sted of F644Y F646Y F667Y F733Y F782Y F882Y and Y639F 4 3 2 Fig 4 20 Table 4 10 0 O0 Table 4 20 p T7 RNAPO O00000 O0 T7 RNAP 00000000 0 0 Ppolymerase 0 0 3 dTP UU PU U U i
51. 5 PCRO O O0 DAU II HU L BE VL ER 0 DE SPEC REGE E EE ops SR DE TS 0 0 O1 8 diam m nooctane0p 0 00 00 00010ng0000 DIA I luu Uu D 1 1 Y Hayashi zaki K M yai K Kato K Matsubara FEBS Lett 188 394 400 1985 2 Y Mirakani K Hayashi S Hirohashi T Sekiya 1991 Cancer Res 51 5520 5525 1991 3 R L Tol man R K Robins L B Townsend J Am Chem Soc 91 2102 2108 1969 4 S Tabor C C Richardson Proc Natl Acad Sci USA 84 4767 4771 1987 5 S Mzusawa S Nishi mura F Seela Nucleic Acids Res 14 1319 1324 1986 6 H Yamanaka D Nakajima O Ohara DNA Res 3 81 86 1996 7 G Sarkar H S Yoon S S Sommer Nucleic Acids Res 20 871 878 1992 8 J F Mlligan O C Uhlenbeck Meth Enzymol 180 51 62 1989 9 J D V tson F H C Crick Nature 171 737 738 1953 10 K Hoogsteen Acta Crystallographi ca 12 822 823 1959 11 S Tabor C C Richardson J Biol Chem 265 8322 8328 1990 12 P P Cunni ngham J Ofengand Bi otechni ques 9 713 714 1990 13 A E Pegg Biochem J 234 249 262 1986 14 J F Mlligan D R Groebe W Wtherell O C Uhl
52. ugagamugagaugagauauuagauuu 644 0 F6670 3 OoHD EE P B p EE BE ED D BE EE DI C HDH U HBH Table 4 3 Relative selectivity of F644Y F667Y and WId Type T7 RNAP f or 3 dNTPs and rNTPs i 3 dATP ATP 3 dUTP UTP 3 dCTP CTP 3 dGTP GTP F644Y 46 08 50 53 83 89 86 11 127 71 130 0141 61 44 10 F667Y 38 76 41 81 68 23 70 32 115 79 118 4334 92 37 10 WI d Type 0 23 0 24 0 31 0 34 0 59 0 61 0 30 0 34 The rates of each 3 dNTPs incorporation to rNTPs are conpared for F644Y and F667Y mutants and wi d type T7 RNAP The template is given in Fig 4 2 and Tabl e 4 1 respecti vel y The 3 dNTPs concentrati ons were 500 u Mand 250p M 4 3 5 T7 4 Fig 4 5000 0 Thyrotoropin cDNA PCR O0 0000000000001 RNAP 0 T7 RNAP DNAD T7 RNAP O0 0 0 H O T7 RNAP 4
53. Hoton 0o 0 6 u MO O PCR 00000000 2 5 units ExTaq DNA polymerase 0 00000 0 PCR I PTC 200 DNA Engi ne M Research CA U S A 0 0 0 0 OPCRO0 I UU O0 94 co 1500000 B U 0O 094 C 0 300 H 55 cC 0 10 H 72 cC0 20 0 000000 300 0 000000 0 0 PCRO O O Dye Terni nator FS Cycle Sequenci ng kit ABI Pri sm 377 DNA sequencer 0 DNA J O LEE EL TEE EEOEPSEL AED BREED DE PRI ED O 644 6 DNA I l 0000 Aa 0 0 0 Pt O O F667Y F733Y F782YU 000000 Xo 0F882Y0 D Xem n iu u ugmmumummuudub panmguuuuuuuuuuuumumumumuadadggaul DNAO O O pI7RD
54. UU U O Template Suppression Reagent 25 H 0 ABI Prism310 Genetic Analyzro0o 00 000 000 0000000001 72 Hoop To BE RE BEP DT M BHI cso 0000D O Big Dye terninator Cycle Sequencing kit ver 3 00 000 Ts0 00000 bpPapnumumaauuauauuuuus csauautalu Prism310 Genetic Analyzer i D DB 0 U U EE EL EE E RE BR ET EJ 0000 O O t JUCSC Genone Browser htt p genone ucsc ed LuduubBmggaudugbspPamngguagumugadadasuuHBHHdb 7 2 2 Q000000 0 0 MA0 0 0O 000000000 Map 0o00 Oo PCROOCOOCOOOOOCOOCOCOOODOOI DA I l 5 ei iuuugamgagagagaguguuau 1 B dq qa
55. HE DESEE STE I DE RAJ O O DBNADDODDDU O R110 CH 3 dGTP Dye G R6G CH 3 dATP Dye A O 3 dUTP Dye U ROX CH 3 3 2 3 2 1 D0 0000000000 paogaosgapgpgggomupogpogudDuPreer nuagagguuuatd 6 carboxy tetramethyl rhodani ne TM3 6 carboxy X rhodani ne ROX 5 carboxy rhodani ne6 6 5 carboxy rhodani ne110 R110 N Hydroxysucci ni m dyl ester Mol ecul ar Probe OR U S A 3 dUTP 3 aCTP 3 dATP 3a3 darPipnguggaagugaaggaagag agagugl TM 3 qUrP Dye U ROX 3 dCTP Dye C R6G 3 dATP Dye A R110 3 dGTP Dye G 0 0 0 DO0O00CO0 0C0000 0 0 0 0O O 7 deaza 3 dATPO 7 deaza 3 aGrP purin 00 70 UD D 3 3 daurP 0p O0 pyrimdine D B 5I 0 0 UU C C CH NH I HI IU N N di methyl f oor rem de JUD DDD triethylam mne rhodamne N Hydroxysucci ni m ester J UU DD DDD JUDU O rhodamine O O carboxyl 18
56. 000 0 M290 p530 0 0000 00000 O R274H SE Ep DR ED SEE EHE p ps TD EE ESRB DIESE EDS d d PCR 0 BH WB mu csmgT uduqgWmu wl U 1 8 diamnooctane IJ UD DNAD B Br DI H H DH H HH TS H Hg csu grm rmy n uu dgomgwu1 1 11 5 3 Taq DNA pol yrerase0 0 O0 00 000 00000 400000000 Uil EET REGE FE DIE REESE LEE ETT ED ER E DE E SDEUDE PE BE pGEM 5 5 99 5000000 0 0 400 bp D lU L TI Ul 5 U L ugmgmuuuauaaacgugbpwapgsauausuuuuuuuuewnmnulllu Ul O umuuugb pwamnmumumnmumiBmultetrrlrvt U Dwag D H Ul Ui U DNA HB DE 1 14 EF ER BEBE EE EE EE BE SEDED DE EISEE ED DIE HOGEBE EE DESEECSSET TE ET EE DNA 0 O0
57. ABI Prism 310 Genetic Anal yzer Appl i ed Bi osystens Tokyo Japan o0 00000000000 0000000000 6 2 3 prs p U UU D 5 T prometer T3 promter j 0 0 8 5 AGC GCG CGC AAA TTA ACC CTC ACT AAA GGG AGA GAG CT 3 5 CTC I CC CTT TAG TGA GGG TTA ATT TGC GCG AC 3 5 CCT GCA GGC TAG CTT GCG CA A GGA TCC TAG GCC TGA AGC TT 3 5 GTC GAC AAG CTT CAG GCC TAG GAT CCT TGC GCA AGC TAG CCT GCA GGA GCT 3 5 GTC GAC GAA TTC ACC CGG GAA GAT CT T GCT TAC GTA CGC GTA CCA TGC A 3 5 TGG TAC CAC GCG TAC GTA AGC AA 63 G ATC TTC CCG GGT GAA TTC 3 5 TTC TCC CTA TAG TGA GTC GA TTA TGC GCG C 3 5 TTC TCC CTA TAG TGA GTC GTA TTA TGC GCG C 3 5 AAT TGC GCG CA T AAT ACG ACT CAC TAT AGG GAG AAT GGA 5 I U DD DD UD U 8 5 O O T4 pol ynucl eoti de ki nase Ni ppon gene Tokyo Japan U 00 000 00 000 0000 00 00 00 00 000 0 0 T4 DNA li gase Ni ppon gene Tokyo Japan o O00000 0000000000000 0000 10 0 DNAQD O0 JU UU UU DAJ JUD DD DU DU coR l 0 A ne T LA E DNA 000000 Eco RI 0 pUC19
58. 5 Hr 0E ORE ED BILE BER CE EE UD DBLOED TE EE BET EP ELGBAC EE BUTS BEES O DINAR 5 64 uuunnnnnnnnnnnnnnnunnnnunnnnnnnnnnuni rhodamne 000000000000000 0000000000000020 BOD PY O0 CO O 0O0000000000000000000000001 lD D F6G44Y 0 0 O 17000 173 000000000000000 T7 delta Feadvyu1n DD D D unmnnnnnnnnnD
59. DNA elongati on conpiex0 0 000 0 DIA Bop BED I BE 00000 00000T7s0g00000000cso0000000000 DAU nn uD uggpaggpagagBBBHBBBBBBBEBBBDS paAn n nnnnnunn DIE UWIJdSHIEBEBU HgHIBHdHHIggBmgdumgbmgBgJdugdgd bNAD H 00 5 5 75 CS TS T promoter Template DNA Template DNA Heat denaturation of template DNA Annealing of T polymerase with T promoter on template DNA me es RNAP Annealing of Taq DNA polymerase with primer and template DNA DNAP Fig 7 1 Conparison of TS and CS reacti on mechani sms for difficult to sequence tenpl at e Fig 7 20 Fig 7 30 Fig 7400000000000000 DNA D D D H TS 0000 DNA UU UU Hu HU li Fig 7 2 00000 0000 Gn H i HHBH HninnnnnHnn DNA U
60. l 1 1 PEEL Do CD EDD REGE Es Eo Y AE CEPS BS BLADI D DES I ES DERE Fr BEC oC 0 0 x9z7 9 Wg ugguggau ggaug 1 5 3 eoti de ki naseg 00 00 00 000000R0000000000 DO0 00COCOCOOCO O0O0C00 00000 0 0 bNAI 00000 E 0000 0 0 19770 00000000 rig 1 1 T T 0 DNA DNA pol yrerase DNAPO 1 0 00000 0 0 DNA DNAPO I deoxynucl eoti de tri phosphate dNTP 0 D D DNAD di deoxynucl eoti de tri phosphate ddNTP I 00000000000 0O 0O O DNAD O I I Ui 1 0 0 DNAI H HD DUH HHI
61. 856 7 Fig 5 80 0 0 T7 RNAPO 0 0 ugaagmgaagugaaguaaguguaaguuaagu 5 8 vat 00000 pBluescript DNA O0 O0 DNAD O O0 O0 O0 O T7 RNAP O RNA cadaveri ne 1 8 di am nooctane O0D0 0000 0 O speermdinep 0o 0000 56 00000 1 8 diamnooctane 4 62 0 O cadaverine l l l 3 4000 59 5 80 n0 RNA I I 1 8 aninooctane cadaveri ne tH Ll I 0000 5 13 diamnopentane 1 9 am nononane RNA
62. L1rrrr zr L 0 1 0O L L 58 Fig 5 10 Effects of 1 8 di ani nooctane on the sequenci ng si gnal s Sequenci ng si gnal s of the TS froma small amount of the template DNA in the absence of any additi ve upper panel A and in the presence of 1 8 di ani nooctane lower panel B 5 4 7 5 5 Hoogsteen ogoogo DNAD OODD 4 5 3000 Fig 5 4 HB HE 5 3 6 6
63. T7 RNAP 000 delta F644Y0 0 00 00 00000 0000000000004000 000042 2 00000 0 T7 RNAP F644Y0 00000 DAD J U 0 0 0 172000 173000000000 00000000000001 T7 PCRO D O T7 delta F 5 TAC AAG AAA GCA TT T ATG CAA GTT GTC GAG G3 PCRO D D O T7 delta R 5 GAC GTG CCC T AC GTT GAG TTG TTC CTC AAC 3 0000 PCRO000000 0 T7 RNAP E ELE DESEE BERE E OH DE EHE DESEE REDIERE 0 0000 PRI AC BOB dH gu dudo wg Hm Wy Wu 000000 T4DNAligaseg 0 0 0 0 0 O O T7 RNAP 0 8 644 T7 RNAP 0 del ta F644Y 00 0000 000000 T7 RNAP 0 delta Fea4Y 0000000 EGFR PCR O0 0 TSO 000 DN0OCOOO0OD0OOCO0O0O0O0O0O0O000I 40 mMMTris HCI pH 8 0 8 rMMCI 5 mMDIT 2 mMsper m di ne HCI 0 4 mM MCI 0 05 Tween 20 1 mMGMP 250 u MATP 500 MITP 250 MCTP 250 u M 5 Br UTP 0 1 u M BODI PY R6G 3 0 2 H M BODI PY FL 3 dGTP 0 4 u M BODI PY 564 570 3 dUTP 0 3 u M BODI PY 581 591 3 dCcT II O0 00000000 DNA J j PCRE Hj 10 ng D D D lU 00000000000 50 units T7 RNAP 0 delta F644Y 0 0 005 units PPase 0 0 0 O0 37 C0 10 D U J D UD D licentri Sep col um Appl i ed Biosystens Tokyo Japan 0 00000000000000 RA0p0000 uUugagagamBg
64. 7 00000000 TSO 0O00 0 O 0O O F644Y0 O0 O 66 DE EL LESE BE EE HAE BE DE SEP ETE EE UL ED TS 5 EDT d LEES CIEL LCD b Se e De E EDU DEESSET E DE EE I3 92 1011 21 1 Tp BEC DIC E DEI E tri phosphate urerrhppuuauuuaaasuuuuuuauaamsBiun inosine tri phosphate ri TPO O 7 deaza guanosi ne tri phosphate c r GTP HEBEL OE RESRD BEBE DNA LU UU UD gt u I
65. 5 3 1 pBS pGoEMI H BEBE STE EP BF E 2I SPUR RE EE ER CREDE TR IE 2E BP EE F EBD IRE E EE RP ED SE ET RU prsi j 700000 co0 000o DN D 5 DE EL DESEE BEES TI HPD hunan hepatitis Bvirus hHBV u uguagaaggbpapnpsgaagammususuggmumuggggumsgBagadmlmumibI 0 100 0 00 00 00 00 0000000000 00000000000 00000000 I 00000 HBVOO0O OOOCOOOOCOCOOOOOCOCOCOCOOCOOOOOODI ETSI Doe HT d 00000 DNAOQOOCOOOCOOOOCOOOOOOCOOOCOOCOOOOOODI OO0OOCOUOOCOUOUOUOUOUOUOUOUUOUUOUOUOUUOUUOUI 6 5 0 e mgggTsegg cegmg TSO 0 0 037 C0 0 000 00 00 000000000000 00 00 T7RNP 789 4 EGARI HHUH D 190 0 00 000000 PCRO 0 O0 pBS oGEMJ UU DDD UU UU DDD TSI 70
66. 5 80 0 0 DNA 00 U U ugmgmuuuaaaaguuulullulLtctrtyttulutltlil EL DESEE EISE DL DE Dn EEG BIST Sp SB O PCROOOOCOCOLDI DAJ O DNA I J D D HABET HUHHHHEHHBUHHBHHHiHBdHliHHuliguuaglH BNAHIT Ehe 5 UUDUdUUDIDDUKEHUDHDUIEL TESTE H UU 1 00 1 M Izawa N Kitamura N Odake F Maki K Kanehi ra H Nemoto M Yamaguchi A Yamashita N Sasaki M Hattori S Kanayarma and Y Yoneda A Rapi d and Si npl e Transcri pti onal Sequenci ng Method for GC rich DNA Regi ons Jpn J Vet Res 53 3 4 159 169 2006 10 K Shibata M Izawa Y Hayashizaki and M Wtahi ki Practical Application of Transcri pti onal Sequenci ng for GC rich Tenpl at es J Struct Funct Genomics 4 1 35 39 2003 1O Y Shibata P Carninci K Sato N Hayatsu T Shiraki Y Ishii T Arakawa A Hara N Ohnsato M Izawa K Aizawa M Itoh K Shibata A Shinagawa J Kawai Y CXa S Kikuchi N Ki shi mto M Miramatsu and Y Hayashi zaki Removal of Poly A Tails from Full Length cDNA Libraries for Hi gh Ef fi ci ency Sequenci ng Bi ot echn ques 31 5 1042 1049 2001 J Kawai A Shi nagawa
67. 70 5 1 lH runs s s snn n s 3 3 NON OG 8 3 3 3 3 3 3 n n 3 3 nn 3 3 3 X3 n n s 3 3 4 3 3 X3 n n 3 3 n n 3 3 3 3 3 3 3 3 3 3 3 3 s 72 2 su s sn n 3 3 3 s 3 3 n 3 3 3 n ns s ns n 3 n B 4 3 3 3 ns 3 3 3 4 3 3 n n n 3 3 4 3 3 3 n 3 n 3 4 s 3 nsn 72 7 2 1 TS EI H Bel Dr D Hs PNATNN SP DD expertes 72 7 2 2 5 SENI su e 73 35 su s n s 3 3 3 3 3 3 n 3 3 3 4 ns s s n 3 3 ns 4 3 3 3 n n 3 3 4 3 3 n 3 n n 3 n 3 3 3 n 3 3 3 4 3 3 3 3 3 3 3 74 kat DNA 74 732 DNA0OO0O00OO00 is e n a a Qe GR KATE Re Cpu e 79 7 3 3 Tcp 0 000 0 0 DAJ JUD RD TRE BE BEER HT Eee Pr pe ESSE 82 4 ra s n n s n 8 3 3 3 3 3 3 3 3 3 3 3 3 3 X3 4 3 3 3 3 3 3 3 3 3 n 3 4 4 3 3 3 3 s s 3 n 4 4 3 3 3 V 3 3 3 3 3 3 3 85 5 ra s n n s 3 8 3 3 n ns nn n ns 3 3 3 3 3 snn n 3 3 3 3 3 3 3 nsn nn 3 3 3 3 3 s 3 4 4 3 3 3 pn n 3 3 3 3 3 a 87 90 U L 0 10
68. HDH Temliphi amplification kit Arersham Bi osci ence Tokyo Japan 00000001 3 Forward 5 GTA AAA CGA CGG CCA GT 3 0 PCR 0 0 0 0 0 M13 Reverse 5 CAG GAA ACA GCT ATG AC 3 0D O0 O 0 5 units Ex Taq DNA pol ymerase Takara Bio Shiga Japan o 0000000000 DAJ D U L 000 Ap DIESE FABER CEEEE BE 000000 0 O0 PTC 200 DNA Engi Research CA U S A UD D D PCR ID IU HD 94 3 9 450 055 C 0 4g7zzZcyamgauguuuuusesuuuuuuuuuz7ecgsmuiuil 000 0 OPCRO 0O 0 0 0 MAQ O0 0 0 0 DAJ O O cso oO O O bNAT O O DuugagugauuuumsugeExonucleasel O0 Shri mp alkaline phosphatase Amersham Bi osci ence Tokyo Japan ugaguummuszcegsoununauauuuauauuesocy 1 5 PCR aresgugpnmuaauuuauauu PCR 000 Ts0 0000 Exonucl ease O Shrimp alkaline phosphatase J UD D DD D D 000Ts0o0O 000000 0000000000 0 0 MA0 OOOO PCRO O DU 00 8 0 00000 TI U U B DE ELE
69. 5 DNA 0 00 5 ugagaggggaguguuga agag beangumgaguagagauadgus suni displacerent 8 5 000 7 2 0000 7 2 1 TSOO000O00000 0 0 5 0000000000 40nMTris HCI pH8 0 8 mMMjCI 5 nMDIT 2 mMsper ni di HCI 5 0 4 rM MCI 0 05 Tween 20 1 250 u MATP 500 H MITP 250 H MCTP 250 H M 5 Br UTP 0 1 u M BODI PY R6G 3 dATP 0 2 H M BOO PY FL 3 dGTP 0 4 u M BODI PY 564 570 3 dUTP 0 3 H M BODI PY 3 dc P 0o 0 000 00 0 DNA 000 5 DNA 100 ng LE TEES DESEE TO BP SE DES 50 units T7 RNAP delta F644Y 0 005 units PPasep 0 0 0 0 a7cpg 10DDUUl 0000 56 nM 0000 ethyl enedi am ne tetraacetic aci d EDTA 99 594 v v EtOH 70 v v Et OH
70. 7 HE EE RNAPO Xo l HUU H 000000 p7R I l o6eg MPCRO 0 0 O 5 CAT CTG GTC GCA TTG GGT CAC 3 PCR O O Xho R 5 CCA AGT GT T CTC GAG TGG AGA 3 PCREH H D O O Xho F 5 CTA AGT CTC CAC TCG AGA ACA CTT GG 3 PCRO O O O O Af l T R 5 CAG CCA GCA GCT TAG CAG CAG 3 O 000 I U I 00 DNAD pT7R 100 pg U 2 5 units ExTaq DNA pol ymerase Takara Bio Shiga Japan 0000 PCR 00000 PCROO OOO0O O O PCROOCOOCOCOOCOOCOOOLOI 0 0 J PTC 200 DNA Engi ne M Research CA U S A J UJU U DO I 00000 94 cC0 100000000 0 0 0 94 C0 300 0 55 C0 450 O 72 C L L 3 50 00 0000 30000000000 72 cC0 50000000000 PeRH pDpuauaggaamgagauagasuuuguu Aee 28 Xol UU UD D DDD T7RNAP 0000 0 DNA J I U 3H 0 DNA ugagaau us erzREg T7 00000000 T
71. GC 85 Uu u U O DNA DNI I IDD DD UU LU lll 7600000000 99 5 0 DNAD uuu uUuuuununununu_nuununununnpununnunnnnunnHnPCcRi O cco U I 0 DNA DNAQ O0 oap I I D l 0 0 0 MAD TSO O0 D 0000 DAU D n UU UH UH DNAD ELO DE DICTI DE EDT E EJ P EI 81 CCCCCRCCLE T TCCHHERHBRH ERE ER CHER HH 8 18 b n ui 38 N ROLE bEGCRCCEEGCGETTCEEEGTCCECRCICD TPEU RE PIT 11 B i y a l A MI MI i 9 d Wi Wm 4 Fig 7 6 DNA sequenci ng el ectropherogramof a hi ghl y GC ri ch product usi ng CS and TS Sample No 2 of MDA reaction in Fig 7 5 was subj ected to di rect sequenci ng usi ng CS upper panel and TS j ower panel CS yi el ded a great y reduced si gnal and finally became unreadable In contrast to the results all MDA cl ones coul d be anal yzed conpl etel y by TSO data not shown
72. RR D P 9 Uy PT RT E Muri AT qe Z m E E ded Q 11 11 U 5 met hyl ec Uu 1 3 lU 1 3 U Dl DNA uuu 1 000000 E tb BOB n H DNAD Ulu DDD H uu 0 Bub RNA pol 85 RNAP DNA D uuu E TESBE DE EE UU Ud HDH E 7 T7 RNA polyrerase T7 lU DA in O T RNAP L L DNA O0 U tri phosphate NTP O O 0 00000 00000000 DAH IU DU L U l Dp agn
73. 2047 2057 1997 D S Osada M Izawa T Koyama S Hirai and 5 Ohno A Domain Containing the Cdc42 Rac Interactive Bi ndi CRI B Regi on of p65PAK Inhibits Transcriptional Acti vati on and Cell Transformation Medi ated by the Ras Rac Pathway FEBS Lett 404 2 3 227 233 1997 0000 0 5 Hirai M Izawa S Osada G Spyrou and S Ohno Acti vation of the JNK Pathway by Distantly Related Protein Ki nases MEKK and MK COcogene 12 3 641 650 1996 nD 0 11 11 1 11 ES DDD RAJ HDD HUU 2005 006578 97 2007 7 Du 98
74. 4 6 387 391 1997 P Carninci C Kvam A Kitamura T Onsum Y Okazaki M Itoh M Kam ya K Shi bata N Sasaki M Izawa M Muirarmatsu Y Hayashi zaki and C Schnei der Hi gh Ef fi ci ency Full Length cDNA Cloning by Biotinylated CAP Trapper Genonics 37 3 327 336 1996 96 H HU uU L u uuu uiuit 2 00 Dual End Sequerci ng 138 20020 30 11111111 111 H HE EBORE EE HO HE EE EDS ERE umumuguusuuuyuugsaudggitlu JUD E HBHWHHWHHBdEHd gouy t p 50200 1999 120 lID ET DE TES DT SRI EE EE SET HHP HH UD uu EP DO E d E RE SEE E p 322g 19985 120 BE I IDD 1 1 HESS 200 3 000 D 5 Osada M Izawa R Saito K M zuno A Suzuki S Hirai and S Ohno YSK1 a Novel Mammalian Protein Kinase Structurally Related to Ste20 and SPS1 But Is Not Invol ved i n the Known MAPK Pathways CQncogene 14 17
75. AA E SC BEP e e a EJ aae E EIE r3 r3 EE EX ES ES Es ESTE E TES zig AER r D b 000g o 2 aand D Da rrrcdocrsrsrt rt O 1 s DL n a gt tp IE ao E ES EE ooo E3353 lt Es Ep EY a El i gornog oono jc Ej 13 N mn aada H a0 n noa0v EEE adu c O 1 2 ja E Ed EJ HE EIE ES qd 1 A r3 is c oEJU n EJ Em m g m 5 r3 Ey E 5 n r3 Ed EE oog rd n a 3 1 m O mci F dcn cd pou r3 EE JE r3 r3 X gt rx up E EE sj E ET T ES r3 E33 0 I AES Ed ERES E ESSE E o ooon E E Er ES E
76. Sibi primer DNA product phosphate Q Fig 1 3 DNAP Reaction Fig 1 40 0 DNP I 0000000 000000 0 dNIP dATP 4 3 3 3 4 d I o dATP P P PpP i o H HH s Lm 2 2 9 H ddATP P 0 rP 20 rP u ll H H Fig 1 4 Structure of dATP and ddATP 5
77. TARDE HA DUHH UU DDBDBHBUJDUUDUHUDUDHDHDHBUDUHUUDUCHDEDHDHBHUDUHEDDHIDEBHEHBUDUINIULDI DNA Fig 1 5000 Repetition 3 5 Template DNA ll Heat denaturat ion 38 5 nnea ling of DNA primer with template DNA 50 C i ALL Initiation of DNA polymerization B0 C 3 Fig 1 5 Procedure of Cycle Sequenci 37 cC0 K enow Fragment T7 DNA polymerase 0 000000 00 000 0 DNAPD Thermis aquatics 0 DNAP Zag DNAPO Oo 0000000000 ugugbwenumumusupuauuggaudgbpwaimmuuuuiulillulu 10 0 DNA 1 L
78. Appl i ed Biosystens Tokyo Japan 0000000000000 loeadi ng buffer 6p Li Bo 2u 10 90 C0 30 U U BOTE DEAE BE 0000000 ABI 5 377 5 4 20 5 3 0000 5 3 1 diTPO c dATPO O00 0000000 000000000001 guaninep gp 0o 00000000 3I Uu Nu0gmgggagaggBgggv tsocickgaupmnguggaggaggma BBIlI ugugaauusuuuaauauudcrPy dl TP lD HDI ES d pr e EP PC EE e BEC e DE E DEO HB s p PE Ep E RR AE BUS HE RE v tson Hoogsteen p 0 00020001 000 0000000000000001
79. Each lane al so had WI d type W and F644Y mutant RNAP lane 1 5 and ane 6 10 respectively 3 dATP concentrations in lanes 1 and 6 500 u M lanes 2 and 7 100 H M lanes 3 and 8 50 u M lanes4 and 9 2 5 H M lanes 5 and 10 1 H M 4 3 4 T7 RNAP O F667Y0 D 0 0 H 0 T7 RNAPH D 3 dNTP D 0000000 Table 4 20 Fig 4 30 00 00000 3 daP0 000000000 7 F644Y0 O0 O F667Y0 B D D O Table 4 30 3 awPpnugmnggumnggduuutunu DNA O bNAD 3 000 T7 3 l T7 F667Y0 3 daNrPOo 0000000 T7 RNPI 0000 3 dNTP O O l 39 J J J OA aoada Er O J J J ESI 0000 O0 Fe44Y 0 00 F667Y0 0 0 3 3 daNrPO 0000000000 0 3 dUTP23 dCTP23 dATP gt 3 dGIP 0 D D purin 0 000 pyrimdinep 00000 3 T7 RNAP O O F644Y 6 6 3 dN PI I B BO BU U D U I
80. invitro 0000000000 PCRO L L PCRO PRODDT in vitroj D DNAD I L L 5600 DNA 0o00 U 5 HT290 0 O0 p530 D D 8 PCR 0 0 O0 0 TS 53 8 H290 0000000000000000 p530 0 000 80 0 0 000 0 0 p530 R274H0 0 HD DL D D DNAD O HH mpm O DNA D PCRO DL CGT DUTSU l 0 00 HT290 0 O PCRO D D D CAT D z L
81. ugguagamgagdgue mguguFeezg Helix ZI Heli x AA 659 aa 684 aal 0000 0 0 loop HH BN BH HB Hog Es p Tod E To BC 3 dNIPI 00000 0 lJ O polymerase 0 0 0 0 UUDdJDUdDBUHBJDHUUDUHUDUUDUHR DO0 O 0 O0OCOOCOC0O0O0O0COOCOO0O0000000 29 9d NEP BE UJU JU H 00000 T RNAP F644Y D F667Y 0 0 0 0 0 HU T7 RNAPO 4 3 dATP 00000000 OE E GERE EE AE EG ES ER ETE HOT Dp Y69 l 3 OH L 3 OH HEP HE EHE 41 EJ rJ J J J UD UU UD DU E 11 111 T7 RNAP F644Y F667Y T7 RNAP O O O Fe44Yv j FeezYiu UU UD UD I 000I 03 dArPOO0O C00 0C00 0000 0C00 000 0 0000 O0F6440 F667 3
82. 20 co000000 4 2 3 T7 RAPO D0000 O0 T7 pol yrerase t D H T7 RNAaP 0000 UU T7 RNAP polymerase 0 00 000 000000I ugagamgagamgmmuagmuuauuutill40nMTris HCl pH 8 0 8 rMMCI 5 DIT 200 u M GMP 250 u MATP 250 u M GTP 250 u M CTP 250 u M UTP 0 2u Ci a gt P UT I U U U D BH 0 DNA O pBluescript SK Stratagene CA Pv4M IDDDDDODD DNA 20 ng D D T7 1 15 PDH UUHBHL 0000000000 Sousa J 0 O O Bonner 0 0000 DE81 0 0 D Matnan Tokyo 88 D81 0 000 0 BECKMAN Tokyo Japan 0 U O0 T7RNAP I HHH HH L 7 Tokyo Japang 000001 uL 4 2 4 T7 T7
83. 250 u MGTP 250 u MCTP 250 u MUTP 0 2 u Ci a P UrPO 0000000 pBluescript SKO 0000 uj DNAOQDOCOOOCOOCOCOCO 2nMri JU D lU 0000 T7 5 units O0 0 O 37 C0 1 00 0000000I uggagmgagagauauu RAID DU DJE 6 M00000 8 4wv op HU U H 000000000000 O BAS2000 Image Analyzer Fuji Film Tokyo 1 ER H DI 46 RE HE HHUH BD EESEE SEHE DI RI 5 2 7 5 TS TL pL dE qu m ppm pm ESTE CERA PEDE BEAT BE TL B 40nMrTris HCl pH8 0 8 MMC 5 mM DIT 2 sperm di ne HCl 2 mM GMP 2 5 nM ITP 250 u M ATP 250 H MCTP 500 H M UTP 0 1 u M R6G CH 3 dATP 0 1 u M R110 CH 3 dGTP 1 H M TMR CH 3 dUTP j 5 u M ROC CH 3 dCTP 1 DB D D DAU PRIUDU10ngnniD unuuul 25 units T7 RNAP Fe44Y 10 units PPase 000 37 cC0 1000 000 0 0 OSephadex G50 col um ArershamBi osci ences Tokyo Japan 0000000000 RA0 0000000000 0 l 0 Centrifuge Concentrator VC 960 TAI TEC Saitama Japan 0000 ABI Pri sm377 DNA Sequencer
84. 400 base long secti ons fromthe first signal 6 4 D D 5 60 5 5 5 Mcro electro mechanical systems 5 DTE ugaggauugagagumggugagersinuuauuaguauuuguuudcusecelu 00000 prs p 0o 000 wspyumn nunu 00000 pomwteri 00002000000 0 0 O0 T7 RNAP J O 48 856 processi ve el ongati on phase UU 0000000000000 initiation phase T7 promoter ugagasggasuuauuauu RA 0000000000 initiation phase processive elongation phase 0 000000000000000 69
85. ET DESDE DEL DES 3 T7 UumggggmeudupbgmumguguubgamBuuuguusuudugagsgscsnseseutuutututl uggmmugumuuauugnansun nnBp5puamuuiuBiugn rmnrttt t 4 ne Hg m B BE BS BP nsp o EE BI BEP RESTER SE 1 1 PP PIE DE PCT E D Db Dope d AED 3 OOOOOCOOCOOOCOCOOCOCOOCOO ns P DE Ho HET EE HIH H DHHI REGE BE E EE DNA 35 24 3 EM MER RR EE MR P EE E AR 000000 0 DAPO DEED gp mp d 9 B 3 5 00 D RNAP
86. F644Y0 667 3 RNAP O Fe46Y 3 dArP0 0000000000000000 3 T7 F644Y 66 3 dATPE H l T7 RNAP 0 F644Y 3 dArPO 0O 0 0 00 6 l 2 Table 4 2 Relative efficiencies of recognizing the 3 OH groups of ri bonucl eoti des as substrate in each mutated T7 RNAP and d Type DUM Fol d reducti on by 3 dATP F644Y 98 24 99 12 F667Y 90 46 90 76 F644Y F667Y 106 24 108 43 F782Y 21 89 23 22 F733Y 20 22 21 14 F646Y 8 23 9 44 Y639F 17 17 18 62 W I d Type 19 12 19 35 For the tenpl ate DNA see Table 4 1 3 dATP concentrations were 100 p M The experi ment was tri plicated The value in thi s table represents the average of the relati ve reducti on of the a P UTP incorporation rate by each RNAP in the presence of the 100 H M 3 dATP 4 3 3 3 dAT P O00000 0 T ZRNAP Fea4Ytu 0 O FeezYgt t T7 RAPO 0000000 Fig 4 30000 M00000 0 reeler coap 0 B B O plasm d DNA 6 uggmgmgaauguugHs3 dATPrung guug T7 RNAP O 0O O F644Y0O 1 U TT
87. Japan 000000 D EE BOE E E 8 20 CT ED PLE DE 2 3 7 DNALigation kit Ver 4 Takara Bi o Shi ga 8 8 uagamgagugugggmumgudgpampgmnmuunmnmsgusmecms 180 0 00000 000000000000 2 3 8 20010 15m 000 427cgp 90 800 p g socpp i 0 0 O 37 C0 ini 000000 3796 dad rm L 2 3 9 000000000 DNAD O D DNA 00000000 0 Gene Quant Anersham Bi osci ence Tokyo Japan I 0000000000000000 00000 000000 16 2 3 10 DNA I B D D LU U U I T7 RNAPO OOOO O PRR OO CSI D DAJ JD 0000 0 0 Big Dye Term nator Cycle sequ
88. M Lauro K Li Y Rogers R Strausberg G Sutton L Tallon T Thomas E Venter M Frazier J Venter Proc Natl Acad Sci USA 103 11240 11245 2006 90 Den 1977 5 csi HET BE EB BT REED E EE EE 0000 000 0 0 0 0PCRO 0 0 deoxyri bonucl ei c aci d tri phosphate dNTP PCR J U D D PD D D D DEC EE B BESTE BR ET p p dep BE d D HE E E BE DE EDT HE daNiPO PRR JII U PU UH Br PE gp pe SEE BE E p bp DE pp SE P HET HE EE EE CEDE LABES Bb DNA
89. M mi MN Fig 5 5 Improvement of peak uniformity by use of PPase Sequenci ng reacti ons were performed i n the absence or presence of PPase Mnus or plus indicates the absence or presence of PPase All peaks i ndi cati ng term nati on patterns with 3 dUTP are shown on the sane scale The arrows i ndi cate sites rel ati vel y sensiti ve to pyrophosphol ysi s a Peak degradati ons in sensitive sites were prevented by PPase b ban 0000Fig 5 60 0 0 TyretroincbDNA 00000000 DNAD FH D DNA0O0OCO OCO COD0ODO0OOOCSOLO O 000000 panel 5 8 HH HEU EU D B Do du ud uua uuu eut uu LJ E E 53 Fig 5 6 Conpari son of TS A w th dye terninator cycle sequenci ng B on the ABI 377 DNA sequencer 100ng of human TSH cDNA was sequenced with both methods Fig 6 Tsng 5 Table5 1 500 ng 200ng0o 0 0
90. Pyrophosphatase 0 000000 Pyrophospatase PPase 0 00000000000000 H PPase Si gm Tokyo Japan I UI PU PD UD U UU uu DU RNase B H 0000 PPase 20 mMTris HCl pH 7 9 1 mM O dialysis Buffer JU DD D UD dialysis Buffer I SP Sepharose Aners Bioscience Tokyo Japan 00000 M 0 1 MOD wedunub l 656 5 QSepharose AmershamBi osci ence Tokyo Japan 0o 000 OM 1M0 56 U 4 U ham 0 sos 0 II UD DOT BE up OE HEBES EEES DE DE EE PPase l L 32 08 20nMTris HCI pH7 9 1 mM EDTA 50 v v glycerol storage buffer U 00000 20 C O00000 45 3 2 4 TSO H 5 I HI gguumumgmuugsggd gagggi1b HIiHg gg sere 5 0000040 nMTris HCI pH8 0 8mMMgCI 5 2 mMsperni di ne HCl 5 2 GMP 250 u M ATP 500 u M GTP 250 u M CTP 500 u M UTP 0 1 H M R6G CH 3 dATP O 1u M R110 CH 3 dGTP 1 u M TMR CH 3 dUTP 5 H M ROX CH 3 dc P 0 0 0000 DNA J HU PCRO O 10 no j pn
91. Q Zhang Nature Genet 26 61 63 2000 13 R Feli S khosia Trends Genet 15 431 434 1999 14 E Heard P Clerc P Avner Ann Rev Genet 31 571 610 1997 15 H I keda J I shi kawa A Hanamoto M Shi nose H Kikuchi T Shi ba Sakaki M Hattori S Omura Nat Biotechnol 26 526 531 2003 M Chamberlin J Ring Biol Chem 248 2245 2250 1973 P Droge F M Pohl Nucleic Aci ds Res 19 5301 5306 1991 16 17 12 H 2H BH 2 1 DD 2 1 1 D000 Escherichia coli JMLO9 Ni ppon gene Tokyo Japan 00000 2 2 2 2 1 BD 1 H B D DNAD O l u uggagagmmugugagausgugggauguuuuagagdg DAU II ID amicillingg gguud L80 0 000 0 37 c0 18000000000 O0 Q Aprep Plasmd Mni kit Q AGEN Tokyo Japan 0 RNase AQ P1 Buffer 0000000000000000 000 DA J H O O RNase A DEAE p BR ET DES Ep DEBERE EE PE pe D Eb pP EH ER PEE Csci 0 0 000000000001 250 37 18 hi mac CR20
92. Structure of pTS1 nulti pl e cl oni ng sites Mul ti pl e cl oni ng si te anked by ni ni mimT3 and T7 promoters i n pTS1 vector Di recti ons of each arrow show directi ons of transcription 6 3 3 5 u gtFig 64 32 00000 0 pfS1 000 DNAD 0000 OTSOO 0O O0 5 pGEM3Zf pGEM 64 5 5 pGEM 0 0 DNA 0 0 DNA U UU UU BD 0 0 UTS U UU U UI ugagumgaagugs5auuaauauuasuauug DAU OOOO 40000000 u ugmumuaaagesseguuuuuuuuuusgb papnpuuuuuuuluIl
93. TAG CCA TGG AGG ATT GAT ATA TGA ACA CGA TTA ACA TC G CTA AG 3 O T7Rpol C 5 ATA TTT TAG CCA TGG TAT AGT GAG TCG TAT TGA 6 3 7 DNAD O O DNA 100 ng 0 0 D Ex Taq DNA pol ymerase Takara Bio Shiga Japan o 0 0 PRIIUI I HUH PTC 200 DNA Engi ne M Research CA U S A I I HHI PCRO0 00000 0O 94 C0 10000 I 0 000 0 094 C0 300 J 55 c0o 450 072 C 2 300000 00000 72 c0 5 PCRO Terni nator FS Cycle Sequencing kit ABI Prism 377 DNA sequencer Appl i ed Biosystens Tokyo Japan 0 PCRE D wcag iD HDI Gene Pure Kit Nippon Gene Tokyo 8 8 E Aca DICERE pTr c99A Amer sham Bi osci ence Tokyo Japan U UU DD DDD D D DD T7 RNAP O00000 pI7RI UD U l 4 2 2 T7 T7
94. Y Sugahara T Tanaka M Wtahi ki K Ozawa E Ohara H Funaki Y Yoneda S Matsuura M Murarmatsu Y Okazaki and Y Hayashizaki I denti fi cati on of Stabl e RNA Hai rpi ns Causi ng Band Conpressionin Transcri pti onal Sequenci ng and Thei r El i m nati on by Use of I nosi ne Tri phosphat e Gene 222 1 17 23 1998 Izawa N Sasaki M V tahiki E Ohara Y Yoneda M Muramatsu Y Okazaki and Y Hayashi zaki Recognition Sites of 3 OH Group by T7 RNA Pol ymerase and Its Application to Transcri pti onal Sequenci ng J Biol Chem 273 23 14242 14246 1998 00 N Sasaki M Izawa M VM tahiki K Ozawa T Tanaka Y Yoneda S Matsuura P Carninci M Muramatsu Y Okazaki and Y Hayashi zaki Transcri pti onal Sequenci ng A Method f or DNA Sequenci ng Usi ng RNA Pol ymerase Proc Nati Acad Sci US A 95 7 3455 3460 1998 uult N Sasaki S Nagaoka M Itoh M Izawa H Konno P Carni nci A Yoshiki M Kusakabe T Moriuchi M Murarmatsu Y Okazaki and Y Hayashizaki Characterization of Gene Expression in Mouse Blastocyst Usi ng Si ngl e Pass Sequenci ng of 3995 Clones Genormi cs 49 2 167 179 1998 00 N Sasaki M Izawa M Shi moj o K Shi bata J Aki yama M Itoh S Nagaoka P Carninci Y Okazaki T NMoriuchi M Muramatsu S V tanabe and Y Hayashi zaki A Novel Control Systemfor Pol ymerase Chai n Reacti on Usi ng a RI KEN GS384 Thermal Cycler DNA Res
95. agggopgs amweggapnadgdaugpng T7 Ho Bb ES EE p ES nd E EP BE BO RNAP HEUTE D BEC DESEE REDE d PEE EP DIC ELDER L1 SEE DESEE SED p pe p ER S p Bu B e b a CD EE EC e EE ERSTE BET RNAP 3 dNrPO NIP I ID ID DD 000000 DNTPU D DI ELTE REEL HDHH DU T7 RNAP 856 DIESE E EZ DES E EE 8 5 1 Cp DEBES E E E EE BER E EE EE RE E SURE Ses NT PD RE BEES EE EE Bp ETE LEES ET 13 3 p o ET E SEES E LESE BE b E DE E EDT TE 00000000 0 O0 e8 uescript pBS DNA 0000 00O DNA DH D Ud D U P JUrP O 0 0 00 3 3 RNAP F644Y 0 0 O0 66 T7 RNAP 204 00000000000 44 F667Y0 3 T7 5 5 3 4 vtren a utut
96. dH T7 3 dATP IO H D RNA DB O D cesset mmmmmmmmmmmmm emt 31 U r3 o 4 2 7 T7 RNAP O F644Y0 n p F667Y0 D T7 RNAPD O 3 dNTP DJ tos sns rss snn nsnsi nsnsi ns n a n ao aoa 31 4 2 8 F644Y0 0 0 O T7 000 RNABBBD jGGGj HRK III I 31 3 DOO B creme meet em m me m IK m I 32 4 3 1 T7 RNAPEDUUOLULU sU e amp R ne mmm i 32 45312 IL D H B T rZ NAP TLS E DIE E e oes pena 34 4 3 3 3 dAT P 0 0000 0 T7 RNAP O 0 O Fea4Yt FeevY D 37 4 3 4 T7 6 6 T7 RNAP 3 dNTP J JT 39 4 3 5 T7 RNAP 0 I O 64 J 1U I 40 4 MT 41 5 ut Fono 3 OR Oa OR RON O3 RO O3 2 O3 2 O3 3 O3 3 2 O3 O33 ORO 2 42 50 T MT 44 2 JI 44 5 2 1 DNA TX 44 5 2 2 ED 8998s 45 5 2 3 Pyr ophos phat aset Dlg0Bga ld ul eene 45 po Nuu pa DEDE CER DIRE
97. exonuclease i J I BB D U 0 ERE D BE EE CE DEED EE BEBE O DNA HET EL EEOBE BERE PEORES BE SUBE EE EE ERE HESSE EERE ETE CREDE ES BE RP REED HEEL BESTE 20 0 DNAQ 10 0 DNA Heg EE HODIE Bru helicase d gg mug uu ge ilg BE DEC EEADIS EE EE bp epo p ET EP E D ETE En DES BD DE uUugugsuauagusuusuuuuususausBugusuususuHuHgudysdeiuslulHllLu tl 8 GC DNA Fig cco DNAD O O O O O CpG island 5 1 1 19 1 DRIED ELE BESTE DU BE EH cyti di ne EST EST EZ 1 ona U CJ L3 Ed Ed Ed C3 O Esi ep eed pcr Es EI Ei cepere pue pem e ent dp Es ai Ee 8
98. rorPp U raseppgagggaagagagggaguuuuuuuuurcecrPppauguuguud TSI U H EL TE BEES BESEE E BUE EHE BEES TS 0 sper dine O0 0000000000000001 E HEC E O T7 RNAP D 5 2 1 0O TyrotroincDNAL PCRO J D 0 1M O O TSH F 5 ACG TTG TAA AAC GAC GGC CAG 3 PCR TI D 0 0 TSH R 5 TAA CAA TTT CAC AGG AAA CA 3 0 H 00 0 DNA I B Tyrotropi n cDNA pBluescript DNA 1 pg O 0 5 units Ex Taq DNA pol ymerase Takara Bio Shiga Japan 0000 PRR UJ UD D D D 0 0 PCRO0O0 00000 0O ps300 000 80 n in EE 0 000 0 O0 HT29 Aneri can Type Cell Collection VA U S A d d U D 0 0 DNA 100 ng 0 0 DNAQD I I 7 PCROO OD 44 P53 T7 5 GTA ATA CGA C
99. 0 uultu 1 J M Prober G L Trai nor R J Dam F W Hobbs C W Robertson R J Zagursky A J Cocuzza M A Jensen K Baunei ster Sci ence 238 336 341 1987 2 L G Lee S L Spurgeon C R Hei ner S C Benson B B Rosenbl um S M Menchen R J Graham A Constanti nescu K G Upadhya J M Cassel Nucleic Aci ds Res 25 2816 2822 1997 25 3 Z Zhu J Chao H Yu A S V ggoner Nucleic Acids Res 22 3418 3422 1994 26 LAE H DL B B DB B B BCH H 1 1 TERNAR TI 4 2 1 T RAPO 0000000000000 Uu pr H T7 RNAP EE BE TEASE d Bu isuu 1 He Hoa T7 RNAP 3 dNrPO T7 L 7 uuu DESEE GE ET E SET 3 dNTP T7 RNAP E BEBE E Hbc REOS EE ESRB DIC ERHI 3 dNTP lUDUUUBUHHUHUUUDJHBBHBH 3HULD OHI 0000 T7
100. 00 000 0 0 0 600000 0 0 DNA 000 925 DNAO0 O0 O0 90 0 0000000000000 O 14 1000 0 00 0000 0 UU LUE BE SI TEC BIS BE EIL BERE D HE PS BEBE 0 bDNA25ngp O0 500 ng0 00000000 1 5000 0000000 10 ng O 1 4000 0000 0 93 0 0000000000000000 00 OTSO 0O 00000 z Lr1rrrrrr5cr rr tr OA oOo Ez Ui o Q LJ EJ PJ E El 54 Table 5 1 Effects of template amounts on Bases Sequencing Amount of 1 101 201 301 401 501 protocol plasmid ng 100 200 300 400 500 600 Dye terminator cycle sequencing 500 99 99 100 100 94 55 200 100 99 100 99 88 100 99 99 100 8 56 50 98 96 98 B 63 45 25 5 3 9 10 nd nd nd nd nd n d Transcriptional sequencing 500 98 100 8 200 96 97 97 94 7A 100 96 9 95 6 50 96 96 097 95 95 83 25 96 92 9098 95 71 10 96 92 9 Jl 50 n d not determined sequence accuracy Fig 57 PERO 0000 DNAD O O DNA uuu TspnuggaupnnuaununaPeRDnDUDH DNAD L3 L
101. 1 1993 16 G Gao M Orlova M M Ceorgiadis W A Hendri ckson S P Goff Proc Natl Acad Sci U S 94 407 411 1997 17 M Valerie P Huberts Nucleic Aci ds Res 24 4845 4852 1996 18 P B Vander Horn M C Davis J J Cunniff C Ruan B F McArdl e S B Samos J Szasz G Hu K M Hujer S T Donke S R Brumet R B Moffett C W Fuller Biotechni ques 22 758 765 1997 19 K A Mookhti ar P S Pel uso D K Miller J J Dunn J E Coleman Biochem stry 30 6305 6313 1991 20 Y Huang F Eckstein R Padilla R Sousa Biochenistry 36 8231 8242 1997 43 H 5 TSDULUDBD 5 1 D D DI DNAD lD DU DE EDD EE DESEE L LEGES DESDE EE ES E Ee TIC DEDE TE DIE eu EE 1 51 15 O RNA EE ER SEE ET LET E I I DNAP uuulutL 1115 E DD EE BE ED Es Bb BE Ebo guanosi ne tri phosphatB 2 inosine tri phosphate ri TPO O 7 deaza guanosi ne tri phosphate c
102. 11 DNA DNAPO 0 0 00 0 0 40 0 0 aNtPO 1 4 DNA O O DNAR EC2 7 7 7 D D 0000 Fragrent U coli U UD UD U TJ U U 0 UD O0 T7 DNA pol yrerase J co itf DNA polyrerase 0 O Klenow DNAPO 0 0 H DNAPO Fig 1 30 00 000 0 0 0 DAJ J 3 HD UO U D 5 lU B EDI EAE Sp E E SE E EE SES DE BE CC 2 o 1 T o 1 SS SS o o F Z m v z i VN lt o T tu o Qum o Cum o oz E o 4 0 o o m NEN aa t SRM 0 IN T Pi a CN OH H Pyro dATP dCTP dApC phosphate Fig 1 2 Reacti on Mechani sm of DNAP Catal yzed Reacti on Template DNA Template DNA HER LOTH lI II n lI NNUS Pyro
103. 448 1991 9 R A Bambara D Uyenura T Choi J Bi ol Chem 253 413 423 1978 10 R A Ikeda A C Lin J Clarke J Biol Chem 267 2640 2649 1992 11 J F Mlligan D R Groebe G W MWtherell O C Uhl enbeck Nucleic Aci ds Res 15 8783 8798 1987 12 C T Mrtin D K Miller J E Coleman Biochem 27 3966 3974 1988 13 D Imburgio M Rong K Ma W T McAllister Biochenistry 39 10419 10430 2000 14 E P Kartal ov S R Quake Nucl ei c Aci ds Res 32 2873 2879 2004 15 G V Papatheodoridis S J Hadzi yannis Clin J Viral Hepat 8 311 321 2001 71 TSTH H TH BT HHB DAH TI H BHH U HH TH 7 1 DD csop 0o BBEIZORBEE HE HEURE EE BEER TE UE HEELARE 0000000 0O DNA I H H 2 DNAD bNAL L ecnuguuagauuuuuugbpanpupaagaagaugggggugguuguugguul
104. 80 co00o0000 n 2 2 3 DNA D l 0000 DNA QAprepPlasidMni TE 10 nMTris HCI 0 1 nM EDTA pH 8 0 0 D HD B D D n p 20 c0 DB ug 2 3 DNA N 3 1 O0 00000000 00000 1 39 1 wv 0o 0000000000 0 Agarose S N ppon Gene Tokyo Japan 0 3 0 0000000000 OD Agarose 21 Ni ppon Gene Tokyo Japan 1X TBE 50 Q M Tris 1 M EDTA 49 Mborate i H d DD D D 1 11 EAE EE EE EE E HET EE 5 EtBr l U U DO U 0 O O Advance Tokyo Japan n Advance Tokyo Japan 00 D E DESEE D EE BILE DE db ET Dp X Mupi d Advance Tokyo Japan 0 00 0 0 DNA EST E E CES 0 0 100VO0 O0 0000000000 UBDUDI l 0000 Printgraph Atto Tokyo Japang D D 14 2 3 2 Ethanol EtOH DNAOD O O0 01 200 O 3Msodi um acetate pH5 2 0 0 0000000000002000 9
105. 9 5 000000 0 0 0 80 c0 5 himac CF9RX Hi tachi Koki Tokyo 8 85 O 18 700 Xg0 150000 0 0 DNA 0000000000 EEOHI o0o0og0g0og0ogog go O 70 EtOH0O O0 O0 O HHH 18 700 Xg0 0 DMA000000 Y00000 DAT DCD O Centrifuge Concentrator VC 96 TAI TEC Sai tana Japan 0 U 0 000000 TE 0000 20 C UJ 00000 2 3 3 PCR PCRO O0 00 u PRR UU B CU 00 0 O DNA pol ymerase Takara bi o Shi ga 8 85 204l 0 0 DD DNA PCR pri mer deoxynucl eoti de tri phosphate dNTP ExTaq DNA polyrerase 0 0000000000000000 PR ggg PTC200 DNA Engi ne M Research CA USA O O SUPREC O2 Takara Bio Shi ga Japan 0p O00 0 00 00 000 20 0000000000 0 PRO0OOCCOCOO0OD 000 000000 2 3 4 00000 DD PNAD D D D TD EE Ene D E dp b s DE d Pp D B opp PP PLE 0 BNACETOBIS REEL SOT p bp DEP EL 2 3 5 pA D 0000
106. A P Bird Nucleic Aci ds Res 27 2099 2107 1999 7 G Egger G Li ang A Aparicio P A Jones Nature 429 457 463 2004 8 H Ikeda J Ishikawa A Hanamoto M Shi nose H Kikuchi T Shi ba Y Sakaki M Hattori S Onura Nature Biotech 21 526 531 2003 9 International Human Genome Sequenci ng Consortium Nature 431 931 945 2008 10 E E Eichler R A Clark X She Nature Revi ews 5 345 354 2004 11 T J Hudson Nature Genetics 33 439 440 2003 12 P Droge F M Pohl Nucleic Aci ds Res 19 5301 5306 1991 13 R Durand C Job D A Zarling M Teissere T M Jovin D Job EMBO J 2 1707 1714 1983 14 A Rich A Nordhei m A H V ng Annu Rev Biochem 53 791 846 1984 15 A Herbert A Rich J Biol Chem 271 11595 11598 1996 16 L F Li u J C V ng Proc Natl Acad Sci USA 84 7024 7027 1987 17 G P Schroth P J Chou P S Ho J Biol Chem 267 11846 11855 1992 18 S L Turner F J Jenkins Bi otechni ques 19 48 52 1995 19 W Henke K Herdel K Jung D Schnorr S A Loeni ng Nucl ei c Aci ds Res 25 3957 3958 1997 20 K H Hecker K H Roux 20 478 485 1996 21 F B Dean J R Nelson T L Gesler R S Lasken Genome Res 11 1095 1099 2001 22 J Yan J Feng S Hosono S Sand S S Sommer Bi otechni ques 37 136 143 2004 23 J I shi kawa A Yamashita Y M kam Y Hoshi no H
107. DNI 6 DpngggvionpmnmnmnauapnngagagnunagggapnpununsangunmunmnpnnpH lu ddATP daGTP j ddCTP ddTTPL HABE EE EE rini i itb CREE DEEP REBEL ip ithal Pi Ipin EH HE HELD EL TH Eb CET E 115 DES LORS 1 E 16 ddNTP ETUDES EE LESE OBL DE BP CREDE D SEE ud CET Eu Hs HE HE E DI H9 DESEE HE pe DI DE D CE DNA pr
108. E DE E S DEEST DE T UU DIS bb PES DIE EE E DEC P BOE DE PE S ESBCRE EAE HE EE B EE ELE SEE IS EDS BE LET EET I helicase IDD UD ul ugagg uaguguHhelicase 20 0 DAJ JI I U 100 DNAD J I l 65 0 TSs00 LJ rJ E 5 J J m 00000 Fig 620 EGRO 000 1900000000 PCRI l tn 190 0
109. EGFR gene coul d be transcribed f rom both ends for 5 m nutes The electrophoresis shows only T7 si de sequence of the GFRexon19 The l i nes showthe I ocati ons of del eti on in NSCLC 66 6 3 2 p s p 0 00000 5 0 0 0 DNA T7 Promoter 7 7 lD E SET ES EE E class I class U T ELE RIDERE d 30 0 T7 prowter 0 0 0000 T7 0000 00 00 0 T7 promter 000 class III T7 promter U U pr T7 promter O 000000000 I U 230 Ul 3 000 class 300000000000 class 5 TAA TAC GAC TCA CTA TAG GGG AGA 3 0O 0 170 0 5 TAA TAC GAC TCA CTA TAG GG 3 T7 promoter 00000 0 07 prowter 0 0 000000 00 T7 p
110. H u ul Table 4 10 T7 RNAPO J U J U H T7 RNAP pol yrerase L T Fes2Y 000 T7RNAP0 0000000 56 882 rNrPOOOOOO NPDOUDUBD 56 7 dap E ET HE EE EE T7 RNAP F882Y0 0 0 0000000000000000 0 polymerase D D E E rJ Ez J J OA 34 Table 4 1 Relative activity of T7 RNAP mutant Relative acti vities of RNAP Mutation Siteactivity corparing with WId type F882Y N D F782Y 0 31 F733Y 0 65 F667Y 0 56 F646Y 0 39 F644Y 0 63 F644Y F667Y 0 45 Y639F 0 12 WId type 1 00 The rel ati ve acti vi ty of each RNAP mut ant was measur ed as the i ncor porat ed rate of a P UTP The reaction was performed i n the presence of 250 H M each RNTP usi ng the plasm d DNA di
111. Hg JU gg 2g 3 H00000000000000000 2 3 6 32 oO0O 0O0 C0COC0C OCOCOCOCOC0CO0O0C0 0000000000000000 3 OH 00000000000000 T7 625 aag 684 aJ dB B d u nnunnnnnnnunn F6440 FF6460 0 O0 66 7 0000000 po lyrerase0 0 0000 0 T7 RPO 00000000000 0000000 0 F7330F782 0 0 0 882 0 0 T7RNAP Y639F0 2 coli DNA polymerase 0 3 polynerase 3 85 NTP 6 3 E EC REDE T3 SEE PE PESE ODE ER DEDE E S BE PE BTE DER EE i 000 300 H000 000 00 000 00000 00 0 T7RP0 000 3 odn n DEOR EE DIESE
112. Hitachi koki Tokyo Japan 0 0 0 4 000Xg0 1 2 5 50 1000000 0000000000010m0 Sol T SEE 1 E RE BEES 100000000100001 00000 7 5m Sol FABZHOHE HH H BREGHOH BOE HOHOBSH 6 2 0 8 4m TE 10nMTri s HCl 1 LJ EDTA 7 5 DNA j UH i H FL EO UD CsC 97 oBu unn et hi di umbrom de 5 ng m 0 84 m DESEE EET H CP 70 MX Hitachi koki Tokyo Japan 0000 40 000 X g0 13 DAI HU uui butani l I D D HJ O ethidium brom de EtBr 0 0 0 0O 0O p EBI HDD nD DU BH TEJ 0000 m000 0000 00 2000 99 5 v v ethanoi ethanol 00 000 0000000 m0000 2 2 2 00000000000 0000 37 cC0o 8 300 1 5 1 50 8094 v v U glycerol 0 0 00000 0
113. I GEI DERE BE ecard afarcinica 52433 DNA UU DD UD 0 O0 Rhodococcus eryhtropol s 0 DNA gp J J DU UD UU UU DD O Acot anasylvestris Micotiana tomentosiformis z ul U ul ul 1 n 0 D ug lenagrac lis DNA fibrillarin inn n nununununi TSTOO0O000000000000 000000001 000000 Aactuca sativa J D OD Lactuca G sco u Neurofi bromatosi s type 1 NF1 Palindromc AT ri ch repeat PATRR O 5 DO000 00 00 0000 00 C0 N10 0 0o Oo 000000000 O 5 DNAT D uggmuuuaaaagaagaagaggsgguuum
114. J JI IU U D 5 6 2 4 5 TSO 00000000 0 DNAD OO0OCOCODOOCOCOCOOODOOO 6 2 2 00 0 000000000 0cs0 000 0 0 0O OBI g Dye term nator Cycl e Sequenci ng kit ver 3 Appl i ed Bi osystens Tokyo Japan 0 BER Bed pl Tsj au gguecesumumuuuuaauuuu O O ABI Pri sm310 Genetic Analyzer 1 O O pBluescript SKO Stratagene CA U S A pGEM3Zf Pronega Tokyo Japan O0 O pTS1 DNA DNA lU CC Bop gg Bs Sp BH 6 3 D D 6 3 1 TSO0O0 000000 00000000 000
115. K Shi bata M Yoshino M Itoh Y Ishii T Arakawa A Hara Y Fukuni shi H Konno J Adachi 5 Fukuda K Aizawa M Izawa 820 Functional Annotation of a Full Length Mouse cDNA Col lection Mature 409 685 690 2001 M Iwata M Izawa N Sasaki Y Nagumo H Sasabe and Y Hayashi zaki T7 RNA Polymerase Activation and I nprovenent of the Transcri pti onal Sequenci ng by Pol yam nes Bi oorg Med Chem 8 8 2185 2194 2000 0000 I K Shibata M Itoh K Aizawa S Nagaoka N Sasaki P Carni nci H Konno J Aki yana K Nishi T Kitsunai H Tashiro M Itoh N Sum Y Ishii S Nakamura M Hazama T Nishi ne A Harada R Yamamoto H Matsumoto S Sakaguchi T Ikegam K Kashi wagi S Fuji wake K I noue Y Togawa M Izawa E Ohara M V tahiki Y Yoneda T Ishikawa K Ozawa T Tanaka S Matsuura J Kawai Y Okazaki M Miramatsu Y Inoue A Kira and Y Hayashi zaki RI KEN Integrated Sequence Analysis RISA System 384 For mat Sequenci ng Pi peli ne th 384 Milti Capillary Sequencer Genome Res 10 11 1757 1771 2000 95 ool M Itoh T Kitsunai Aki yama K Shi bata M Ilzave J Kawai Y Tomaru P Carni nci Y Shi bata Y Ozawa M Murarratsu Y Okazaki and Hayashizaki Y Automated Filtrati on Based Hi gh Throughput Plasnid Preparati on System Genome Res 9 5 463 470 1999 O N Sasaki M Izawa
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117. NA D L 7 T7 RNAPO O00 0000000200000000 T7 0 0 0 ph5o 0 0 O 0O O Chamberlin 0 0 0 O Zawedzki 0QD 00000001 000000000000000 JU DDD DUH HDUDHUDEDHDHUDHDBUD 000 D5o 00000 amicillinggg 0 OD oo ll 0 40 isopropyl B D thi ogalactoside IPTO0 000 0 4 8 000000 1000 0 20nMTris HCl pH 8 1 130 nM NaCl 2 nM EDTA Wash 0 100 0 50 mM Tris HCI pH 8 1 100 mM NaCl 0 1 mM EDTA 5 mMDIT 0 1 mMbenzani ne 30 H g m phenyl methyl sulfonyl fluori PMSF 10 u o m bacitracin j O sonication buffer O00 00000000000 0000 Sonifier 450 Branson CT U S A 0000000000000 T7 O Hapari n Sepharose Q Sepharose Arier sham Bi osci ence Tokyo Japan 0 0 0iMj g 0 64 M acip I I UU DU UU DU DU T7 55 7 RAPO OOOO O 100 kap JU DU D U DUH 29 5094w v glycerol 20 nM KH PO pH 7 7 100 nM NaCl 1 mM DTT 30 ug m D storagebuffer0p 000 160000 0
118. O OO O L 2o JHILUH LHDN HUU Fig 5 20 lanein i 4D I I D H 000000 rcr U 000 riTPO 200 u MO O 1600 Oo r3 Oo UU HU c rGrPP c rATPH 800 ETE EE Lane 5 800 l L1 L3 r3 r 600 U lane 9 c7 T7 RNa 0 0O 0O 0O O rITPI OLD c raro 00000 DNA LS DE DEBERI EE DE E BISHER BI NUM MER rA P 0000000000 m m GOG m EJ E ESL EE IE LJ t J EJ EJ COL LE uBbgBgaBmuuBugaganugugugpbpBuguusgBBuBdgdg 49 LJ 1 q J J EJ Ed EST E E CT 1 LIE EJ
119. TC ACT ATA GGG CAC CTA CCT GGA GCT GGA GC 3 00 0 PCR E B O G2 5 CCA AGA CTT AGT ACC TGA AG 3 0 2 MD JUD DDD 0 5 units Ex Taq DNA pol ymerase Takara Bio Shi ga Japan 00000 PCRO 0000 p30 000 9 Dp HB BH PCRO 000000 0 PTC 200 DNA Engi ne M Research CA uU s A0 0 0 00 00 0 PCROO0O OCOOCOCOCOCODO O 96 cC0 10 0 0 55 co 300 0 CEDE BOB 96 c0o 300 0 1 D D UD DDD 24000000000 xesy gigugg uR mgt 5 2 2 T O rIlITP Yamasa Tokyo Japan c r GTP JU DE E DTE REDE EDGE EDT 0000000 c rATPE Tol mn 0000 40 nMTris HCI pH 8 0 8 mM MgCl 5 mM DT 200 u M GMP 200 u M ATP 200 H M CTP 200 u MUTP 1 H Ci a 32p UTP 0000 Thyrotoropin O PCR 10ng0 0 0 5 0 T7 RNAP 25 units 00000 0O 37 cC0 7 5 w v Long Ranger 0 0 HUU DU BU 0 0 H O0 BAS2000 mage Anal yzer Fuji Film Tokyo Japan 0 0 0000000000000 IBI PAESE LE 5 2 3
120. TG TGG CAC CAT CTC ACA A 3 0 0 O0 PR O00000 E19 R T3 5 GGC AAT TAA CCC TCA CTA AAG GGA GAC AGC TGC CAG ACA TGA GA AA 3 000 0000 D O DNA Prornega Tokyo J apan 200 ng 0 5 units GeneTaq NT Ni ppon gene Tokyo Japan 0000 PCRT 000000000 PRI IHH PCROCOOCOOCOOCOOOODOOI PTC 200 DNA Engi ne M Research CA U S A 0000 0O PCR J 000000 94 c0 3 4c 450 0 55 lt I 4500 72 C0 3 5 6 2 2 EGFR PCR 0 0 TSO 0 0 0 BODI PY FLO BODI PY Rea BODI PY 564 5700 BODI PY 581 591 N hydroxysucci ni m dyl ester ecul ar Probe OR U S A O0O 0 003 00 00000311 000000000000 0 0 0 BOD PY FL 3 dGTPO BODI PY R6G 3 dATP BODI PY 564 570 3 duUTPO 62 BODI PY 581 591 3 0000000 0 BOD PY FL 3 dGTPO BODI PY R6G 3 dATPO BODI PY 564 570 3 dUTP BODI PY 581 591 3 dc1P0 0 0 000000 80906050000051 T7 RNAP F6G44AY 0000 1720 00 13
121. TS TIU eee eror 46 52 5 45 BL TS H BB a DNATE STI PPTP detenta rin eise 46 52 6 GEI BUDI T BE passe apana s 46 527 TS BU iu TE BOUE ED B B B B B BM H B B HB 2 Saga e e 47 3 BB D B crrrrmmmmmmmmmmmmmmmm m mmm 47 5 31 ev 47 5 3 2 ut FOR s s ns n ns ins 3 9 nn nr 2 3 3 2 2 2 2 9 9 9 9 2 3 9 39 3 3 3 3 3 3 3 3 9 3 9 3 3 2 3 3 3 3 3 3o 3o ao 3 as asas aos son ron on 50 Tet HS spp D mun pp Spade teer aun 52 5 3 4 56 ut TJI 59 5 IO 60 60 T TITO 62 2 correre mmm IRR 62 6 2 1 DNAD Q cere mAMMM MIMj 62 6 2 2 E R PRO 0 0 TSEHU HB 62 6 2 3 5 1 IJ 63 6 2 4 CSI Wie d aiii RU C sau guar i 64 3 BOB creo mme e mme mememmmmmee tm 64 608 1 a s ete ce ehh 64 6 3 2 5 1 I 67 6 33 TS D B B DLE DEG BE Poet e 68 4 rs s a rp FOR RR OR ooo FON Og og o9 93 nsnsi no gogo nono nono asso asso aono on n n 69 5 ut IT 70
122. a IDEAE DESDE ERE T D ELE EET Dp EGER TDI rITPU D H 25 units T7 RNAP 0 0 F644Y0 10 units PPese 0 O0 O0 O 37 CO 10 Sephadex G25 col um Amer sham Bi osci ences Tokyo Japan U U U D Centrifuge Concentrator VC 96 TAI TEC Saitama Japang 0 0 00000000001 RNA ABI Pri sm 377 DNA Sequencer Appl i ed Bi osystems Tokyo Japan uUuggmgagaagg uBgH loading buffer 6pl D I D LUI 000 2nu l uj ocn 3 ABI Prism377 DNA Sequencer T THEE HEAD EET Fl RNA D ulnn 20 n nuuutl 5 2 5 TS HHgguuagrnlnuIgugHste2 2iu d g erem Hu mq 0000000 cs hO O O O O De terninator FS Cycle Sequenci ng ki t Appl i ed Bi osystens Tokyo Japan 000000 0 DABI Pri sm377 DNA Sequencer O 00000000000000 00000000000000 DNAO HH J pBluescri pt SKI Stratagene CA U S A H U D 5 2 6 T7 uugumgdamuuaumutlli 40 mMTris HCI pH 8 0 8 nMMCI 5 nM DIT 200 u MGMP 250 u MATP
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124. c Natl Acad Sci farci ni ca DNA USA 101 14925 14930 FMI0152 2004 Rhodococcus Gap regi on genomic DNA Environ Mcrobiol 8 er yhtropolis 334 336 2006 Ni cot ana I nvert repeat of genom c Mol Gen Genom cs 275 syl vestris DNA 367 373 2006 Ni coti ana torentosi form 5 Eug ena gracilis Lactuca Cisco Hunan Hunan si RNA expressi on vector Repeat region at the vicinity of Fibrillarin gene Gap region of plasti ds DNA Pali ndrom c AT ri ch repeat of Neur of i bromatosi s typel gene GGC tri pl et repeat of Androgen receptor gene si RNA regi on of Kr ppe li ke f actor KLF5 gene Nucl ei c Aci ds Res 33 2781 2791 2005 Trans Res 15 205 217 2006 Hum Mut 26 332 342 2005 J Investig Dermatol Synp Proc 10 293 294 2005 J Biol Chem 280 12867 12875 2005 DTE DE REEL AE FS DT ABL RECTE o DEBE ET ABE ELE BEBE DE EE SEE BEBE UE BE 84 DNAD O0 O0 DR DESI DHH EH
125. cond Edition Cold Spring Harbor Laboratory Press 1989 2 0D000000000000 00000000000000 0 0 1993 3 M A Innis D H Gelfand J J Sninsky PCR Strategies Academ c Press 1995 4 1994 5 W M Barnes Proc Natl Acad Sci USA 15 2216 2220 1994 6 HAGSEBE BM Ig dmg dd 3000000 0000000 0 O 141990 17 EIBNAPOBBDHOBOH OH DR B B D HH B L D UD 3 1 D T7 RNAP EE THE DE ER RNA OL 3 5 5 EF EGG De OE
126. d EE E DESEE DEEST TT EDT EIE EDI EI D EE ET EDI ugugugursapbpwapnpgupnmuugugamnmuugganmnudggagbbcu 1 F W Studier Mol Biol 79 237 248 1973 2 R M Horton H D Hunt S N Ho J K Pullen L R Pease Gene 77 61 68 1989 3 M Chanmberlin J McGrath L V amp skell Nature 228 227 231 1970 4 V Zawadzki H J Gross Nucleic Acids Res 19 1948 1991 42 5 G Bonner D Patra E M Lafer R Sousa EMBO J 11 3767 3775 1992 6 S H rotsune T Takahara N Sasaki K Hirose A Yoshiki T Ohashi M Kusakabe Y Mirakani M Muyuramatsu S Watanabe K Nakao M Katsuki Y Hayashi zaki Nat Genet 10 77 83 1995 7 S Tabor C C Richardson Proc Natl Acad Sci U S A 92 6339 6343 1995 8 Y Hayashi zaki K M yai K Kato K Matsubara FEBS Lett 188 394 400 1985 9 R Sousa Y J Chung J P Rose B C V ng Nature 364 593 599 1993 10 P A Osum Davis M C de Aguilera R W V6 ody A Y V6ody Biol 226 37 45 1992 11 A Y V6ody 5 S Eaton P A Osum Davis R W V6ody Bi ocheni stry 35 144 152 1996 12 R Sousa R Padilla EMBO J 14 4609 4621 1995 13 R Sousa Trends Biochem Sci 21 186 190 1996 14 L S Beese V Derbyshire T A Steitz Sci ence 260 352 355 1993 15 L S Beese J M Friedman T A Steitz Biochem stry 32 14095 1410
127. enbeck Nucl eic Aci ds Res 15 8783 8798 1987 15 D G Blair Int J Biochem 16 747 756 1984 16 D G Blair Int J Biochem 17 23 30 1985 17 J Pelta F Livolant J L Sikorav J Biol Chem 271 5656 5662 1996 61 H ep TSH HI B BE B H BB HHH TL IH H 6 1 I 5 PRI HUU Eso EE EE CE PLE EI EE TI EE EB BITES E DIC EE SEES EE uut 5 5 5 5 EE 1 15 6 2 6 2 1 19000000 PERO 0O T7 promoter 0 T3 promoter 0 O0 H 000000000 03u MPCR J 0 E19 F T7 5 GGC TAA TAC GAC TCA C TA TAG GGA GAC A
128. encing kit ABI Prism Taq dye term nator cycle sequencing FS kit Applied Biosystens Tokyo Japan I 00000000 0000000000000 ABI Pri sm377 DNA sequencer Appl i ed Bi osystens Tokyo Japan U UU UD DD D D DABI Prism377 DNA Sequencer 0 O0 00000000000 loading buffer ABI Prism 310 Genetic Anal yzer Applied Bi osystens Tokyo ui U PD iD U j O0 O0 O0 O Tenplate Suppression Reagent Appl i ed Biosystens Tokyo 84 85 DNA BIG BRE DEBE EO Prism377 DNA sequencer j Hn Bn B H1 1 B D UL Prism377 DNA sequencer HI EEH DT por m gs Wd ugs gg d ggg Prism 310 Genetic Analyzer 00000000 000 u POPe Appl i ed Bi osystens Tokyo apan E E Dres pe Do E E EE E 10 RESPECTE V EE SEL BEBE E REC E EE EP ERES BL BT 6 uut 1 T Maniatis E F Fritsch J Sambrook Molecular Cloning A Laboratory Manual Se
129. gested by Pvull as the tenpl ate ND indi cates that no activity was detected Total reaction aliquots were spotted onto DE81 paper and after washi ng the retai ned radi oacti vi ty of DE81 paper was counted 4 20 T7 RAPO 000000 reeler coap 1 HL HH B plasnid DNA go HUE HE EEGEEEBSET E EDDY e DEO E ET DE E DESDE DEEP DE E CBE 4 200 0000 000 rz T7 6 7 F646Y 639 11 BE BE BEES DIE 7 RNAP O 0 O0 88 7 F882Y0 T7 RNAP 345 HE S EEOBEZ BS PRSE ETE EA EE EP ES EP BEER BI ETE 79 35 Ig q Uva Fig 4 2 Processivity of T7 RNAP mutants The assay method used in this study was descri bed previ ousl y The processi vi ty of each RNAP was measur ed by the incorporati on of a P UTP usi ng a circular DNA as the tenplate In this reaction mi xture 10 H mol of cl osed circular reel er cDNA cl one wa
130. gogo go DNA paggagnsguagggaagadad rig 53 TS 50 ungugagaasgaggagagagagagdadg 0000Fig 5 400000000 53 800000000 PCRO Pane DNAD PCRO Panel BD O O 0000 5 4 Panel A SUUDBUBBDUBBUHBHUUBHUHUHHBHDUHBUDUH 10 2 O00 30 0 0 0000000000 200 HE RETE 4 ACUAA 5 O0 cco l lI I lI I O O H U O GC GAGGUAW GO O O O Fig 5 4 Pane BI H H H 00 CAC I H U ll UU DUH DUH HBH cuch II H 0O O O Gon I 0 H riTP I I U l Ho EE ED E DESI DESEE E DEEP DICERE DUE DICE BE b BE DE EE Ts IE 54
131. mnge pg T7 RNP j Fig 1 70000 DB dU RNA polyrrerase RNAP EC2 7 7 6680 H DB DAJ IJ DU UU Ruanmuuutuu U uu HI TH BDgIHuSgggHuddu u pol Template DNA TM RNA ien OH OH OH a PPP p PPP P CTP Fi g invitro D uH d D uuu 0o00 nucl eot i de RNA Product 1 7 RNAP Reacti on U RNA G G G OH RNAP OH oH oH oH oH phi OH OH ppp np Pyro phosphate T7 RNAP 0 0 0 0 882 00 000 00 RAT ID DU UL yrerasel 0D 0000000000000000 O0 D O 100 KDa 0 0 ETE DE D DE E ERR EHE BIS E EE UE URDU SEE ET EET DEBERE ETE TS T7 T7 230 RNAP 10 DNA DN polymerase Reaction RNA polymerase Fi g Reac
132. nnnguggangunapmnagugnmnR HH HE gd ELSE SHE HE HE OECD 6 6 56 55 52 LJ rJ EJ J J OA e B t re E oononononon o J J O cti uupnpnauupPeenpna uuaguanauagagnauagganaggnagunnuut aTTTancaa aT Tgcaa TTaTaTcacTaTTa a PPase H NI da PEERI E H Hr Wo Pone qus oM ME lcd Ww J Ut b PPase t Hi hn ie f Jil Hip Wo
133. nunnHnn L Lykanov p 7 7 preproparathyroid T70 I concatemr junction CJ B HH B D DD DNA CI HH BB SH dd dg g mogeoog mou iu mNT D g m ED pop Dr m REEL DI LESE 0 000 00 000 000 000 7 RNP 000 8 4 5 4 5 5 polynerase BEBE EE DESEE ES Ho RNA JI UU JUD UD DD CSJ U j 7egDNAP DNA 0000 000O0 O0 O0 O T7 RNA 8 7 gbpwPeg 60 00 0 0 000 T7 u 240 0 0 0 00 40000 0000 000mr00000000000001 coli RNAP co i 4000 0 n ni 500 0 0 00000000 T7 Ra g0p00 00000000000001 0 O7aq DNAPO 7 5 eeuuumuuug bpwanmnnnrntlbiI EE
134. oduct with f luorescent labeled ddNTP l Sample loading Minus charge Computer with base call software Electrophoresis Sequence chromatogram CCD element or Photo multiplier tube Laser irradiation Fluorescence Detect ion f of fluorescence Base calling Sequence result Plus charge Fig 1 6 Mechani sm of Automated DNA Sequencer 1 3 csgogo0oo00 csumngugupapgumumugaumg8mguugumRmuuuusnamumuuuumgmul DNA DNAD l UD IIUDTDIDHDEHBDHDHDJBHBH H TH BE AREE BEES REGE IEEE DUHBHBJUHUBHDUDUDUHN Ho BE EI DE GRE DNA SEE SEED HEATER O O DNA uUuggpeanmgamgugggggggguggggeegagugaaggaggEgag
135. ol ynerase 0 DD HER 40000 0 6 carboxy tetramethyl rhodanine TMROO 6 carboxy X rhodam n 5 car boxy rhodani ne6G 5 car boxy 91 rhodam ne110 R110 3 dUTP 3 dCTP 3 dATP j3 dGTP I C1 HH D I TMR 3 dUTP Dye U ROX 3 dCTP Dye R6G 3 dATP Dye A R110 3 7 DLE DEDE EE HET DE EAE 0 0O T7 n vitro U D 00000000000 0 0 O0 Dye u0 O0 0 6 5 00000 00 0 0 0 Dye A0 O0 O0 ecponmaamuagmmuamltI 7 RAPI ID D D I uggagmguuggaauuuuuuus awP5 5pgamgaggguagdaadut 000 0 O0 CH 0O O CH O0 O0 CH 0 00000 0 0 0O Dye A n4 Dye Cn A0 0 00 000000000000000 0000000401 Da
136. romter H HH Hgt a B segs class III DU ugguuuu mu u sauuuullutu t 00000000L T7 6 T7 promter O promoter T7 promoter MSIJU U D ass m 3 D E B ERN E DE D ul pBS 0 DNA Aetl 5 GcG GCC G3 nH 000 000i 0000 NAQD I I i J i T7 RNAP 0O O0 O0 RNA a B RW BBB B B BB 8 B B i B Fig EL Msi 63 7 67 T3 promoter Hind VII BssH II SacI PstI Nhe I BamHI Stu 5 ACGCCAAGCTGTGCGCGCAAATTAACCCTCACTAAAGGGAGAGAGCTCCTGCAGGCTAGCTTGCGCAAGGATCCTAGGCCTGAAGCTTGT T7 promoter linc Il Eco Smal Bgl s GTCGACGAATTCACCCGGGAAGATCTTGCTTACGTACGCGTGGTACCATGCATTCTCCCTATAGTGAGTCGTATTATGCGCGCAATTCACTG 3 Fig 6 3
137. s used as the tenplate The fi nal product was subjected to electrphoresis on 8 polyacrylamide gel cont ai ni ng 6 Mur ea and autoradi ographed ane 1 WId Type lane 2 F644Y lane 3 F667Y lane 4 Y639F lane 5 F646Y lane 6 F733Y lane 7 F782Y lane 8 F644Y F667Y lane 9 F882Y Table4 20 pBluescript 00 000 211 l DNAD O O O T7RNAPO 0000 0 T7 RNAPO 3 dATPE H D 00000 S sd ATP RNA pol yrerase 0 0 0 0000000000000000 D RNA O00000 0 0000000 3 dATPI H polynmeraser T7 3 uuu 7 T7 RNAPO O O Fea4Yt O O F667Y 36 L 000 T7 3 T7 F644Y0 T7 O 20A 00000000 I T7 RNAP O O0 F644Y0 F667Y NIPO 3 NTP 000000 T7 RNAP 0 0
138. tion p DN polymerase Primer DNA product Z Single stranded DNA region The direction of reaction process RNA product goon RN polymerase ogona p Double stranded DNA region The direction of reaction process 1 8 Conparison of DNAP Reaction with DNAP Reaction T7 RNAP pol yrerase 0 0 O DNAP O polymerase i UU B B B D D Fig 1 8000 0 T7 RNAPO J I I L helicase d UD UL 000000 DNAD 10 0 37 bNAT RNAP RNAL D D DI DNAQ T7 RNAPO DNA 10 0 00000 CCI o H O d GC s DNAD O0 0 0 z0 00000 DNA O O RNAL I OD 0000000000000000 000 0 Template DNA Template DNA Template DNA
139. ugcsuiuuuzd DAI JU UJU H VL SEES DNI Androgen receptor 00 000000000000 GCI DU HD uui ugangususuaaguuumuumssucecuuuunmuuBult pNA O l DAU JD DU ETE E LEEES D E EECDU BD DE P E BT p RD E EE DIESE RD E ER DR d BESIDE EDT unun S 000000 BUDE HERE B HH 83 O p C P0000000 X00 Fragile X syndrore ynuguugagagagmgamugusnauauagunungamuuil Fragile X mental retardati on 1 FM P 1 0 000 5 00000 CGGU t 6 9 FR 1 000000000000000 Fragile X DO 0OC0OO0OO0O 0O O Fragile Xsyndrowel 00000 uggumumumgecenuuug 5 Table 7 1 DNA sequence anal yses by TS method for diffi cult to sequence tenpl at e DNA origin The type of difficult Ref erence to sequence tenpl ate Aocar di Gap region of genonic Pro
140. uul Dye G RNA LI Dye C Dye A 0 Dye C pex p TM UUUDUI 511 1 Dye A n Dye U n 24 Dye C n 4 Dye A 4 k sas ud mf aee zu Soong EX El El Sl Eo rr ge r3 c TR ESups EC ELE E ESPIET EE EI 000 Dye u0 JHI lil nei Dye Q n 4 23 E74 449 56 TES 9 19 m v ET TE Fig 3 3 Effects of linker length on the termnation patterns wth R6G 3 dATP CH and CH linker for R6G 3 dATP were tested Each lane had R6G CH 3 dATP and R6G CH 3 dATP l ane 1 5 and 6 10 respecti vel y R6G 3 dATP concentrations in lanes 1 and 6 100 H M lanes 2 and 7 50 H M lanes 3 and 8 25 u M lanes 4 and 9 10 u M lanes 5 and 10 5 H M 3 4 nf BI LAE FS ELTE EGET E EE AE DE E BE DT ERE CDI
141. yrotoropiincDNAL 0 PCR J D 10 ng 325 5 T7 RNAP 0 O O F644Y n J l E T7 RNAP O0 HUU 0 0 37 1 Sephadex G50 um Amersham Bi osci ences Tokyo Japan o 0 UU DDD D D D J J RNA J O O Centrifuge Concentrator VC 98 TAI TEC Sai tana Japan 00000000000 31 ABI Prism 377 DNA Sequencer Appl ied Bi osystens Tokyo Japan 0000000000000 loeadi ngbufferegp I D D l 0000 2 90 C0 3 ABI Pri sm377 DNA Sequencer Q jin unn _udnunpaunnpaununnpaununpunuHninpunpnn_nununnumD upnpnuRann 4u ng 20 C rtt 4 3 HD Hd D 4 3 1 T7 Fig 4 1 Ho HPBTHBBIdmuggy ugar RAP I JUD D DU NIP ugmgmuadgs ounugagguuugBguuuuRePenpupgagagguguul EE EROR Eb PED EE p EB EE E EE BE EP RB INI 3 7

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