DCode ユニバーサルミューテーション検出システム 操作取扱説明書
Contents
1. DCode LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI U U U LL TU U U U 170 9080 170 9104 LU U U 0 L DGGE CDGE TTGE SSCP Ver 2 2 U U U UUUUUUUUUUUHUU Ee oi dan 1 ille O O O 2 TCO Id EKO EE LA an EE ea 3 30 DGGE Denaturing Gradient Gel Electrophoresis 7 4 DGGE Perpendicular Gradient Geli OD l l 9 SH DGGE s Parallel Gradient Gell El gt saa i ioci CiO eo cen D ch Ev a Ce Dose cud Ee cc S Coa us 17 6 J EDGE constant Denaturane Sell TJ cra 20 70 TTGE Temporal Temperature Gradient Gel Electrophoresis T 22 Sa TEGE HAM EMM weete en eeen nnen 23 NICE lin 24 10 SSCP Single Strand Conformational Polymorphis ms 26 EJ Cpu pipu O O EN 27 LZ EEE EEE EL EL DSGECDGE TGE SS GP E LE ar 27 CIR 30 Wei alan 31 Gil 2 1 6 N B JOD U U U U LI 1 0000 DCode 0000000800080 000000000000 0000000000000 D D 0 0 0 DU U n UUUUUUUUUUUUUUUUMUUU UUUUUUDDUDUUUUUUUDDDUUUUDUDU DC HK DE D ELF DEL EEF EIEH EL DEL EB HIT E R IRH E HAD ee pin TH nn AA A BBA nan h nn an Mn E TL ST SD nun UUUUUUUUUUL O 0000 oovpc 00000 6000 U U D 0 0 D 0 D 0 0 D 0 0 D 0 D U D D ACT D D 0 D 0 D D 0 0 D 0 D 0 0 D 0 0 D 0 D 0 0 DC LD EE hiii i RST SH A EO i in ST RTS ESE E IB NAH LETTER Ede de E EI EE HEED EDIB EE EA yup BH nhai UnIUIUDDuUuUuDDUUUDUuuUuDDUguUDunuUunDD2. 0 D D D 0 U But ada SHER upi RES DST H De EN EEE EEE EEF EB EDEN PE PF cd ed FE ED ETE EE BYDH MI D D D 0 0 0 0 0 0 D D D 0 U D 0 D D D 0 LD LOWDENST 0000000 00000 TOP FILLINGI 81 I UUUUUUUUUUUUUUUUUUUUUYUOUUUUUUUUUUUUUUU 191 M P000000000000 7000000000 00000000 000000 M00000000 Ee Ep EE aaa 00 10 00 U U D D 0 D 0 UD D 0 0 D 0 0 D D 0 D 0 0 D 0 D 0 U 0 D 0 D 0 U D 0 TOP FILLING D D D D D U 0 U U D D 0 0 0 0 0 D D D D D 0 0 0 0 0 0 D D D D 0 0 0 0 I lI D Fig 54 Fig 5 4 200000000000000000 2 D D D 0 0 gaga D D D D 0 0 0 0 D D D D 0 D UD 0 0 0 0 0 0 0 0 D D D 0 0 0 0 L 22 0e D 0 D UD l 6 Constant Denaturant Gell JnIUIDNUIUIUNDUUNUUIUUUDUUUUDUUUDDUUDDUDUUDUUUUUUUD T 770 1x TAE 0 0 D D DCode 0 0 0 D D Fill D Runleve line D D D D D 0 0 0 D D U L DCoOde D D 0 0 0 0 D D 0 0 0 0 D D D D 0 0 0 D D D 0 0 0 0 D D D 0 0 0 0 D D D 0 0 0 0 eq U U U 0 DU D D D 0 UD 0 D D D 0 0 0 0 0 D D 0 0 0 0 D D D D U RR U 0 0 D D D D D D I I I D 200 C mt DO O DO ODO ODO OD RHS TO T STS O EO UO TEC U D U D 0 0 0 D HD 150 0 D D D D 0 0 0 D D 0 0 D D 0 0 0 D 0 D D D D 0 0 0 D D D 0 0 D 0 0 0 D 0 0 0 D U HT EMO LED lak Kd JAN dade fogao AAA ARO TEE ARE EED EDE Ia 210000000000 00000000000 ET aa ARIA aT ST EET EET EE EI B n ET EL EE EDD hi oo EE SH DEEP EI RR E EEE AC AEDES nM AB El EEE EA JED AE VEE 00000000000 EB BEDD DEB U U D D 0 0 0 0 L Figel Fig 6 1 300000000000 00000000000 HBD SN E
3. 0 D U Ram Rae D D D D U U 0 RR D IU Il 0 D DU 0 0 0 LU U D D D 0 D 0 0 D D D 0 D D D D D D 0 D D D 0 TTGE O U D Ram Rae 00000000000 BL A DO DO O DO ER UR Ia T Buih ht SSCPH I LD D D D D D 0 0 0 D D 0 0 UD UD D 0 D D 0 0 0 1015 0 U tutu 2000000000000 0000 000000000000 sSCP D D DO D 0 0 0 0 0 D 0 D D 0 0 D 0 D 0 0 0 D 0 0 0 0 D D 0 D 0 0 D 0 0 D 0 0 D 0 0 U L E E Se AE EE EE 1 T 31 00 DU DDD 0 D 0 D 0 D U D SSCPII D D O 0 D 0 D 0 D 0 D 0 D U D 0 D 0 U 0 0 0 I 0 1000 0 U 3000 D D D D D 0 l HER DST ST HS EU E TEU UH SDSU H ENE aT A OL iB ph BE BD 04000181000 0000 00 004000009000 8 08 EL BE ELE EDGE PR E ET ELE EE IB BLE Pi PED 1400000000 L UUUUUUUUUUUUUUUUUUUUMUU QUO 2 0100000000000 DDD 0000000 JnUUDDIDDUDUUUNUUUDDUD 3 00000U00000000000000 DCode lid stand 00000 w ODE EE EEE ED EDE Ed TRE hhi ED EE EE UD gt UUUUUUUUUUUUUUUUUUUHUU UUUDUUUUUUUUUUUUUUUUUU PT hp EA EE U000000000 Fig 14 1 Fig 14 1 e pnuguupbuuuiuuttubiuttltubtultLt 72 IUUUUIUUUUIUUDUUUUUUUDUDUU EDE EEE 8 ODUUUUUUUUUUUUUUUUUUUUU UD D D Fig 142 2 0000000000000000000000 NUUUUUUUUUUUUUUUUUL 10 DODO DO 0 0 DD OD DO D D 0 0 000 Aaa EE EA AC NL IL 0 D 0 00 0 D 0 D 0 0 0 U Fig 14 2 1500000000 DGGE CDGE TTGE U 0 I ELE aaa aaa 2 EtBr U 50010000000 10mg m EBri 0 Z DO 0 SS 00000 L 3 0000000404529 D 0 0 0 0 U A AN BAN OUE DD OOO HHT OOOO LB Bi I BHD L qilib n SHREE SSCP
4. D 0 0 0 0 0 0 00000000000 BANA E WA DNA BADAN NG EA AA AA ANA D NANANG RAH lUIUDIUUUDUDDUDUDUUDUDUUWDOU ED EE Ea RH DH 040080000 Fig 414 ZD D 0 D 0 0 D 0 D 0 D 0 D 0 0 BD phu ha NEE ED NT ARE pipa puna uu ee L U U U U ITH IT A DDD AA EH H H H H UI Q04 5000000000000000 uuuuuuuuw GEE EE DUDU M475 DUDUDUDUDUDUD 0 75mm Bul 75x10cm 16x10cm 16x16cm 7 5x10cm 16x10cm 16x16cm 7 5x10cm 16x10cm 16x16cm nota BED EF E EEE EL HEDD itu paq ph hhi NOUUUUUUUUUU ELE SH ET EE ARA NE RNA NANA M hhi mid i uii ICE EP SIE 9 ECH EB ETT lI ARR t i aO FEE DU EEE BERKA 3 5 6 5 9 5 5ml 8 ml 1 1 ml Volume adjustment screw Lever Volume setting indicator VOLUME 10 ML SYRINGE Syringe holder screw Syringe sleeve Syringe AN Luer fitting E 7 a icc 0 e amp o Syringe holder Tubing Y Fitting PAN lt WA Plunger cap Plunger cap screw A Cam d DELIVER CT MODEL 475 GRADIENT DELIVERY STEM ER THIS SIDE FOR HIGH DENSITY SOLUTION BOTTOM FILLING LOW DENSITY SOLUTION TOP FILLING 22 DD D U D U D D 15 5 gm 0 00 21 0 UD UD D D D 0 0 0 0 0 0 D 0 U DODODO HS T aT Da U U 0 0 0 0 0 0 D D D D D D D D 0 UI 0 High 0 0 Low D D 0 U DU DADO DO BD ODO TT TTT TT HITS LB Ee DU UUUUUUUUUUUUUUUUUUUUUUUUUUUU LEFEL EEF EF ELE EFE ELEIELEFEL EL EREN SEED GE BEBE BE BET ELE BEE NA NAHAN ARA e
5. D D D D D D 0 D 0 0 0 0 ut DCode TRB TSS Ecco Li Ja kade EEA 99H NEE I El U U U 00 AU ZC I I UU 0 TT 000000 3egqo000000000 e DUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUL 0000000 RampRate 22 hr i 0 D D D D D 0 0 0 0 0 0 D D D D 0 0 0 0 0 0 D H 7 UU UD U 0 DD 0 000 D 0 0 DD UD UD D D 0 0 D 0 U 0 0 D D 0 0 D D Sa TT III ELS EE EEE EEE BEE ia ihhh Han EE 11 SSCPU IU D L U U UD UD 0 D D 0 0 0 0 0 D D D TTGE D D 0 0 D D 0 0 0 0 0 0 D D D 0 UD LL PIS 120 U U UU UT DBGECDGETIGESSCPIT eh AN ANAN E E A ANAN L 00000000000000000000uo0ooo0oo0o00oo0oo0oo0o0000000000000 0000000000000000000000000000000000000000000000000 00000000 2 lDUUIUIUUUDDUUUUUDDUUUUUUDUDUUUUa 0000000000 0000000 IA iq AA O OG AAA DIETE EISE So EI EB EEE 3 IIUIUDDDDDDDDDUDUUUUUUUDUUUUDDDDDDDUDUUUUUUUDUguuut DD BBB hhi hh ht gd ud gu puli miB Lp B HID HHE DSS Tin EI U U D Locating pn D D D D 0 0 0 D D 0 0 0 0 0 D D 0 0 0 D D 0 0 0 0 D D D D 0 KA D 0 0 0 0 D U Pa B tE pi thi DR Ee iu i Ll phi aaa E A RR 0 000 B EED DEED NANA UE JH ED EDA EM ICE Ell EE EMO EL LEV a Ad EV BAN EVO NN U U D D D 0 0 0 0 0 0 DD D 0 D D D 0 0 D D 0 0 0 0 0 0 D D D 0 D D D 0 0 D D 0 Fa 12Z n 0 0 D U YHS aT ish Tp TE A E ST STS LY BB hiii iii Pip B 0D netchi B hiii HSR DS Fig 12 1 SDU DD 0 DUD D 0 D 0 D 0 D D 0 D D D D 0 0 D D D 0 0 0 LA D 0 0 0 D 0 D 0 U Bin 535 NN HES E E Th Th THB EP E BRE BIETER B gaga go EERE Cl EEE AE E E P HSP DR Ea EE EL N
6. EA iH Fig 4 21 Fig 4 22 39 600000000000 1m00000THtrdq000000000000 0000008000000 5 DGGE Parallel Gradient Gell EL PIB hi hili WEE A BE EEE AE EE Ph ii T A 1x TAE D D D D DCode 0 0 D D D Fill D Run level line 0 D D D D D 0 U U D U 0 L DCode O O 010000000000000000000000000000000000OOOQQ000O 60 9 D 0000000000000 TT Td TO 0000000000000 D 09 RR 0 0 0 0 U U U U U D 200 qI 0 0 D D D 0 0 gaga D D D D 0 0 0 0 0 D D 0 0 0 0 D D D D 0 l 1004000014 1500000000000 000000000000 000000000000 DI D B UOC Td TT TT DD OD DO BOO ODUOLLL LL A BE AA ARANA 210000000 000000 00000000 phi hua oa 51 9551 RIO E NEE EEF PH EA ew EE DIG GD EO EE ESSE E E EE E AA WA HAH DODO D hu ph DO DO D RA RA Lit i U U00000000000 Fig S 1 Fig 5 1 ETEN ITL ITL ITL TL TI TITI PTI D T AE ENE ITB 91 EEE EEE AN AA E AAN CG B Bi A naaa Fig 5 2 4000000000000000 DCodel EE EM Tit B h U000000000000000 Fig S 3 AN EEE nn na a a EE ELE AV EE ERE in T T 910 E ELE RN EEE 91 ETE BEER OE Bi hin Ep B i EE EHO EIEH IH EE EF ED E BIET SET PED IEEE EEE JNDUDHOUDUDUUUDUDUIIUDUUDUNUUUUDUDUUDUNUUUIUODUDUUUDUDUDUD TREST HSE TSH a EE ii iii TYS SH narani iii D Qa Eh 0d DD OD ODO UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUHULU BADHA EI RH DEE phu aaa aaa TD BER PE E EE A OPE Drip ENER 11000101100004110000100000000000 0 E B EE EAA PB RER EIGER ENE E E EERE BIS thi EE iB EEE EEE naaa S So ata ao EEE EEE E e A E
7. J U U U L TID TDBB OOO DD DDD ODO L iB Y HSS ST HDD RT RTD TTT NANG HYD mg B i B D U BRID D 0 0 0 D 0 0 0 0 D D E0 D 0 0 D 0 EGET 2 Radiant red stan 7000000 000000 1703122 SYBR Greenll Molecular Probes Inc U U 0 0 UD D D D D 0 0 0 0 D D D 0 D D 0 D 0 aT 0 0 D D D U ai 00000000000000 8M 0 0 0 D 0 0 D 0 D 0 0 D 0 D D 0 0 0 D D 00 0 D D 0 0 0 0 D UD 4 UW D 0 D D 0 0 0 0 D TT 0 0 D D 0 0 0 0 D D D 0 0 0 D 0 0 0 0 0 U D 0 0 0 D 0 0 L BRIF DEE B lm Ui n YI BD E JED LD TID EEE puri B hip EE at E E ST il LOI 00000000000 l ii GEER EERE EI NENDE PED EF BH An EEE ANO EME EF HOUUUUUUUY L U U U U U UU 00 U L L U LU 00000 HOU L U U U UD U 0 U t JUIUIHIUDUDUHIUUUDUDU UUUUUUUUUUUUUUUUUUUU IA Aa U U U QD 0 D 0 0 D 0 0 D 0 0 U JUIUIuUIHUDUUUUHDDOL SIII STS TSTS A DANA E ANGE ANA DA BA HHDH RHIW FEDD ED EE ED ED BAHAN ET B PPD i Bl UOUUUU DGGE L U U EVER HPH Ee h Ph EI EE EE i Tipu U U U D LD 0 0 0 0 0 D 0 L Moa NAN 10000000000 EE ELTE EFEL EF 0002000000 L U U U U DU 0 DO 0 DU U U U 0 U Uu Uu UU UU UU UU H ww g i L U U U 0 U HH E rB ii Bi LEET H UU H D DD H HBD D HD UUUUUUUUUUUUUUUUUUUU UU J U U DUD U 0 U U DU U U U EEA HDHH u HHH HD ESET ENE D BHL HI DI L1 DI DI DI L1 DI DI L1 DI DI DI Co DI C4 DI Co DI DI DI FAXU 0 M HD ITT FAXO OO HU ITT UI M3119 9905
8. U Run level lne II D D D D D 0 0 0 0 0 D D DCode I 0 0 0 D D 0 D 0 0 0 0 0 0 D D D D U U U D 0 D D 0 0 OD DD 0 0 D D 0 0 0 0 0 0 0 0 0 0 0 TT 0 0 0 0 0 0 0 0 0 D 0 U U 6 0 0 utu U U U GR D 0 0 0 D D D D D 0 0 0 U 20 07 D 0 0 D 0 0 0 0 0 D D D 0 0000000000000 HOU UDDUUUY 1500000000000 0000000000000 00000000000 0000000 A A A AA an nn a EL EE EOL AA EE OC 21 UUUUUUUUUUUUUU AAA gaga ERE BADAN A DADA HAHAHA HAHAHA NA AAN U O E E E E E E E E TR E e E E DD E E EEE E B E B EF c a EEE IA DCH A U U U U U U 0 U 0 U 0 0 U U EBRE 95H ad NENTEN AKAN ANAA NG Fig 4 1 Fig 4 1 AGN pa OENE Sl EE Al AE BE DANG HL Aa EEE A ANO nD SIR m T n AE EHH YH HR a fnn EEE aa Fig 4 2 Fig 4 2 4000000000000000 DCode Hl hh BL En a ann B aa EU EEE EEE 0000000000 000000000000 UUUUUUUUUUUUUUUUUUHUUU NAPA DADAN DARANE AA NA DAH ANA NANA e E NADA RAN LIB ahi EERE AA aaa EEE EE EEEN aA ED EEEN EK NOUUUUUUUUUUUUUUUUUUHUU UUUUUUUL U U U 0 0 D 000 DDD 0 0004 500000 000000 Fig43 ENE BEEN RYD E0 GE Ip itu Ha kta Bia rh up i iqu Aaa Aroa a EO EAV RRE BEER D OD UL 1 Wem U D HL D 0 D D UO UO DO D 0 D 0 00000 Q000000000002 000000000 Q000000000000d00002000 EROE DAHA BEBE HE BT i NANA Q00000000000 Fig44 ef EE EEE ARA E EE EEE P i H EED EEE OE HT 6919 rd UUUUUUUHUUUUULU 0000 Fig43 Fig 4 3 Fig 4 4 Fig 4 5 90080000000 0000000000000 A EEDE EE EI BH ESD ST BB BIER
9. atada e a aaa s U0000000000000 Fig415 3100000000000 00 D D 0 Sam 0 D 0 U UD 0 0 D 0 0 0 0 U U U D D 0 0 D 0 0 0 0 D 0 D D D D D D D O 0 0 Fig A 16 Fig 4 15 Fig 4 16 AIIDIUUHUDDUIUUUDADDUUUUDDUUgDYDUUDDUUUUDDUUUOUDDUDUUUUD HOR OAN O o A A DUDU DU 0 0000000000000 DUD 0 0 0 0 0 0 0 0 0 0 ga D 000000191 0000000 00009 vvI l TEMED 009 0 WW IU D DO D D D D 0 I CI i D D I O O Dye Solution 0 0 U D D 0 D 0 0 0 D D D D D D D D 0 0 0 0 0 0 D D D D U 0 emi 0 0 0 1004 D DD D D D D L 281000000000 0000080000000 000000 000000000 000000000000 HOUUUUUUOUUO 4 ET le EEA E E E E E E El ESET BI LE EEE EEE EE Del 2000000000 000000000 00000 0090000 HIGH DENSITY 70000000 U OO D U BOTTOM FILLING 0000000 DD DH EF DUE UE DEE ED e Hai pi EE AD EI ED ET NUUUUUUUUUUUUL U U U U 0 UD 0 0 UD 0 UD U 0 UD U UD U U 00 U U THER NO ia EE hah Phi i 0000000000 FigA417 Fig 4 17 2000000 00000000000 000000 000000000 LOW DENSITY 000000000 BOTTOM FILLING Fig 4 18 Fig 4 18 3000000000000 000000 DO doo Ya 00000000000 LU U U U Fig 4 19 30 O0O0D0 000000000000 000000 EL EB CET ED MH OH tada ELD NE ELE ED ahi ERES BE E U O Ll bottom filling 00000000000 END EERE Bee ee EEE Eyle a ENEN AAA Ee sz U U UD UD D D D D D 0 0 0 0 D D D 0 0 D 0 LUL DADO DO DD ODO DO Bh T B I U DD 430000000000 Fig 421 33 Titrod 000000000000 00 Fig4 22 4D0D0000000000000 0000000 cana E
10. de go ED ga ope fode EY JE NA E gago ES ETE EEE ED EEDD EEE EEE EE A EED EE E El U U U U U U U DU BED 0 0 0 0 D BI D loading lid D D 0 0 0 U0 D 0 D D 0 0 0 UO C OE D CE D H A DUDU DU 0 0 0 0 DD D UD U D UD D D D D 0 0 0 0 0 D D MI 0 0 157 D D D 0 0 L 5 UU DUD DU U 00 U U 0 RP U 0 D D U U U U U ramprae DDU 0 D D D D D D U 0 0 0 0 0 U U ramprate 5 10 I Iii 0 0 D D U D 0 0 0 0 0 GRAND D D D L Heteroduplex CSGE PITT L UD U U DU BB U D U D E CE D U U D U U LU D U 0 U 2 UU DUDU D D D D D D 0 0 0 0 O DCode lid stand 0 0 D 0 D D 0 0 0 0 0 DD D D D D D 0 D 0 0 D D D De AE PE ED RD HES Er AEEA aT BR ELE ELE EL NE hip hi Ea EEP A A E DEDEN EDE BE E 8 20 00000 L 00 loading lid 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 U HB EE EEEE 2 000000000600 O sequencing loadingtpUU0OO0OO0O0U0000000000000000000 cantan ateo DD FYD 3 000 loading lid D D D 0 D 0 0 0 D 0 D D 00008000000 8 200 0000000000000000000000000000000 Power Paco 000300 0000000 13530000000 ODGGE CDGE D 0000000000000 00000 D 0 0 0 TT 0 0 U 0 UD U D D D 10451000L L L an EE Ta VEE CFE 00 EE EE D EU EC DEDE DE B DH H U U UD U U BM D 0 0 0 0 D 0 0 0 0 0 0 D D D D D 0 0 0 0 BMI D 0 D 0 0 0 D 0 0 0 0 0 D D 0 U TTGE OO D D D D 0 0 0 0 0 D D 0 0 0 0 11 1000 300 0 0 30010000000 U U U 0 D OMI D 0 0 D D D 0 U U U U TTGED I I II PMI D D 0 0 0 D D TT 0 UD D D 0 0 0 D D D 0 0 000000000000 UU 4UU000000000 0 RRO D D D
11. 0 0 D D D 0 0 D D D LI peralef 00000000 CH PH e e E E e ENE e ee ge DEE AD BERE E EE ENA E U U U D D D D D D D O mutant wid type DNA GO D D 00 DOO ODD D D 0 0 0 0 C C D mutant wild type DNA D I 0 UU DO DEO UO D D D mutant wild type DNA O 0 0 D D D D 0 0 0 0 0 0 D D D PCR D 0 0 0 0 0 D D 0 D 0 U UnIUUUDUUUUUUUPNAUIUUDUUUUUUUUDUUUDUUUDUUUUDUUIUUDUUUDUL 0000000 mutation 0000000 S UUUUL DCode D 0 D D 0 0 D D D 00000000000 0000000000000 0000000000000 UDUIUUUDDDUIUUUUDDDPNAUUDDUDUUUDUUDDDDUDUUUDUDDOD 0 100 20 70 U U U D D D D D 0 0 0 0 D D U 0 U U U D 00 IW 0 U 0 0 D D D D O IDO UDD 4H D D D D D D 0 0 0 DGGE D D D 0 0 0 0 D D D D 0 0 0 0 0 D D D 0 0 0 0 0 U 0000000 1709150 DCode Electrophoresis Reagent Kit for DGGE CDGE EEEN T B E LE CES o DGGE CDGE I I lJ Fig 3 2 A Perpendicular denaturing gradient gel in which the denaturing gradient is perpendicular to the electrophoresis direction Mutant and wild type alleles of exon 6 from the TP53 gene amplified from primary breast carcinomas and separated by perpendicular DGGE 0 70 denaturant run at 80 V for 2 hours at 56 C The first two bands on the left are heteroduplexes and the other two bands are the homoduplexes B Parallel denaturing gradient gel in which the denaturing gradient is parallel to the electrophoresis direction Mutant and wild type alleles of exon 8 from the p53 gene separated by an 896 acrylamide bis 37 5 1 gel with a parallel grad
12. 000000 000 U 902951 O UU UU DNA DD 0 UO UO DO UO D D D D D DO 0000000000 2 DU DD U U ASC 0 0 D 00000000000 00 0 D 0 0 D 0 0 D 0 0 D 0 0 D 0 0 D 0 0 D 0 0 D Ul Bapu Hii 300000000000 000 5149 00000000000 000000000000 0000000000 UUDUDUUUUDUIUDUIUUUDDPNAIUIUIUUUUUUDUDDDUDUUUUUUUUUUUUUUUULD d DD 0 0 D D 0 0 D 0000000000000 000000SscPr 0 27 D 0 D 0 0 D 0 DU 0 D 0 0 U U U U UD D D D D D D 0 s104 0 D D D D U 81 DD DDD U UD 0 0 D D OO 05x TBE I 0 LOx TBE D 0 U U U U U DCode D D 0 0 0 D 0 0 0 0 sesa 000000000000 SP D 0 0 DD D DUO I U OU 9 HD D dd B Ti Pili S PHH EI LT li Bi Epi EEE A EE EE ee EEE E E E EPE i JEN p tii DR EE hii B the 20q000000000000000000 QOUUDO sSCP 000080000000 000 00000000000 SSP DU UDD 0 D D 1709172 UOUOUUUUUUO EE BDE 35T REE a ih h hi B ELE ELTE DD BE BC B BI KABER Eh BU ELF L DCodel O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D 0 D 0 D 0 D 0 D 0 D 0 D 0 D 0 D 0 D gt 209000 NOUUUUUUUUU LG UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUMUL 0000000 NESLAB RTETIT RTE 21T RTE 22 D D D D 0 0 0 0 0 D D 0 0 ODD D e HTH aT NE NAN U UUUUUUUUUUUUUUUUU SMI E E E ET IH ET HDE DECOTE 3 DCodej DOOO0O000000000000000000N00000000U0U0D00000UUDUU0OO TERRE sah hi STT Y hi ht hi SHR iL iii O i PS ET Eo Y EEE EEEN EEE EN EE HOUUUUUUOUUO 2 UUUUDUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUHUU U U 0 DU D 0 D 0 0 D 0 D 0 0 D 0 D 0 D 0 D D D D 0 D U U 0 LG 0 0 D 0 D 0 D 0 D 0 0 MU U D D D 0 D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 DD D D D D D D D
13. 1 bases GC Clamp Fig 8 1 U D D IMO DO 0 00 U melting temperature C D D D D D D J D D D O Fig8 1 U melting temperature U D 0 0 D 0 U GC D BZ 0 D 0 0 U D D 0 D D U 7 D 0 0 0 D D 0 D D 0 0 D 0 0 Serg 000000 TTGEU D e D D D 0 D D D DO D APG 0 met profile 0 0 0 0 iu 0 0 Ii D D DU 0 D D DE EEDD EL 00080 mutation II Ui Uu 0 0 0 D D D Z O 0 0 0 D D D D 0 0 0 0 0 0 0 D D D 0 0 0 0 D 0 U TTGE 000000000000 40708 DELE I TIGEN UU 0 D D D 0 0 D D 0 D 0 D D 0 0000000000000 1709171 TrGE 000000000 T iii lD PSE E EB E A RATA DA NARAH A JnUUDDUDDD DCode D 0 D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 UU D D D U 0000000 Ram Rae II D D 0 0 D 0 0 D D 0 0 D 0 0 D 0 0 0 D D 0 0 U NOUUUUUUUUUUUUUUUUUUUUUUUUUUUU CQ RRG U L 2000000000000 0 0 DD D 0 0 00 H 90 TTGE 00000 BRT OD DO DO ODO ODO ODO ODO OD TOTO TR ODE EEUU LI D Y LD D D 0 0 0 0 0 D D D D DCode 00000 Fill Run level line 000000000000 OOOO DCode 0000004848000 00000000000 00000000 0000000000 DBA poo DB O DD OOOO ODO ODO LLIPH DDD 000000000 CRRIDODLULULULULULU LU U U U 2009000000000 000000 000 H 0000004 1570 0000000000000 0000000000000 0000000000000 JnIUIUNUIUIDNDUUUUUUDUUDUDUUDDUDUUUDUUUUDUUU a H EAR D BBT EEE EEE h bhi U Bae EEEn EAE RAR EE T a AA NARA NARAH LU U U U U 0 0 UD 0 0 D U D 0 D U D UD U 00 L HOUUUUUUUUUUUUUUUO Fig 9 1 Fig 9 1 300000000000 00000000000 A ANA 0400000000 Fig 9 2 4000000000000000 DCodel B
14. D L U U EEP EE DE EE EEE Br ST BST 0100 0458 TSP DST 1111111 DCode 1000 D D D D 0 0 0 0 0 0 D 0 D D 0 0 L uBpugggugugugmugugpuguggmuausggbpuggaguiuuubiututrisesqs iuuututlutltu A A EEE at aT EST D ERR ER BA BARADHA NAN o U U UU 0 LU U U 0 0 0 0 0 DNA D II DNATOT ODDO D D D Leman rn GO Dd dg 0 D 0 00000000 BIR DDT DE Eee HDS Til E EST D B DR CH EEF EE EE UUUUDIUUDNAIUDUIUUUUUUUUUUUUUUUDUUUUUUDUUUUUUUUUUUDUDUUL DNAjUDUUUIUUUUDUUIUUUDDUUUUUUDUDUUDUU U U U D 0 0 DD D D 0 D 0 0 0 0 D 0 0 D D 0 D 0 D 0 0 D 0 D D 0 0 D 0 0 0 DNAT DD 0 00 D 0 0 0 U UNS Shy NABA ikili JEL GCI LIT EEC EN yi SUS Pinu EEE 2 DCodel DG D D D U D D UD D D O D D U UL UUUUUUUUHUU 1 D00 E 0000000000000000000000000000000000 00000000000000000000 Fir 0 Maximum 0 0 0 D T 00000000000000000000000000000007000 wi ma 2 DBDUIUDLDLULULULU LU Hi UUU000000000000000000000000000000000000Dcodeu0000b NUUUUUUUUL UDUUUUUUUU ENANA EE EIEH HA Bc EE EH RE Aa UUUUUUHUULU U U U 00 U UU 0 L IAN AAA UUUUUHUUHULU RENESA MENERA HAH EU EE all UUUUUHUUHULU JUlnUUDUUUDUDU RAN 0 80 Mata aa a DB DU EEE EL EEE EE EU BE ET LEE BH N 8 UUUUUUUUUUUUUUUUL ze AE BE EE EE EE EE DEBE ELE UU BL O E e O DO O DO ODO B iW pi BgddulblbllL Ph EI E E HB BL El DS i hi li EDENU LEDD ERR pe EEE thi DES DR PHD k Thi i ti BNR PL hias ea hiii LIL hb t AA A O DS ha hili l i hi B th Bu NODUUUUUUUUUUUUUUUUUUHUU B REA E RB O AA EVA AREA O BEST P E
15. D 0 0 0 0 D D D D D D D 0 0 D D D 0 10m II I D D D 0 0 0 0 0 15m UnnguuunsemlU I Ti ui i uu 000000 000000000 0000000000000 ETHEL BEE x AEE EE CHEI BE EE NEE LE E UDDUIUIUUUDUDDDUUUUUDDUDDDIOmUDD3OI IU UD D D D 0 0 D D 0 U L e Bf c e E N e a E i ED EA CE EE E hi i hI EE e eE Bd El E e ET EE eE EEE gt MEL EG EF i ds sh E EE EN EEE EE EEN ED AE e E SE aT EE a EE EF E BRI c DO DD OD DO DO DO DD O Ee aE TO eE TO TOT E OUDEUODULDLULDLDL LU U U 0 U 008 00 08 00 add A EE SE EEDE Ee THEE ED EF e e E E E ee EE LPTK Ph i ll LIT Dl EE EEDD EE ELE Ph EE Dirt h iii U U U HDD Ba ADE EE Ja KAN T RE RE T PE aT EEDD BE DER EEE RENEE EFE El BERSIH ED DE LET EHI OE s gt Oe A EE E NE R D lUIUDUUDIDUUUUDUUUDUDUDUUDUDUUDUUUDUDUDUUUDUDUDDDUDDDU JnUUDUIUDDUDUUUUUUUUUDUUDUDUUD e DESTE EEE E EE e E cE E S aT RH EER EE EE ERE e e STTS REE RUST aT B Bi OENE Eel OE PUP i ES ii aii k s SBS T HSS STT i i HB i lUIUUIUUDIDUUUUDUUUUDUDUDUUDUDUUDUUDUDUDUDUUUDUDUDDDUDDDU DEE AN EE IH 8159 STS DEE EEE E ET EDE E B FI Fl QOUUDO BIB p BO DO D DI D UNnIUUUDUIUUDUUUDUUUUUUUUUUUUUUUU 55m O0 0 D cm D 0 0 D 0 0 D 0 0 U UUIUUDUIUUUUUYDDUUUUUUUDUUUUUUUUUUUUUUUUUUDUUUUUUUUULl nen OU A DANA LAA HA UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU DI 10 YJ UU utut 11 YJUUUUDUUIUUUUDUUUUUUDUUUUUUDUUUUUUDDOUL nUUDDnUIDNDUDNUUUNUDUL U D D 0 D D D D 0 0 D D D 0 D D D D 0 D D D 0 0 10M 30m 0 0 D D D 0 0 D 00000000000 BSE RRIT RST Hp E hi
16. ERBOSE Q00000040000000 e POD00000 0000000000 00000 000000000000 a UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUHULU EE EDI RESIS N e PLESE i E N E EE B BA BA ENA O e aa Bahia hh nn NET an a H hi hi ANA RT AA iii VA AED hi a E FED EEDD SDE HE BC BI ET ihhh EEE TERE EEE 0000000 M475 00000000 00000 0000 0000000000000 0 75mm 16x10cm 5ml 6 5 16x16cm 11ml 9 5 1 00mm 16x10cm 1 1 ml 9 5 16x16cm 16ml 14 5 1 50mm 16x 10cm 16x 16cm 3900000000 BSem D D D 27 0 0 D D D D D 0 0 0 0 D D D 0 D UD 0 0 D 0 0 0 0 0 D D D U 19 0 0 D 0 0 D D D D D D D 0 D D D I I D High 0 0 Low D i U 0 0 0 0000000000000 JUIUNDIUIUNDUDUDUDDUDDUUDDUDDDOUDUUUUUUDUDUUUDNDUUDUUDUUDDODDUUD Ben PB B ap GT AT EE EE L B PETERE EE gare B T D EE una li DARA HAHA DEE ABA tti bhi HE KREE EAR REDER DU D D D UD D 0 0 0 D UD D 0 Sem 000000000000 00000000 0000000000000 UU 127 DD 0000000000 197 D D D 0 0 0 0 0 D 0 0 0 0 0 U B1000000000000000000 UD D U D TTT TT TT D D U 10 D 0 D D D 0 D D D 0 97 DU WV U TEMED 0 09 0 vW D 0 D D Ii Ii I Ii i id D I U O Dye Solution 100ml 0 0 JUIUDIDUIUDDDUDUDUUUUUUUUUUUUUUDDOUDDUIDUUDUUUNUUUUDDUUDU JA HAHA 000 00000000000 B1000000 000000000000 000000000 JnUUDIDUIDDUDUUDNUUUUDDUOL 181 000000000000 MD 0000000000000 0000000000 000000000000 EEE AE AA A BB EE BAN ANANA BABAD 1810000000000 00000 0000000000000 HIGH DENSTY D 0 D D D 0 0 U 00000 TOPFILUNG D 0 0 0 0 D 0 0 0 0 D D D DDD Td D D D D 0 D D 0 0 0 0 0 0
17. O ODO OD ti iti TH TL DU DADO BOBO OD Td TTT TT TT TO TO TTT TO TO TO TO DO DO OD ODO DODODO TRENUJE H EE ye AA EEE EE E OA la Phi DCode 100 U D D 0 hr U U D D 0 U D 0 U U temperature ramp re OOO 0 D 0 0 0 D 0 0 0 D 0 0 L LU U U 0 UD 0 D 0 UD 0 U 0 D U U ramp rate D U O O U U U U U U U U U U U U U U U U U U U U U UI UUDTTGED III I 71 nup Fig 7 1 Temporal temperature gradient gel Amplified mutant and wild type alleles of exon 7 from the cystic fibrosis gene Separation by TTGE run at 130 V for 5 hours in 1 25x TAE buffer on a 6M urea 6 acrylamide gel 37 5 1 using a temperature range of 50 60 C and a ramp rate of 2 C hr Lane 1 mutant allele 1154 ins TC lane 2 mutant allele G330X lane 3 mutant allele deltaF311 lane 4 mutant allele R334W and lane 5 wild type allele Samples courtesy of L Silverman Division of Molecular Pathology University of North Carolina School of Medicine o TTGED UUUDUUUUDU TIGEN 0 D D D D D D D 0 0 D D D 0 D D 0 0 0 DU Mac Melt 0 0 DNA Melting Software D melting profile 10000000 U 0 0 D 0 D D U I LI D melting doman 000000000 DGGEI 00000 U O PCR primer 304098 GCI D D 0 0 0 0 0 0 D D D D LI U D D 0 0 0 D D D D 0 0 0 D D D GC 0 0 D D U D 0 0 0 DNA sequence D 0 0000000 melting temperature melting profile graph I 0 I I Ii D D D I 0 D Fig81 00000000 O melting temperature D I UU I Uu i Uu Ul UD U U O ODO 0 U iu UU U UU LU U Displayed 1 do 191 of 19
18. PI DE ELE EEDD DD ED T i ha hp ELE HE B n EI E LU U U U A HD El D D D D 0 0 D D DO BO D D DO D 0 0 0 0 0 0 0 0 D 0 0 D UD 0 0 0 0 D 0 0 D D D 0 D 0 D 0 D 0 DOB po p TTT TTT TT TT TTT TT TO TT ODO 11 T H Jan E I oad dea ss Lamm 000000000000 000000000 BB DEB BO ELE U AO BEBE B EE DEED BET SIS HEDE HSP ER BRE EE EEE C BEI U U U D UD 0 D DGGE OU U D D D D 0 perpendicular II iu 0 D D D D D D 0 0 0 0 0 D D 0 D 0 0 0 0 0 0 D D D U EE S ERER Ee EE VAE JUIUUIUUUDDDUDDUUDDUUUDDDUUUUDDD DE Ra na a i DEER Mn nn nn Eg I nu E uan EIER RER AER EE AA aan E JUIUUUUUDDODDUUUUUUUUUDDDUDODNDUDUDULU Hii A XE SEE 3 UUUUUUUUUUUUUUUU lUIUIUDUUUUDDUUDDDUUDUDDULU UOUUUUUUUUUUUUUUUHUU 40 BED Bp JnIUIUDDUDDUUDDUIUNDUDNUUUNUUUUUUUUD 11 0004 500000000000O LL EHE ASTE DE DEBE DEDE HHY EE DEDE DEI il HI II TH TUDI n nin D UU LU LL Volume adjustment screw Lever Plunger cap Volume setting indicator cap screw VOLUME 10 ML SYRINGE Syringe holder screw Syringe sleeve CUN Luer fittin Tubing di Nen d A Y Fitting lt Syringe holder BIO RAD lt MODEL 475 GRADIENT DELIVERY SYSTEM THIS SIDE FOR HIGH DENSITY SOLUTION BOTTOM FILLING LOW DENSITY SOLUTION TOP FILLING BEB A MEL Ls LU U U U 10m 30n D D 0 0 D D
19. SEDE a i AA REA REA PERA EL RUDD ELE OF EL EF 00000000000000000000 Fig48 100 D 0 0 D 0 D D 0 D D 0 0 0 0 D 0 0 D 0 0 UUUUUUUUUUUUUUUUUUUHULU HOOOUOUOUUUOU Y40 000000 Fig 4 6 10000000000 000000000000 UOO0Cd0 00000000000 1410000000000 000000000000 LU U U 0 DO DO 0 U DDD U UD 0 pi Ll UUUUUUUUUUUUUUUUUUUHUL RE OO REGE sa EN AN Er AO qiyu H EP AA B EE E EE Phi RE ih NET KANAN A Fl Fig 4 7 Fig 4 7 LA PD BIB EF EE ED EE EH ELE EF B D uguuuiuliuululiltitiii8i Hh Bu LLL umlmgmmgiium uBudgmuugtumsa i ut LU U U U uuuiuiuuiliuiliilibitlLitLliLLtL Fig 4 8 Fig 4 8 IT 0 D UD U D 0 0 U D UD U U D 00 D UD U U T HG L U UD D UD 0 0 U 0 U 0 U D UD 0 D U 0 U 0 U 0 D U Fig 4 9 1510000000000 0000000 Fig 4 9 16 0000000000000000000 Tilt rod 000000000000 Tit rod 000000000 Fig410 Fig 4 10 171 UUUUUUUUUUUUUUUUUUUUUL ION hii a E T ARE EA R NA PEREM ER AREA RT RE EH BRAVA EPEE E AN B RA RAIER A MEDA NAD E EMEA NUUUUUUUUUHUUUUUUUUHUL AAA ANA AE PAD OPE AE ENDE Hu B PA nn P E i nn A ARE E L gaga U U U D D 0 0 D 1P D D 0 D D 0 D D 0 0 U Pipu pu iii B ia EEE EL NA NAH Fig4 11 Fig 4 11 181000 0000000000000 0000000 O U D D 0 0 0 0 0 0000000000 Fig4 12 19 Tilt rod 1 0 0 D 0 75x 10 000000 Tilt rod ID Z D 0 0 0 D 0 000000000 EE EFE BE DEBE Ce Ee EE UDDDDDU 20000000 U 16x 16 D 0 U D 0 0 D D 0 D D 0 D 0 0 L U D 0 0 0 0 0 D D D D D L Fig4 13 20 D 0 D D
20. T iii rh h BEA LEE nuu ERR MASS ORK B ti ia 000000 Fig 6 2 4000000000000000DCode D 0 U JUIUUIUUUUDUUUDUDUDUDUDU U D 0 0 0 0 0 0 D D D D D D OU D Figos Fig 6 3 al DD ED TRT E ELE EE DG EE EEE DE BE D DEBE EDI ETO EEE EEE E0 EVE EL EDGE E ET HEH HDD Pl iii EE EY E iii bia pipi SS Bibi EEE Hillu DO DO DD ODO ih LTU ID ID OD ODO DO TO TO ITB HSHH BP nB Biri B TT TTT TTT aj HAN e AA E GE TE ET DEI EE EE EEO ET EI GDB po gado E1 ii hili EEL EHE AED EEN ED ET TEH nT TEE ERLEBT OEE DESDE T ET RER ECT T D hu ST RT n EE EE Di L ITT BLE hi BEST 8 bh TEN iW li ii PE ge AAA R Z UNA JUIUUIUIUUDUDUUDUUDUDUDDUUDODDDUUDUUUUUDUUUNDUUDUUDDUUDOUDDD UDDUDDDDDDDDDD49 o II 0 0 O TT TO Uu D D D D D D UU UU UU 00000000000 800000000000 0 0 0 H 0000000000000 000000 UUUUUUUU 16x16cm 16x10cm UDDDDDDDDDD perpendiculart I D i D D D D 0 0 0 0 0 0 0 0 0 D 0 D 0 D D D D 0 D 0 0 0 U U U 0 0 gg D D D D D 0 0 0 0 D 0 0 U UUDUUUUUDUDDUDUUPSUDDUDDUUDL 91191 000000000009 vv TEMED F U 009 wy D 0 0 0 CUI U C l Fig 64 Fig 6 4 19 000000000000 000000000000 00000 00000000000 00000000 ES LD Z EE HE DB ET EI B E BD EEO ANA ST ENEN AE ET EUO EDD EME ans NA WA BA A EE EEE UUUUUL 2 0600000000000 7 TTGE Temporal Temperature Gradient Gel Electrophoresis TTGEU DGGE D 0 UDUUUDUUDUDUUUUUDUDUDUUUUUDUDUDUDUUDUDU D Gene mutant wild type DNA 0000000000000 000000000000 0000000000000 cama i EE EE OD O D
21. ak RN aii ED ED EEE EEDE Le D E E NANA NA NAHAN O HHH H BED BC SE DG EL BEU EHO ED HDH EB HIE VEE NAN 0 00 000 B hihi hiiia AN EEE KA MAN AA hiii hiii AN ED a Phi hiii i MEE EEE Edi 7 00000000000uu0003omU00000u0uUU0UUU000000000000000000 JnIUUDUDUIUUUUUUUDUUUUUUNUUUDUUDUDUIDDDUIDDUIUDUDUUUDUUUUDUU U U BAT U UD D D D D 0 0 0 D D D D 0 UD UD 0 0 D 0 LAT 0 0 0 0 0 0 U DGGE CDGE SSCPU I J LUDU DU DD DD D 0 D 0 D D D 0 D UD D UD D D D 0 0 0 0 0 D D D 0 0 0 D D D 0 0 0 0 0 0 0 D 0 0 D D U 2 UU DUD 0 D DD D I 0 0 O DCode lid stand D 0 D D 0 0 0 0 0 0 D D D D D D 0 D 0 0 0 0 U EE Ea Ge EKO DEE EEEN 0100 04 ERE DSL EA H d EE EE EE U 0 0 0 0 0 0 0 00000 DD DD D D D D D D D D 0 D 0 0 0 0 0 0 0 0 0 0 D 0 D D 0 D D U Spe Ea EIEH ED EB EE phai th LP TL TL ET RTH EE L HP HEB GRE DOE 155 aT EEE EE EEE E EL ELA BERE BEP EA EEE EEN EEE A EEE EE EHO EFE hBi Itil TDP ELS aE ED ae EE AED lD BIS b PE CE HE ELE 1 Tl el mene cp E eb n DES DC BE BS DESDE EE DEC BEI D ELE ED HI loading lid D D D D D 0 D 0 0 U D 7 0 D 0 0 0 D CE D H 4000000000000 D UD U D UD D D D 0 0 0 0 0 0 D D OJI 157 D D 0 0 0 L TTGE LUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUL 2 000000000 I 0 O DCode lid stand 0 0 D 0 D D D 0 D 0 0 0 0 0 0 D D D D D D 0 0 U EB E BET T P hl anh iii TE I hh thi hhi BTE hihi B h EE BED Bul bu phi EE tiyu Ec LE STET 99T EE 9P EU ED EE ER EDB OBE NEE HEH EY Pu ET AE phili A A EB BU Plc e ET EEDE E EIRIO ET S E paga
22. i gn nan hiii ld E hp lah L BRE U D D D 0 0 0 0 0 0 D D D D D D F3 a ao ENE OE EEE REE H EET EIS EL BOE ER E RR ARO ED ERE EL BEBE ee ELE A P ET EP E EE BIB SHS REA EAV Ph a A B E E JDIDUIUDDIOUUDDUDDUDHUUIUUUUDUUDUDUDUDUDDUDUUDUUDNUUDUUUUUDUL UUNnUUUUDDUuuUUtuuUUUDUUnuuUuUuUDuuuuasIUuUUUDUuuuuU GEI EE ELE dada do Cencio ET i i ELLE EL EOB EMOD TE ED EE EEE MOUDDO ODO BO DO OO BO DO OD OD ODO DDD OOOO D DH OO TTT 00000000 0000004000 Eee OEE EE eeh EN LEE EE EEE r HS ED EDE EED ER TEN EP BI CE AE NOUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU UV 00000 FB ADRA UNO EE DDE EU Fl 8 SON I 0 i Du DU UD D D D L U UO UB BL BL ELO U 16x16cm 00 0 75 mm 25ml 1 00 mm 30ml 1 5 0mm 45ml g1 0 D U 003 vv 0 TEMEDI 0 OD wW I I UI D D da 0 0 D D D 0 UU 0 Dl 10 100000 iB Bi T TP 0000000000 Pi 1 9998 Th i B 1 iB B a Ph hi hit B u HB Phi DEER iti RH 10 OUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUL 0000 HET 1911160000 0000 1 O SSCP Single Strand Conformational Polymorphis ms ELE NAG EE DEI EEF T EF EE EEF EF ELE Ed EFE EF ELE EFE EEE ELE EI p440000ssePgoo000000000000000000000000000 wild type mutant DNAjUUIUUUUIUUDUUUDUUUDUUUUUUUUDUUUUDUUUUUUIUUDUUUDUDUUUuD U U D D D 0 0 SSCM 000000000000 000000000 0000000000 000000000 Hb aB i RE aH SPR Ll EE E ELE ENE EE DE E BP En Bu hh EN AAA la EO nn an H aT RE am EH HARA nn i EM AE ri BAN 1 DNAIUDUDID D D D D D D D D D D D D D D D 33081000000000000000
23. ient of 40 6596 denaturant The gel was run at 150 V for 2 5 hours at 60 C in 1x TAE buffer Lane 1 contains the mutant fragment lane 2 contains the wild type fragment lane 3 contains both the mutant and wild type fragments CDGE D 0 0 DGGE D wid type mutant 1000000000000 000000000000 000 LIE ED ED ANA oo En D oo 20 70 perpendicular gradient gel D D 0 i 0000000 LU U U 0 0 0 0 LLL Fig 3 3 Example of perpendicular DGGE gel used for determining the optimum denaturan concentration used in a CDGE gel The distance along the gradient where the maximum split seen between samples is 5cm The denaturant concentration of the gragient at this distance is 51 Therefore the CDGE gel should use a denaturant concentration of 51 HOHUOUUUUUUUUUO PL B ii a EFE El U wild type mutant DOO DE AED AA AN D EH NEE Wn gn MI Aa nn E BIR T S H T 895 U II Fig 3 4 Constant denaturing gel Amplified mutant and wild type alleles of exon 8 from the p53 gene Separation by CDGE run at 130 V for 2 5 hours on a 10 acrylamide gel in 51 denaturant at 56 C Lane 1 mutant allele lane 2 wild type allele lane 3 mutant and wild type allele 4 DGGE Perpendicular Gradient Gel U U UU ED Wem D U D D D C C 21 0 4 D 75x 10cm Perpendicular gradient gel D LU U U 0 00 U 0 0 U APV ABD EM EEA EEE LHR TSE DD 0 0 0 D D D D 0 0 0 0 0 0 D D D D D 0 0 0 0 0 D D 0 OU ITA 0 0 0 0 DCode 0 0 0 0 Fill
24. li hii EE S JO AN E EN YE Ee Ee EENE E E E Ud TT TT TT O TT DOT TT OG TT TTT TOGO TONDO 3 DGGE Denaturing Gradient Gel Electrophoresis U U 0 U D D U D U 0 U U 0 0 U DGGE Denaturing Gradient Gel Electrophoresis DNA 0 0 XI DO 0 U U U D D D D D 0 D 0 I O DGGE 000000 Fisher Leman 1 I I i 0 0 0 D D 0 0 D D D 0 0 0 U Q000000004000040 DNA d AA BAB UUUUUUUUUUUUUUUU HUU Q000000 00400094400 TM D 0 D D D D 0 D 0 0 0 TTT 0 D D 0 D 0 0 0 D D 0 0 Tm U U U 00 D DO O DD DO 0 0 D 0 0 0 0 D 0 D 0 0 DNA DD 0 0 0 0 D 0 D DNA 0000 EIE DEED phili h DST aT RST SH UNAS Tm D D D D 0 0 0 0 0 D D 0 D 0 0 0 0 0 0 D D D D D D D D D 0 TT 0 0000000000 mutant wild type D D UI 0 0 DNA OO D D 0 D 0 0 0 0 D 0 DNA D D D D D D 0 0 0 0 0 0 D LI D Fig 21 Gco D D 0 0 D D 0 D 304000 TO O 0 0 D 0 0 0 0 D D 0 D 0 0 D 0 0 D 0 0000000000000 HOU GC D 0 D 0 PERI D D 0 0 0 D 0 D 0 D 0 D 0 0 0 D 0 D 0 0 D 0 DNA D D TM D 0 D 0 0 0 UI UOUUUUUUUUUUUUUUUUUHUU DEE R EEA EL EA EE ii Ll Denaturant 100 mur Partially melted Wild type Partially melted Single mutant strands mur Wild Type mur Mutant siseJoudoJ199 3 imm Double strand Fig 3 1 DGGE 0 5665100000000 00000000000 0000000000000 000000 MO 00400000000 100 D D 0 0 0 0000000000 000000000000 0000000000 00000000 perpendiculal 0 I D IJ D 0 D 0 D 0 D 0 D OC 0 0 0 2079 D i i D i D D L U U D D D D 0 0 0 D 0 0 DGGEIJ 0 D D 0 0 0 0 D D
25. nuuuUnDuuUDUnuguUDACUUDUDUUD U U U DC 0 0 D D D 0 0 0 0 2 0 DU 0 D D 0 D 0 0 U O 000000000000000000000 0 0000DCok 0C0000C0000C00000C00000000000 O 000000000000000000 D 00000000000000aAcg peg 000000000 00000000000000 0 ACI DC 00000000 300000000 lB hab hihi NA BABA NABA HSP Thi EDE EA laen in na B ii Fil 0000000000000000000000000000000000000 Mec 0000 LU U 0 0 U BH nu nM EEE EEE aaa AA U Dcode D 0 I I 0 0 0 0 0 Ii i 0 D D L A LU D DUD D LI U U U U DD D 0 0 D D D U 0 U D DC ACI D D D 0 0 D D 0 D 0 D D D 0 0 0 D D D UD 0 D D 0 0 0 D 0 U U U 0 D 0 D D D D 0 0 0 D 0 0 0 0 00 BRT Y RIRI DO ODO lh hhi HE HR i h a Dll U D 0 0 0 0 0 0 D D D D D D 0 0 0 0 D D D 0 D I O DCodelidstand D D D U D D C D 0 D U ED ECE 95T 95 PARA EE HR nn 1000 00 L D U U U 0 U U D 0 U O U U U D U U 0 0 0 U D U U U U U U D D 0 U 0 0 0 U lI LU U U U 0 0 0 0 DO OD U U UD D U U U U U 0 0 DDD 0 U U U U UD D U U U DNA D U U U U 0 0 000 U LU U U D 0 0 0 0 O UD DO DD 0 0 D UD U 0 0 0 D 0 0 UD D U 0 0 D 0 0 0 U D U 0 0 D 0 O U SingleStrand Conformational Polymorphism SSCP Denaturing Gradient Gel Electrophoresis DGGE Carbodiimide CDI Chemical Cleavage Of Mismatch CCM RNase Cleavage Heteroduplex Analysis HA Protein Truncation Test PTO D 0 0 0 D D 0 U U D D 0 U U DU U U D 0 0 U U O0 Temporal Temperature Gradient Gel Electrophoresis TTGE 0 O0 0 0 0 LU U D 0 0 0 D D 0 0 DCode D 0 0 D 0 D 0 0 0 D U 0 0 0 0 D D 0 D 0 0 0 U D 0 0 0 D D 0 D 0 0 0
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