RECOMBINANT DNA TECHNIQUES and PROTEIN ENGINEERING

1. RECOMBINANT DNA TECHNIQUESandPROTEIN ENGINEERING Methodstostudygeneexpression Nextgenerationsequencing

2. RNA level: northernblot RT-PCR gene-chip differential display FISH, etc Protein level: enzymatic activity western blot 2D elpho – MALDI MS affinity chromatography Immunprecipitation TAP immuno histochemistry • detection of DNA-proteininteractions: • electrophoretic mobility shift • footprint • methylation protection • methylation interference

3. NORTHERN BLOT

4. HuIVTry leader actin pankreas cortex cortex cortex cerebr. fehér putamen cortex marker HuIVTry pro HuIVTry signal Distributionof trypsin in differentbrain regions RNA isolation Trizol RT-PCR

5. Comparison of RNA expression level

6. Electroforetic Mobility Shift Assay

7. Footprinting Cleavage: DNase I Hydroxyl radical Methylation protection DMS, NaOH Methylation interference

9. DNase I footprint of CRP (CAP) and lac repressor with lac promoter DNA. • Lane a: Sequence ladder. Lane b: No DNA-binding proteins added. • Lane c: CRP (CAP) only. • Lane g: Repressor only. • Lanes d, e, and f: CRP (CAP) and repressor both added. • Science274, 1930-1931 (1997) When CRP binds to its site, repressor binding at O3 is shifted upstream by 6 bp. This puts CRP and repressor on opposite sides of the DNA.

11. Reporter gene assay

12. CAT assay

20. Roche 454 sequencer

21. Steps of thesequencingprocess • Generation of DNA fragment (nebulizer) • Ligation of adapters, singlestrandsboundtobeads • Emulsion PCR foramplificationonthebeads • Sequencingbysynthesisinpicolitervolumewell • Lumimetricdetection of pyrophosphaterelease

22. dsDNA fragments Bio P P A B Ligation Bio Fill in Bio Bio Bio Capture on SA-Beads & Wash Alkaline Elution A B • DNA Capture Beads • N-hydroxysuccinimide ester (NHS)-activated Sepharose HP • (5’-Amine-hexa-ethyleneglycol spacers CCATCTGTTGCGTGCGTGTC-3’) • hybridization at limiting dilution (A seq.) • Amplification in emulsion (ePCR) • 0.625 µM forward (5’ - CGTTTCCCCTGTGTGCCTTG-3’) • 0.039 µM reverse primers (5’-CCATCTGTTGCG TGCGTGTC-3’)

23. 44 um in diameter • 55 um in depth • 75 pl • 480 wells/mm2 • 1.6 million wells Incubation of DNA beads(25 um) with Bst DNA polymerase, Large Fragment and SSB protein Dynal enzyme beads UltraGlow Luciferase and Bst ATP sulfurylase were prepared biotin carboxyl carrier protein (BCCP) fusions

24. Principle of pyrosequencing

25. Substrate (300 µM D-luciferin and 2.5 µM adenosine phosphosulfate) • The flow order of the sequencing reagents • PPi flow (21 seconds), followed by 14 seconds of substrate flow, 28 seconds of apyrase wash and 21 seconds of substrate flow – calibrationstep • first PPi flow was followed by 21 cycles of dNTP flows, each dNTP flow was composed of 4 individual kernels: (dNTP-21 seconds, substrate flow-14 seconds, apyrase wash-28 seconds, substrate flow-21 seconds); an image is captured after 21 seconds and after 63 seconds.

26. Life Technologies -Iontorrentsequencing • Samplepreparationsimilarto 454 – basedon PCR amplification • Sequencingbysynthesis – sequentialadditon of dNTP-s • detection of released H+ - CMOS transformedintominiature pH meter

27. Helicos - tSMS • Truesinglemoleculesequencing – no PCR amplification • ImmobilizedoligoTon chip • polyAtailing of fragmented (~100 -200 base) DNA – last A fluorescent • Polymerizationusingvirtualterminatornucleotides • Paralellreadingbillions ofsequences • Oneday – 1000 $ genome • Bankruptcyin 2012 

28. PacificBiosciences - SMRT • Singlemoleculerealtimesequencing • Phospholinkedfluorescentnucleotides – polymerasecleavesofflabel • ZeroModeWaveguide – nanophotonicvisualizationchamber – illuminatedvolume 20 zeptoliter (10-21) • single polymerase molecule bound to the bottom ofthe chamber - real time sequence reading

29. PacificBiosciences - SMRT

30. Oxford Nanopore Technologies Nanopore Sequencing

31. Introduction to nanopore sensing A nanopore: a nano-scale hole. • Biological: a pore-forming protein (e.g. α-Hemolysin) in a membrane (e.g. lipid bilayer) • Solid-state: in synthetic materials ( e.g. silicon nitride or graphene) • Hybrid: formed by a pore-forming protein set in synthetic material

32. Nanopore sensing Ionic current passed through membrane by setting a voltage across the membrane. • Disruption in current detected when analyte passes through the pore • or near its aperture. • Characteristic disruption indentifies the molecule in question.

33. Nanopore DNA sequencing • DNA polymer or individual nucleotides pass through the nanopore. • Detected by • a adaptor molecule ( e.g. Cyclodextrin). • Tunnelling electrodes based detectors. • Capacitive detectors • Graphene based nano-gap or edge state detectors.

34. Nanopore DNA sequencing • Strand sequencing: • Sequencing in real-time as the intact DNA polymer passes through the nanopore. • Exonuclease sequencing: • Individual nucleotides pass through the nanopore by the aid of processiveexonuclease.

35. Strand Sequencing Snapshot from movie at http://www.nanoporetech.com

36. Electron-based read out Four different magnitudes of disruption which can be classified as C, G, A or T Modified base, e.g. methylated cytosine, can be directly distinguished from the four standard bases

37. Strand Sequencing • Hairpin structure: • Sense and anti-sense sequencing • Advantages in Data Analysis Snapshot from movie at http://www.nanoporetech.com

38. Exonuclease Sequencing Snapshot from movie at http://www.nanoporetech.com

39. Exonuclease Sequencing • Adapter molecule (cyclodextrin): • Accuracy averaging 99.8% • Identification of meC Snapshot from movie at http://www.nanoporetech.com

40. Working strategy • MinION: a miniaturised sensing instrument • Portable. • Field-deployable. • Requires minimal sample prep. • Compatible with blood serum, plasma and whole blood.

41. Working strategy • GridION system • Uses single-use, self-contained cartridge. • Can be used as a single instrument: Node • Can be used in a cluster, connected through network. • Low power and space required. • Permits scheduling and multiplexing.

42. Workflow versatility • No fixed run time • Can be run one or more nodes for minutes or days. • Data analysis takes place in real time. • Longer run enables collecting more data points. • Run until... sufficient data • The GridION system enables users to run an experiment until sufficient data has been collected to reach a predetermined experimental endpoint.

43. Advantages over present sequencing technologies • Real-time sequencing strategy. • No strand amplification needed. • No bias due to sequencing amplification. • Low cost: trying to fulfil the target of $1000 per human genome. • Lager read size: read size is limited only by preparation. • No requirement for large amounts of high-performance disk storage. • Large-scale structural variation can be detected at lower depth of coverage. • Enable long-range haplotyping. • No need for expensive and time-consuming mate pair library construction.