Moderní analytická instrumentace pro genetický výzkum, lékařskou diagnostiku a molekulárni identifikaci organizmů Karel Klepárník Oddělení bioanalytické instrumentace Ústav analytické chemie Akademie věd České republiky Brno Jac.N Drno Q Capillary electrophoresis CE Capillary electrophoresis scheme inlet electrode chamber Q A) electronic ^ detection electroQsmotic window detail ©B) electroosmotic electrophoretic mobility high voltage outlet electrode chamber M Hk91|| WE KB H.St &fl IHI Jorgenson, J. W.; Lukacs, K. D. Ana Jorgenson, J. W.; Lukacs, K. D. Scie James W. Jorgenson Department of Chemistry Venable and Kenan Laboratories The University of North Carolina at Chapel Hill Chapel Hill, NC 27599-3290 USA . Cnem 1981, 53, 1298-1302. nee 1983, 222, 266-272. Prof. Miloš Novotný Indiana University, Bloomington Department of Chemistry Why capillary electrophoresis? Q, =UdI = U'2wdrKlL Qc = 2mLÄ- AT = T -T ĽS.1 i0 1 R E2R2k 4Ä Miniature capillary: 1) high resistivity O low current at high voltage O low heat production 2) efficient heat transport O low temperature difference inside the capillary DNA electromigration K. Kleparnik, P. Boček, DNA diagnostics by Capillary Electrophoresis Chemical Reviews 107, 5279 - 5317, 2007. DNA primary structure íc A 1 , ) T r 5"**wJ —f HOCH, „ ■O-P-O a ; A iff V" ' H H- C li o .j$j f"1 *V^ '°r~ fe L -p— P—ŮH— 3' and O DNA electromigration reg n of spaces in a random network of rigid to a spherical molecule ods Ogston regi me Ogston (1958): * distributic available v * penetration probability \, / PD>r = eXP- (InvLr 2 + 47TVT3 3 y< v average density ofnumbero fibers >rl Sv 2L fiber length y^m \ D „pore" radius -7*-S- ' X r particle radius Rodbard Chrambach (1970) P =ü = ü 1 D>r ,7 = exp- (K C) í; el. n M> fre obility electrolyte el. mobility Va ac essible volume KT~(r + df" -3 V0 void volume Kr retardation coef. Ferguson plot (1964): log// = log /^ -Krc d gel concentration Fergusson plot log p = log ft-Krc ft = 6nrrj Ferguson plots of DNA molecules in agarose gels. The logarithm of the mobility, extrapolated to zero electric field strength at each gel concentration, is plotted as a function of agarose concentration, %A. a) b) J..... ONA Biased Reptation Model 1 q {K) L tube legth l friction inside x field direction 1 u = -!-x 3# 1 N e1 + — 3_ E«1 :M?^^^ qEa 2kBT N reptation segments tjy e scaled el. field q segment charge c) /****ÍV-^ a segment length /Mž$»l 1/(3N) , / M'Mo ~ \ E2/9 n«e-2 N» E-2 #H^ Pierre-Gilles de Gennes R 1932-2007 ole de Physique et Chimie (Paris) Nobel Prize in Physics in 1991 ' nee of DNA electrophoretic mobility on molecu ) i s*«^Ogston sieving logü Mo Rsm a b c log M Jean-Louis Viovy Curie Institute Paris, France Garry W. Slater University of Ottawa Poymerase chain reaction PCR amplification Kary B. Mullis born 1944 La Jolla, CA, USA University of British Columbia The Nobel Prize in Chemistry 1993 For his invention of the polymerase chain reaction (PCR) method PCR amplification scheme DNA Primer DNA dissociation annealing synthesis Correct copies 1st cycle: n=1 2nd cycle: n=2 3rd cycle: n=3 N=2m1 - 2(n+1) 22 - 2-2 = 0 23 - 2-3 = 2 2* - 2-4 = 8 Human Genome Project J. CRAIG VENTER, Ph.D., PRESIDENT, CELERA GENOMICS REMARKS AT THE HUMAN GENOME ANNOUNCEMENT THE WHITE HOUSE MONDAY, JUNE 26, 2000 Mr. President, Honorable members of the Cabinet, Honorable members of Congress, distinguished guests. Today, June 26, 2000 marks an historic point in the 100,000-year record of humanity. We are announcing today that for the first time our species can read the chemical letters of its genetic code. At 12:30 p.m. today, in a joint press conference with the public genome effort, Celera Genomics will describe 'I the human genetic v from the whole genome shotgun sequencing method. tlS ago on September 8, 1999, eighteen miles from the White House, a small team of scientists headed by myself, Hamilton 0. Smith, Mark Adams, Gene Myers and Granger Sutton began sequencing the DNA of the human genome using a novel method pioneered by essentially the same team five years earlier at The Institute for Genomic Research in Rockville, Maryland. The method used by Celera has determined the IC Code OI five 1HC ... There would be no announcement today if it were not for the more than SI billion that PE Biosystems invested in Celera and in the development of the automated DNA sequencer that both Celera and the public effort used to sequence the genome... J. Craig Venter The I nstitute for Genomic Research (TIGR) The first president of Celera Genomics The completed sequence of the human genome was published in February 2001 in Science. Venter, C. J. etal. Science 2001, 291, 1304-1351. DNA sequencing ■ -rr.i:. rtiiLiiisiitu DNA sequencing strategy Primer walking li linu iiiiiiiiir consecutive primer selection Shotgun Fluorescence chemistry Fluorescent lebels "COO* ~xxxx Fluorescein Rhodamine CXJXX'J H'°' "O i ^^j^g^O-Nj Texas Red BODIPY "'" '°rY*i—ŕ ^—CY"'°' í 0,K KAJ^^XXJ xttP NBD Cy3,5,7 | Sequencing primer attached to Fluorescence Resonance Energy Transfer | 5'-TTTTCCCAGTCACGACG-3' PRIMER SEQUENCE Dideoxy terminator attached to Fluorescence Resonance Energy Transfer Prof. Richard A. Mathies University of California at Berkeley Department of Chemistry Berkeley, CA srsjir <$><& LIF detection Four channel LIF detection arrangement band pass 570 ntn PMT band pass blocker ss „ r & objective blocker beam band pass 540 nm ^20 nm AS 40x; 0.65 520 nm splitter 610 nm lens ^-----^ separation capillary ID 50 mm SCHEME OF CONFOCAL DETECTOR PINHOLE OPTICS BEAM SPLITTER SENSOR | \JB/| amy -JU2S MICROSCOPE OBJECTIVE v—7T~ ~zs rpo^i Prof. Edward S. Yeung Ames Laboratory U.S. Department of Energy Iowa State University. Sheath-flow cuvette excited sample electrode chamber electrode chamber polymer filled capillaries open tubings I Prof. Norman Dovichi University of Washington Seattle, WA, USA -■>-('■-: Tiblrurm-nl llfaiy Prof. Hideki Kambara senior chief scientist Hitachi Central Research Laboratory Tokyo, Japan m e< i ABI PRISM® 3700 DN, 96 active eight reserve capillaries \ Analyzer ABI PRISM® 3700 DNA Analyzer id ^ *l 1*1 FW. 5*v| **.*»* ftiiS»!*!^^! ui ^.--vy- |c=*«vvř* | i .;« DNA sequencing record ■U.)W..l|,M^ň fft. w\^uy\uM _/:. JÍLUJME ,(^mhm?f4\tl\éu\;i\A JWMiw 7 PE Applied Biosystems Molecular Dynamics ABl PRISM 3700 MEGABACE 1000 accuracy > 98.5% to 550 base accuracy > 98.5% to 550 base 96 samples per run in 3 hours 96 samples per run in 2 hours laser Ar-ion 488 and 514.5 nm laser Ar-ion 488 nm detection in sheath flow energy transfer dyes concave spectrograph and cooled CCD confocal scanning with 4 filters and 2 PMTs DNA sequencing over 1000 bases in 1.5 hour Separation matrix: LPA 2.0% (w/v) 5.5 MDa E: 150 V/cm, T: 50 °C ÍĚM^k^MkM^MM^^ 'fefti ft. tiwMwefí fct ťj%^ iÉLSĚ&ÉMmíMmi i^MiJM Barry L. Karger Director, James L. Waters Professor of Analytical Chemistry The Barnett Institute Northeastern University Boston MA DNA sequencing up to 1300 bases in 2 hours Separation matrix: LPA 2.0% (w/w) 17 MDa, 0.5% (w/w) 270 kDa E: 125 V/cm, T: 70 °C iWíifXS1^^^^^^^^^ lion time (min) DNA mutation analysis Restriction (amplification) fragment legth polymorphism RFLP (AFLP) *'rľU^!lw,l,*,w* dlMll. a*-|iicu*-|-ii",tra*4. ■i^'|---|*rCO*-TtAT*TCTJ**00*Q*- | "T £ 1 1 1 1- Size based separation of ds or ss DNA fragments Resolution: ss>1000 ds > 400 h*:ců*rir*i:TcuAůDnwi | «*-** " t «TMnaUmCAOTI HMOTU UMTI ITC |ATBUlTCJlTMC»iUULiaD#LCIIIl | **"¥—•"—-—" ^ dxl|AT»AT|1C^TCT«MC4A|irAlC |AfiůjhtfCAr*tcTOAAaoAaTATc - r- r. ■ II 1 |aTC**TTÍ1*.T ||*TCM;**CC*n*T | íl í« 'LJULí J\. 8 Minimum Separation Time Effect of Injection and Detection 1 / sample hr = hf 'space separation capillary Miniaturized CE system with capillary heater and LIF detection Capillary effective length 2.5 cm IfcttT beam B elíclTopfcpríffliisrurt-topvíew C3= Effect of detection, denaturing and temperature AD Capillary 30 cm 2% agarose 0.1 M Tris/TAPS + 7 M urea 173 V/cm LIF Capillary 2.5 cm 4% agarose, 0.1 M Tris/TAPS + 7 M urea 600 V/cm ■ i;r ■ ... im, [JK] Effective transient ITP stacking Polymorphism of CT, CA, GC repeats in endothelin 1 gene Multiplex PCR amplification: heterozygote: 207, 217 and 201,211 homozygote: 203 Denaturing: sample dissolved inlO.05 M NaOHJ Separation medium: 4% agarose BRE (EMC) in 0.1 M Tris -TAPS and 7 M urea Fluorescent dye: SYBR Green n (492/513 nm) Capillary: 2.5 (5) cm; 50 Jim ID Temperature: 60 °C Electric field strength: 600 V/cm Injection: 5 sec, 600 V/cm 42 44 migration time (sec] DNA denaturing DNA separations under alkaline conditions • effective denaturing - DNA sequencing • fast separations - high effective charge of polyanions • effective stacking - maximum mobility of OH ions • compatibility - fluorescent labels and sieving media Effective denaturing Alkaline CE of DNA sequencing fragments electric field strength: 200V/cm I I G-dR110(540nm) __^1MjuUiA1^^__.___ , , A-dR6G(570nm) T-dTAMRA (600nm) i C-dROX(620nm) Hydrogen bonding contributes little to the stability of the double helix. Hydrophobic forces largely stabilize DNA secondary structure. igh effective charge of DNA fra Migration of DNA sequencing fragments at pH 12.6 and 8.3 Sample; mixture of aH fragments labeled will dideoxy tarminators G-dR110 (HS nm], A-dRSG (570), T-dTAMRA (595), OdROX (625i Capillary: L 19.5 (25) cm, ID 50 um, A) uncoated, B) PVA coating 4% LPA H 5 MDa) In A) 0.04H HaOH. Bl 0.1 H Trig-Taps * 7M urea 30°C;200V/cm 540 nm i fpH^n j£t>jío-:ú Dissociation of DNA bases pKa' H Hypoxanthine 8.94 12.10 G Guanine 9.20 12.30 A Adenine 9.80 T Thymine 9.90 13.00 C Cytosine 12.50 High effective charge of RNA fragments 2-deoxy ho-ch^ ribose ribose Migration of RNA and DNA fragments under alkaline conditions Sample: size standards RNA and DNA Uncoated capillary: ID 50 nm; L 30.0 (34.6) cm Electrolyte: 2% agarose in Q.OSM NaOH T: 40°C; E: 145 V/cm I pH 12.7 I 234 LLJÜJL- migration time [min] Mixture of DNA wild type and two different mutants melting temperature mid type mutant wild type - mutant ) mo duplex homoduplexes hetero duplexe s migration time mutant - mutant heteroduplexes 10 [°C] 85 65 Theoretical melting curve mutation GC clamp base pairs Mutation detection by CDCE Fragments amplified on p53 gene mutation: 2 substitutions A>G at different positions. Separation conditions: 14% solution of LPA (180 kDa) in 50 mM Tris - TAPS buffer at 79*C; Lr - 55 cm; L„ - 40 cm; E - 360 V/cm. —l—■—!—■—l—'—l—'—l—■—I—■—I—'—r 15 20 25 30 35 40 45 50 | ^^^^^^^^^^^^^^^iTTiaration time [min] Principle of SSCP technique native dsDNA denatured ssDNA native environment wild type -► -------► ----------------► ^ __^_ point mutation % o o 32 o Phenylketonuria a) health homozygote SSCP analysis Detection of point mutation C > T in phenylalanine hydroxylase gene on chromosome 12 Separation conditions: 2% solution of agarose SeaPrep in 1xTBE with 10% formamide T - 30#C LC- 55 cm LD- 50 cm E-a) 183 V/cm, b) 135 V/cm. u b) heterozygote dsDNA ■ time SSCP analysis! in-capillary alkaline denaturing affected homozygote Separation medium: 2% LPA in 0.1 M Tris -TAPS Electric field: 280 V/cm Capillary: length 15 (25) cm; ID 75 urn Temperature: 27°C Excitation: Ar-ion laser 488 nm Injection: 5 sec, 200 "V/cm Denaturing: 0.1 M NaOH, 90 sec. 11 Single Strand Conformation Polymorphism (SSCP) Cystinuria type I A disorder of amino acid transport characterized by a renal cystine transport defect resulting in urinary tract calculus disease. Genome: The most frequent is point mutation M467T, a substitution of Thymine for Cytosine in position 1400 in SLC3A1 gene on short arm 2p of human chromosome 21. Proteome: The mutation causes the substitution of Methionin for Threonin in position 467 of transport protein rBAT, a 90 kDa glycoprotein. Sample: PCR fragment of 317 bp. The mutation is in position 146. Sense primer is labeled by fluorescein (520 nm) on 5'-end. Antisense primer is labeled by TAMRA (610 nm) on 5'-end. SSCP analysis! in-capillary alkaline denaturing Point mutation M467T causing Cystinuria (heterozygote) substitution of T for C at position 147 in 317bp fragment Separation n nedium: 4% LP A in 0.1 MTris -TAPS Electric Held : 280 V/cm sense Capillary: le ngth 15 (25) cm; ID 75 um fill ^1 Excitation: Ar-ion las er 488 nu antisense Injection: 5 e c, 280 V/cm TAMRA610nm Denaturing: 0.1MNaOH,90 s 7.10 7.15 migration time [min] Comparison of migration order with theoretical structures FkfUr+P. Th*Oi$r>SiJ 1*-O-(Jim*rt*i>rt0l UrGVKlIin/ hC**A ittxictu»! ol complonianlary standi |3l 7 nlj amphh+d on • and carrying mutation MlEľT - A, C. The rrwtnlkin ait« n1 powtrVi 1501» Indk»t4<] by arrow». Cíici*»t«l t1ruíii»tt in »oUEianof Ü.Ü1 m Ma' at a 1«mp*r,n!un ol 27 C using ¥ar**n 3.Q MfOUD SOUrt-íir* Single nucleotide primer extension Minisequencing SNuPE S N u ľ1 E reaction SNuPE producb .u wild Lv p* ■. ľ :; ud <-.<. v UT"TF> CAATAcfl -----*" CAATAC -atgccatgIcJtc g -b] pmnt i ľ.i ľ i: . 11 CAATacFI ----* CAATACT -at6ccatc[aJtcg -■ľ 1 repetitive n uc leutidci ___dT'TP CAATACÍrH -----" CAATACTT^ -ATGCCATg|A ft[c6-(fr tonger primer n dT*Tŕ TACAATACT' -----+■ TACAATACT' -ATÜCCATujAJTCfi-electron herogram ■ b c d llll Jonathan V. Sweedler Department of Chemistry University of Illinois Urbana, IL Director of the Biotechnology Center Associated with the Beckman Institute, Biotechnology Center, Neuroscience Program and Bioengineering Program Ml jr. n á py. ~ ■I a Ir^tT i:. 12 Single cell analysis VII PT2%* Typical eucaryotic somatic cell 1 -#^"B diameter: 5-10 u,m volume: 500 fl total mass: 500 pg DNA (É^ - l^aíÉĚ nucleus: 20 % of cell mass \ '• . ' faj^l DNA mass: 5 pg (MCF 7 cells) ^Hfi^^^iii^H Proteins 10% of cell mass = 2 fmol "^ Esľif cone, of a protein: 200 zmol (1 zmol = 600 copies) Microtechnologies "lab-on-chip" ■ á ! ^ Prof. Dr. Andreas Manz HeadofthelSAS Dortmund J. Michael Ramsey Minnie N. Goldby Distinguished Professor of Chemistry Department of Chemistry The University of North Carolina at Chapel Hill Chapel Hill, NC USA CD microfluidic device - Gyros AB Confocal microscope detection system PMT CCD camera laser 532 nm objective 25 x 0.65 lightproof curtain 610 nm BP 550 nm LP CD assembly CD device assembly and detection system Separation of DNA restriction fragments ■ Separation medium: 2% LPA (10 MDa) inlOOmMTris-TAPS ■ Channel length: 4 (8) mm ■ Electric field: 75 V/cm wAuA* | 0.68- 1 *»«4«nJJ^w«.w*!;/*s>*i.i »njji 16 18 20 22 24 26 28 time [min.] Alkaline cell lysis Cell lysis in 0.1 M NaOH after 2 min Intact cell nucleus ■ Cardiomyocyte cell nucleus stained by ethidium bromide LIF microscopy: excitation - 532 nm; emission - 610 nm Alkaline cell lysis CI are beeing replaced by OH Electric field strength 6 DNA fragmentation in apoptotic cardiomyocytes treated by doxorubicin (2 ng/ml) I hJU-JJwvu^, 0,0 0,5 1,0 1,5 2,0 2,5 t time [min] Cell lysis: 3 min Electrophoresis: 2 % LPA (10 MDa) + 0.1 M NaOH electric field strength 60.2 V/cm temperature 25 °C Separation channel: 10 mm x 50 um x 20 um effective migration distance 6 mm LIF detection: excitation 532 nm emission 610 nm DNA stained by EtBr K. Klepárník, M. Horký, Electrophoresis 2003, 24, 3778 - 3783. Nanotechnologies "cell as a test tube" Single molecule detection Single molecule imaging Membrane proteins CD58-Cy3 (green) ICAM-1-Cy5(red) in a glass-bound planar phospholipid bilayer under two PMA/ionomycin-treated Jurkat cells. Total Internal Reflection Fluorescence Microscopy Disadvantages: ♦ Photo bleaching ♦ Molecules at the cell surface ♦ Two lasers: 514, 633 nm Parallel single molecule sequencing by synthesis Helicos The HeliScope™ Sequencer 2. KPb/day 109 reads/run 25 - 55 bp read lengths 'f J^ SCIENCES Genome Sequencer FLX System 3.1 (ŕ b f day 100 Mb/7,5 hour run 400 000 reads/7,5 hour Solexa llumina Genome Analyzer 6,10^b/day 3. 10s b/ 5 days run 50, 10s oligo clusters 200 - 300 bp read lengths 36 - 50 bp read lengths The HeliScope™ Sequencer Photocleavable dideoxy nucleotides f»j. 1. tHV. (ffoiliKt P UIPIRUHIÚ bKrtm, lormtd bf mwpoťJbnj a iŘJtP-Ťí ĚO.H r\t&■* prvTKf n jpotvrr*»i/x rniítum «id iti pIkTkV^w*^-. pioducMig UNA 1ijqiiwit F4B1M»mlPt-ROtMW. mafcoJn wn^iL 15