Karel Klepárník Oddělení bioanalytické instrumentace Ústav analytické chemie Akademie věd České republiky Brno Moderní analytická instrumentace pro genetický výzkum, lékařskou diagnostiku a molekulární identifikaci organizmů Ústav analytické chemie AVČR Veveří 97 60200 Brno www.iach.cz Polymerase chain reaction PCR amplification The Nobel Prize in Chemistry 1993 Kary B. Mullis born 1944 La Jolla, CA, USA University of British Columbia For his invention of the polymerase chain reaction (PCR) method PCR amplification scheme DNA template DNA dissociation 90 ºC Primer annealing 62 ºC DNA synthesis 72 ºC Correct copies N=2n+1 – 2(n+1) 1st cycle: n=1 22 – 2∙2 = 0 2nd cycle: n=2 23 – 2∙3 = 2 3rd cycle: n=3 24 – 2∙4 = 8 DNA primer DNA primer DNA sequencing Analysis of Sanger sequencing fragments DNA sequencing strategy Separation methods Capillary electrophoresis CE detection system outlet electrode chamber mobilityelectrophoretic electroosmotic B)+ mobility electrophoretic electroosmotic A)- high voltage inlet electrode chamber purge pressure detection window detail injection point separation capillaryBGE BGE capillary effective length (LD) capillary total length (LC) Capillary electrophoresis scheme Why capillary electrophoresis? T L R   4 22 0 RE TTT R  solid – solidair – solid LrdrUUdIQJ /22  dr dT rLQC 2 T0 TR ΔT Miniature capillary: low R => fast separation 1) high resistivity  low current at high voltage  low heat production 2) efficient heat transport  low temperature difference inside the capillary DNA electromigration K. Klepárník, P. Boček, DNA diagnostics by Capillary Electrophoresis Chemical Reviews 107, 5279 – 5317, 2007. DNA primary structure Homogeneous polyelectrolyte DNA electromigration regimes in sieving media Size separations of homogeneous polyelectrolytes are impossible in free solutions Short DNA fragments Low concentration of media Long DNA fragments High concentration of media log M log Ogston sieving reptation without stretching reptation with stretching Rs  m m  1/M Rs < m Rs > m a b c 0 m m Dependence of DNA electrophoretic mobility on molecular mass 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 the first assembly of the human genetic code from the whole genome shotgun sequencing method. Starting only nine months ago on September 8, 1999, eighteen miles from the White House, a small team of scientists headed by myself, Hamilton O. 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 genetic code of five individuals.... …There would be no announcement today if it were not for the more than $1 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 Institute 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. et al. Science 2001, 291, 1304-1351. Fluorescence chemistry Lloyd M. Smith Born 1954 A.B. 1976, University of California - Berkeley Ph.D. 1981, Stanford University University of Wisconsin - Madison Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, C. R., Heiner, C., Kent, S. B. H. and Hood, L. E. Fluorescence detection in automated DNA sequence analysis Nature, 321, 674-679, 1986. N C S O- O C N+(CH3)2O(CH3)2N NH2-R NO2 NH(CH2)5 C O O N O O NH2-R CH2CH2 F F B N N H3C H3C C O O N O O NH2-R -O3S SO3 N+ N O O O O N NH2R n n=1: Cy3 n=2: Cy5 n=3: Cy7 N N+ O SO O Cl NH2-R N C S C O OH OOHO NH2-R Fluorescein Rhodamine Texas Red NBD BODIPY Cy3,5,7 Fluorescent lebels Cy3 488 nm 610 nm ROX ACCEPTOR DONOR PRIMER SEQUENCE Sequencing primer attached to Fluorescence Resonance Energy Transfer NH 5'-TTTTCCCAGTCACGACG-3' (CH)2(CO) NH (CH2)6 C O COOH O N+ N O O N N N N O NH2 O O -O P O (CH2)6 NH O C (CH2)5 N+ CH3 C2H5 O CH CH CH O N CH3 N(CH3)2(CH3)2N O CO2 - Cl Cl O NH O NH OO -O -O2C O H N O O NH ON O 3-HO9P3O ACCEPTOR DONOR ddTTP TERMINATOR 595 nm 488 nm Dideoxy terminator attached to Fluorescence Resonance Energy Transfer Prof. Richard A. Mathies University of California at Berkeley Department of Chemistry Berkeley, CA LIF detection Ar-ion laser 40 mW separation capillary ID 50 mm objective 40x; 0.65 blocker 520 nm beam splitter band pass 610 nm PMT blocker 520 nm band pass 540 nm band pass 590 nm band pass 570 nm 50% 488 nm 50% 514 nm lens Four channel LIF detection arrangement Spectral filtering SENSOR LASER PINHOLE OPTICS BEAM SPLITTER MICROSCOPE OBJECTIVE FOCUS SCHEME OF CONFOCAL DETECTOR Space filtering Prof. Edward S. Yeung Ames Laboratory U.S. Department of Energy Iowa State University. excited sample laser beam polymer filled capillaries sheath-flow cuvette open tubings electrode chamber electrode chamber Sheath-flow cuvette Prof. Norman Dovichi University of Washington Seattle, WA, USA Prof. Hideki Kambara senior chief scientist Hitachi Central Research Laboratory Tokyo, Japan 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 Barry L. Karger James L. Waters Professor of Analytical Chemistry The Barnett Institute Northeastern University Boston MA 96 active eight reserve capillaries ABI PRISM® 3700 DNAAnalyzer Sheath flow cuvette ABI PRISM® 3700 DNAAnalyzer ABI PRISM® 3700 DNAAnalyzer DNA sequencing record PE Applied Biosystems ABI PRISM 3700 accuracy > 98.5% to 550 base 96 samples per run in 3 hours laser Ar-ion 488 and 514.5 nm detection in sheath flow concave spectrograph and cooled CCD Molecular Dynamics MEGABACE 1000 accuracy > 98.5% to 550 base 96 samples per run in 2 hours laser Ar-ion 488 nm energy transfer dyes confocal scanning with 4 filters and 2 PMTs DNA mutation analysis Restriction (amplification) fragment legth polymorphism RFLP (AFLP) Size based separation of ds or ss DNA fragments Resolution: ss > 1000 ds > 400 Single Strand Conformation Polymorphism SSCP wild type point mutation native dsDNA denatured ssDNA native environment Principle of SSCP technique dsDNA ssDNA dsDNA relativeabsorbanceat260nm a) health homozygote time ssDNA 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. Phenylketonuria b) heterozygote Single nucleotide primer extension Minisequencing SNuPE Next generation sequencing Single molecule detection Single molecule reaction monitoring Helicos The HeliScope™ Sequencer 2 . 109 b/day 109 reads/run 25 – 55 bp read lengths Genome Sequencer FLX System 3 . 108 b/day 100 Mb/7.5 hour run 400 000 reads/7.5 hour 200 – 300 bp read lengths Solexa Illumina Genome Analyzer 6 . 108 b / day 3 . 109 b / 5 days run 50 . 106 oligo clusters 36 – 50 bp read lengths Parallel single molecule sequencing by synthesis Photocleavable dideoxy nucleotides Single molecule real time sequencing (SMRTTM) Pacific Biosciences Next generation DNA sequencing DNA sequencing – DNA polymerase RNA sequencing – reverse transcriptase Codone-resolved translation elongation by single ribosomes Tens of nucleotide peaks in 1 sec Read length 1 – 15 kb 80 000 detection points 15 min/genome: 50 n/s * 80 000 points * 15 min * 60 s = 3.6 Gb DNA polymerase 529 processivity 20 kB – 400 b/s Some enzymes are not processive $ 100/genome 49 PacBio RS instrument 50 Single molecule real time sequencing 51 Pacific Biosciences Read Length 52 Pacific Biosciences Read Length 53 Pacific Biosciences Single molecule real time sequencing SMRTTM www.pacificbiosciences.com DNA sequencing – DNA polymerase RNA sequencing – reverse transcriptase Codone-resolved translation elongation by single ribosomes Tens of nucleotide peaks in 1 sec Read length 1 – 15 kb 80 000 detection points 15 min/genome: 50 n/s * 80 000 points * 15 min * 60 s = 3.6 Gb DNA polymerase 529 processivity 20 kB – 400 b/s Some enzymes are not processive $ 100/genome Hydrogen ion is released as a byproduct when a nucleotide is incorporated into a strand of DNA by a polymerase High-density array of micro-machined wells. Each well holds a different DNA template. Beneath the wells is an ion-sensitive layer and a proprietary ion sensor. If a nucleotide is added to a DNA template and is then incorporated into a strand of DNA, a hydrogen ion is released. The charge from that ion will change the pH of the solution. The world's smallest solid-state pH meter—will call the base. The sequencer sequentially floods the chip with one nucleotide after another. If the next nucleotide that floods the chip is not a match, no voltage change will be recorded. If there are two identical bases on the DNA strand, the voltage is double, and the chip records two identical bases. Ion Torrent The Ion Personal Genome Machine (PGM™) sequencer Different templates in microwells Washing steps by individual nucleotides G, C, T, A The world's smallest solid-state pH meter Digital output http://www.iontorrent.com/ Single molecule passage through a pore Oxford Nanopore Technologies Schematic of the nanopore device. DNA sequencing development 2001: Genome draft of 5 individuals in 9 months – more than billion $ 2015: Complete human genome in an hour – ~100 $ 63