MOLEKULÁRNÍ BIOLOGIE II

Transkript

1 MOLEKULÁRNÍ BIOLOGIE II Interakce DNA/protein DNA/protein

2 Imobilizační/migrační techniky Filter binding Southwestern MacKay DNA affinity Gel Shifts Gel Filtration Sucrose Gradient Interakce DNA/protein Elektronová mikroskopie ds x ss DNA; DNA unwinding DNA wrapping around proteins Protein conformational changes Protection Assays (positional information) Protection Exo Protection Modif-Interference Genomic Footprint (in vivo) Indirect Endlabeling (in vivo)

3 Interakce DNA/protein Crosslinking Affinity enhancement for EM or immobilization/ migration assays Identifying specific contacts on protein or DNA In Vivo Hints One/Two/Three-hybrid Allele specific 2nd site suppression Atomic Level Analysis Crystal Structure NMR

4 Filter Binding (DNA/pure/partial/rapid) Pure protein immobilization by adsorption to nitrocellulose filter Labeled DNA passes through unless bound to protein Useful for quantitating binding affinities and kinetics Note: all proteins retain binding ability when adsorbed to filter * * * * * proteins protein-dna complexes * * * * free DNA

5 DNA Affinity Chromatography (protein/crude or pure/partial/slow-but very high on rate) DNA immobilized at high density to matrix by adsorption or covalent linkage Bound proteins elute at higher salts At lower salts high DNA conc favors rapid reassociation whenever protein dissociates Useful purification step (proteins monitored by activity or western if Ab available) Specific binding proteins separated from nonspecific binding proteins either by loading column in competition with free nonspecific DNA or on the basis of higher salt elution required for specific binding proteins Variation: bind proteins to biotin labeled DNA before immobilizing DNA to streptavidin matrix lose advantage of large excess of DNA low salt load high salt elution

6 Southwestern (DNA/crude or pure/partial/slow) DNA binding domain of protein renatures enough on nitrocellulose filter (after SDS-PAGE and Western) to allow for specific recognition of labeled DNA Useful for identifying which protein in a crude extract or in a tightly bound complex is responsible for DNA binding (proteins that do not bind offer additional specificity control) Assumption: a single polypeptide is sufficient for binding activity * probe with labeled fragment autorad.

7 McKay (DNA/crude/partial/slow) Immobilize protein by immunoprecipitation Labeled DNA bound to protein coips Specificity determined by preferential coip of specific DNA out of mixture containing nonspecific DNA Variations: cross-link before IP to counter problem of dissociation during washes detect DNA by PCR * * * * * * * * * * fragment with binding site input DNA IP d DNA

8 Gel Retardation with Antibody Supershift: Use to identify proteins in gel-shift complex - Ab + Ab * supershifted DNA shifted DNA free DNA

9 Gel Retardation with Protein Induced DNA Bending: If bending is induced, circularly permuted fragments will gel shift differently. The closer the binding site is to the fragment center the greater the shift. shifted DNA free DNA

10 Gel Filtration (protein/pure/partial/slow) Large DNA molecules elute in void volume with any bound proteins Free proteins elute in included volume Protein DNA + Bound Protein Free Protein Fraction V o Included Volume DNA DNA-protein VOID VOLUME Free protein INCLUDED VOLUME

11 Sucrose Gradient (DNA/crude or partial/partial/slow) DNA coated with proteins sediments faster than naked DNA Useful for very large protein DNA complexes or extensive coating of DNA (e.g. chromatin association) protein-coated DNA naked DNA DNA increasing density Increasing Size Fraction dripped from tube bottom Sucrose Sedimentation Gradient

12 Gel Retention/Retardation/Mobility Shift (DNA/crude or pure/partial/ fast ) Protein binding retards motility of labeled DNA Once complex has entered gel caging effect favors rapid reassociation whenever protein and DNA dissociate (effectively reducing off rate) Useful for following DNA binding activity during purification Has also been used to quantitate binding affinities and kinetics Multiple complexes can sometimes be seen as different shifted species Note: not all complexes enjoy caging effect or are fully stable during time it takes to enter gel Simple Shift ex: Multiple Shifts shifted DNA * free DNA * extract amount

13 Kvasinkový dvouhybridní systém návnada lovec

14 Kvasinkový dvouhybridní systém pter&i= &s=09000&n=00020&o=%7c09000%7c

15 Bakteriální dvouhybridní systém Bacterial Adenylate Cyclase Two-Hybrid System Katalytická doména adenylátcyklázy (CyaA) z Bordetella pertussis se skládá ze dvou vzájemně se doplňujících fragmentů T25 a T18, které nejsou aktivní, když jsou fyzicky oddělené.

16 Bakteriální dvouhybridní systém

17 Fágový display -PHAGE DISPLAY SYSTEM

18 Fágový display Plant Molecular Biology 50: , 2002.

19 MOLEKULÁRNÍ BIOLOGIE II Sekvenování, NGS Od restrikčních map k NGS selvenování

20 Fyzikální mapování DNA Konstrukce restrikčních map Restrikční mapa schématické znázornění poloh a vzdáleností restrikčních míst na molekule DNA

21 Sekvenování

22 Strategie sekvenování dlouhých segmentů Sangerovo sekvenování, jednostranné čtení cca 600 (800) bp Genomová knihovna (BAC), klonované segmenty cca stovky kb Primer walking První sekvenační primer je navržen z okrajové části vektoru, sekvenujeme cca 600 bp, navrhneme v této oblasti nový sekvenační primer a sekvenujeme opět cca 600 bp a tak postupujeme až přečteme celou sekvenci. není potřeba jednotlivé fragmenty klonovat sekvenci skládáme postupně, překryvy dílčích sekvencí jsou známé, repetitivní sekvence nejsou problém nevýhodou je doba analýzy (postupné navrhování a syntéza primerů) maximální délka sekvenovaných fragmentů je 5 10 kb Shotgun sequencing sekvenuji se vysoké počty (miliony) krátkých úseků původní DNA zaklonovaných do vektorů k fragmentaci DNA se používají RE nebo fyzikální metody (ultrazvuk), vznikají tak krátké fragmenty, které se z části překrývají. Díky překryvům je možné získané sekvence poskládat do správného pořadí výhodou je rychlost a nízká cena

23 Primer walking

24 Shotgun

25 NEXT GENERATION SEQUENCING

26 Molekulární biologie včera a dnes

27 Sekvenování příští generace

28 Next generation sequencing

29 Next generation sequencing

30 Next generation sequencing

31 Next generation sequencing

32 Emulzní PCR

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34 Bridge PCR Cappelletti_NGS09.pdf

35 Bridge PCR

36 Bridge PCR

37 Next generation sequencing

38 Pyrosekvenování Adaptors essential for purification, amplification and sequencing are added to both ends of the fragments. If the sample is double stranded one strand is removed and the remaining single strandes are used in the following steps. Aided by the adaptors individual fragments are captured on their own unique beads. A bead and the bound fragment together with a water-in-oil emulsion form a microreactor so that each fragment can be amplified without contamination via the so called emulsion PCR (empcr). The entire fragment collection is amplified in parallel.

39 Pyrosekvenování The empcr amplifies each fragment several million times. After amplification the emulsion shell is broken and the clonally amplified beads are ready for loading onto the fibreoptic PicoTiterDevice for sequencing. The PicoTiterPlate is loaded with one fragment carrying bead per well and smaller beads with the enzymes necessary for sequencing.

40 Pyrosekvenování DNA polymeráza ATP sulfuryláza luciferáza apyráza adenosine 5' phosphosulfate (APS) luciferin DNA Polymerase: used to elongate complementary DNA strand. ATP Sulfurylase: converts pyrophosphate (PPi) into ATP in the presence of adeosine 5' phosphosulfate (APS). Luciferase: Uses ATP to convert luciferin to oxyluciferin, which is a molecule that emits visible light. Apyrase: Degrades unincorporated nucleotides.

41 Pyrosekvenování

42 SOLEXA/ILUMINA

43 ILLUMINA sekvenování syntézou Cappelletti_NGS09.pdf

44 SOLiD sekvenování ligací Mardis, Annu. Rev. Genomics Hum. Genet., 2008

45 SOLiD sekvenování ligací

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68 SOLiD sekvenování ligací

69 SOLiD sekvenování ligací

70 SOLiD sekvenování ligací

71 Ion Torrent

72 Ion Torrent

73 Helicos - True Single Molecule Sequencing

74 Helicos

75 Helicos

76 Pacific Biosciences Single Molecule, Real-Time (SMRT) Sequencing

77 Oxford Nanopore

78 Oxford Nanopore