Methoden der Molekularbiologie Nachweis von DNA - Agarose … · Seite 1 Riedel | Molekularbiologie, Biotechnologie, Gentechnik 02 | WS2015/16 Methoden der Molekularbiologie Nachweis

Seite1 Riedel|Molekularbiologie,Biotechnologie,Gentechnik02|WS2015/16

MethodenderMolekularbiologie

NachweisvonDNA-Agarose-Gelelektrophorese


DNA-Analyse:Agarose-Gelelektrophorese

MethodezurTrennungvonDNA-MoleküleneinesGemischesanhandihrerGröße

Grundlage:

  DNAdurchPhosphatrestenegaPvgeladen

  DNAwandertdaherinelektrischenFeldernzurAnode(+Pol)

+ + + +

− − − −


DNA-Analyse:Agarose-Gelelektrophorese

Agarose:PolysaccharidausDimerenvonD-Galaktoseund3,6-Anhydro-L-Galaktose

Prinzip:TrennungnachGrößedurchRetenPonineinemAgarosegel(NetzausAgarosefasern)ineinemElektrischenFeld


DNAQuanPtätundQualität

Prinzip:TrennungnachGrößedurchRetenPonineinemAgarosegel(NetzausAgarosefasern)ineinemElektrischenFeldDurchdasGelwerdendabeigrößereFragmentestärkerzurückgehaltenalskleine.

+ + + +

− − − −



Prinzip:TrennungnachGrößedurchRetenPonineinemAgarosegel(NetzausAgarosefasern)ineinemElektrischenFeldDurchdasGelwerdendabeigrößereFragmentestärkerzurückgehaltenalskleineZugabevonFarbstoffenermöglichenesdenEndpunktderElektrophoresezubesPmmen

DünneGele=geringereDiffusion=schärferBanden



Prinzip:TrennungnachGrößedurchRetenPonineinemAgarosegel(NetzausAgarosefasern)ineinemElektrischenFeldDurchdasGelwerdendabeigrößereFragmentestärkerzurückgehaltenalskleineZugabevonFarbstoffenermöglichenesdenEndpunktderElektrophoresezubesPmmenDieAgarosekonzentraPonbesPmmtdiePorengrößeunddamitdieAuflösungdesGels

%Agarose Auflösung(Bp)

0,6 20000-1000

0,7 10000-800

0,9 7000-500

1,2 6000-400

1,5 3000-200

2 2000-50

1%Agarose=150nmPoren

0,16%Agarose=500nmPoren



NacherfolgterAufrennung:SichtbarmachungderFragmentedurchFärbungmitz.B.EthidiumbromidundBetrachtungunterUV-Licht

Ethidiumbromid


DNAQuanPtätundQualität AbschätzungvonGrößeundMengedereinzelnenFragmenteProtocol for Loading

Step 1: Mix gently Step 2: Load 1 µl per 1 mm gel lane

Recommendations x Do not heat before loading. x Dilute your DNA sample with the 6X DNA Loading Dye

(#R0611, supplied with the ladder): mix 1 volume of the dye solution with 5 volumes of the DNA sample;

x Load the same volumes of the DNA sample and the DNA ladder;

x For quantification, adjust the concentration of the sample to equalize it approximately with the amount of DNA in the nearest band of the ladder.

x For DNA band visualization with SYBR® Green, GelRed and other intercalating dyes, do not add the dyes into the sample, use gel staining after electrophoresis or include dyes into agarose gel to avoid aberrant DNA migration.

NOTICE TO PURCHASER: LIMITED LICENSE This product or its use is covered by at least one claim of U.S. Patent Nos. 5,834,201, 6,680,378, and/or 7,132,520 owned by Invitrogen Corporation. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product in internal research conducted by the buyer. The buyer cannot use this product or materials made by the employment of this product for Commercial Purposes. Commercial Purposes means any activity for consideration and may include, but is not limited to: (1) use of the product in manufacturing; (2) use of the product to provide a service, information, or data; (3) use of the product for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product, whether or not the product is resold for use in research. Further information on purchasing licenses under the above patents may be obtained by contacting the Business Development Department, Invitrogen Corporation, 5791 Van Allen Way, Carlsbad, CA 92008. Email: [email protected].

GeneRuler 1 kb DNA Ladder, ready-to-use

PRODUCT USE LIMITATION This product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals. Please refer to www.thermoscientific.com/onebio for Material Safety Data Sheet of the product.

© 2012 Thermo Fisher Scientific Inc. All rights reserved. SYBR is a registered trademark of Molecular Probes, Inc. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.

Anwendungen:  AufrennungkomplexerGemische  AufreinigungundNachweisvonbesPmmten

Sequenzabschniken  RestrikPonsanalyse

  ...  SowohlanalyPschalsauchpräparaPv

AnimaPonAgarose-Gelelektrophorese



ModifikaPonvonDNA:RestrikPonsenzyme


RestrikPonsendonukleasen

BisherigeMethodenfürArbeitenmitDNA:  IsolaPon  QualitätskontrolleundQuanPfikaPon  VervielfälPgung(PCR)  Agarose-Gelelektrophorese

WeitereArbeitsschrike,diefürdieKlonierungvonDNAwichPgsind:  Schneiden

RestrikPonsenzyme=molekulareScheren,dieDNAschneiden


RestrikPonsendonukleasenEcoRI:eineRestrikPonsendonukleaseausausE.coli

  BeiBakterienundArchaeen

  ErkennenundschneidenspezifischeDNA-abschnike(DNA-MoPve)


R-MSysteme

  TeilderRestrikPons-ModifikaPons-Systeme(R-MSysteme)

  DienenderAbwehrvonFremd-DNA(z.B.Phagen;„RestrikPon“=Abwehr/HemmungvonViren)

  EssenPelleTeilevonR-MSystemen:  Methylase-  RestrikPons-  Sequenzerkennungsdomänen  teilweiseaufunterschiedlichenProteinen

bbif1095 bbif1094 hsdM hsdS hsdR bbif1089

2,000 4,000 6,000 8,000 10,000 12,000 bp

integrase type II Rease bbif0712 type II Mtase

1,000 2,000 3,000 4,000 bp

TypIR-MSystem

TypIIR-MSystem

EcoRI:eineRestrikPonsendonukleaseausausE.coli

XhoI Isoschizomer


R-MSysteme

  Methylase(MTase)und(REase)RestrikPonsendonukleaseeinesBekteriumserkennendieselbenDNA-MoPve

  MTase➜MethylierungdesMoPvs

  REaseschneidetnurwenndieerkanntenDNA-MoPveNICHTmethyliertsind

  BeigleichzeiPgerExpressionvonMTaseundRease:eigeneDNAgeschützt,Fremd-DNAwirdabgebaut

M 1 2 3 4 5

NachweiseinesXhoI-ähnlichenR-MSystems:

M Größenmarker(1kbLeiter)

1 PlasmidX

2 PlasmidX+XhoI

3 ChromosomaleDNA

4 ChromosomaleDNA+Xho

5 ChromosomaleDNA+EcoRI


R-MSysteme

Bislang4UnterklassenvonR-MSystemenbeschrieben:TypI-IV

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R-MSysteme

Bislang4UnterklassenvonR-MSystemenbeschrieben:TypI-IV

TypI TypII TypIII

AuuauEnzymkomplexausHsdS,HsdMundHsdRtypischerweiseM2S2R

MTaseundREasebeidemiteigenerErkennungsdomäne

ModundResbeidebenöPgtfürSchnikModalleineakPv

Erkennungs-sequenz

2-teiligasymmetrisch

4-8Basen(u.U.mehr)Meistpalindromisch(OTTO)

5-7Basenasymmetrisch

Spaltstellebis1000bpaußerhalbderErkennungssequenzUnspezifisch

InnerhalbodernahederderErkennungssequenz

5-20BasenvorderErkennungssequenz

ATP-Bedarf Ja Nein ja

TypIV:schneidetnurmethylierteSequenzmoPve

InteressantfürmolekularbiologischeAnwendungen


TypIIRestrikPonsendonukleasen

  „Schneiden“derDNAdurchDurchtrennendesZuckerphosphat-RückgratesanbeidenSträngen

EcoRI

Erkennungssequenz 1.Schnik

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G




  AuurechenderWasserstozrückenzwischendenkomplementärenBasen(SchmelzenderBindung)

EcoRI

Erkennungssequenz 2.AuuruchderH-Brücken

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G

3‘

5‘

5‘

3‘G A A T T C: :C T T A A G




  AuurechenderWasserstozrückenzwischendenkomplementärenBasen(SchmelzenderBindung)

  TrennenderEnden

EcoRI

Erkennungssequenz 3.TrennungderEnden

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G

3‘

5‘

5‘


3‘

5‘

5‘




Einfacherdargestellt:

EcoRI

Erkennungssequenz Schnikstelle

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G

5’-GAATTC-3’3’-CTTAAG-5’

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G

5’-GAATTC-3’3’-CTTAAG-5’

^

^



EinigeBeispielefürhäufigverwendete(TypII)RestrikPons-endonukleasenundihreSchnikstellenund-muster

EcoRI

XhoI

PstI

NdeI

ClaI HpaI

SmaI

PvuI

KpnI

SacII

JenachEnzym:

  3‘-oder5‘-Überhängevon2-4Basen(„sPckyends“)

  KeinÜberhang(„bluntends“)

AccII AscI




  MancheEnzyme:gleichErkennungssequenzaberandererSchnik

  MancheEnzymeunterschiedlicheErkennungssequenz,abermitmitkompaPblenEnden(Überhängenpassenzueinander)

XhoI

SalI

kompaPbleEndenGleicheErkennungssequenzandererSchnik

KpnI

Acc65I

WichPgfürdieAuswahlvonRestrikPonsenzymenbeiderPlanungvonKlonierungen




XhoI

BbiI

GleicheErkennungssequenzandererSchnik

KpnI

Acc65I

Neoschizomere Isoschizomere

GleicheErkennungssequenzgleicherSchnik

EnzymaberausanderemOrganismus

M 1 2 3 4 5

M Größenmarker(1kbLeiter)

1 PlasmidX

2 PlasmidX+XhoI

3 ChromosomaleDNA

4 ChromosomaleDNA+Xho

5 ChromosomaleDNA+EcoRI


RestrikPonsverdauinderMolekularbiologie

InderPraxis:AnsatzfüreinenanalyPschenResrikPonsverdau:

1µlDNA-lösung(1µg)2µl10xPuffer1µlRestrikPonsendonuklease(2U)16µlH2O

InkubaPonfür1-2h,inderRegelbei37°C

TypischerPuffer:40-100mMTris-HClpH7.5-8.5100-500mMNaCl/KCl10mMMgCl21mMDTE(Dithioerytriol)

Pufferbedingungen,InkubaPonstemperaturund–dauerkönnenjenachEnzymvariieren

EnzymaPscheAkPvitätderRestrikPonsenzymehängtstarkvondenPufferbedingungenab

MancheEnzymesindbeifalschen(nichtopPmalen)PufferbedingungeninakPvoderhabeunspezifischeAkPvität(schneidenfalsch)

➜Star-AkPvität!!!

AkPvität(Menge)derEnzymeinUnits(U)Standard-Enzym:1U=1µmolSubstrat/min



RestrikPonsendonukleasenwerdeninderMolekularbiologievorAllemeingesetztfür:

1.   AnalyPscheVerdaus(physikalischeKarPerung),DNAMolekülekönnenanhandihrerRestrikPonsmusteridenPfiziertbzw.karPertwerden.

cence are critical (e.g., in comparisons of wild-type and mutantstrains), relative levels of luminescence or copy numbers ofintegrants should be verified. Eryr colonies were checked forlight emission, and the integration of p16Slux was confirmedby PCR using primers 16S_rev_XhoI and 16S_fwd_int (5!-ATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGG-3!).16S_fwd_int anneals upstream of 16S_fwd_Econew in the 16SrRNA gene sequence and yields a 1,150-bp PCR fragment onlyif p16Slux is integrated at the correct site in the chromosome.By using this protocol, the following organisms were renderedbioluminescent: E. coli DH10B, C. rodentium ICC169, Salmo-nella enterica serovar Typhimurium UK-1, P. aeruginosa PAO1,Enterobacter sakazakii DPC6440, Shigella flexneri 2a ATCC700930, and Y. enterocolitica NCTC13174 (Fig. 1B). In all lux-tagged strains, plasmid integration was stable in the absence ofantibiotic for at least 50 generations. All lux-labeled strainswere tested for differences in growth on complex media in aSpectraMax M2, 96-well plate reader (Molecular Devices,Sunnyvale, CA). At the time points indicated in Fig. 1, lumi-nescence was measured in relative light units (RLU, in photonss"1) with the IVIS100 imaging system. As shown in Fig. 1C forC. rodentium and S. enterica serovar Typhimurium UK-1, noneof the tagged strains exhibited a significant difference from thewild-type strain in growth rate and final optical density at 600nm on LB media (Fig. 1C and data not shown).

All gram-negative strains presented here produced intense lu-minescence (#1 $ 107 to 5 $ 107 RLU) in LB broth in 96-wellplates (Fig. 1C and data not shown). Luminescence in the gram-negative strains was approximately 10-fold higher than the lumi-nescence observed in L. monocytogenes EGDe::pPL2luxPhelp,

which harbors the same luxPhelp construct as a single-copy chro-mosomal integration (10). Also, luminescence in the gram-nega-tive strains was about 10-fold higher than that in the gram-positivestrains containing p16Slux replicating as a plasmid (data notshown). Additionally, while luminescence in L. monocytogenes(10) and other gram-positive bacteria (data not shown) decreaseddramatically in stationary phase, luminescence in all gram-nega-tive bacteria tested remained relatively stable throughout the en-tire growth curve (Fig. 1C and data not shown). Whether thesedifferences are due to the availability of the substrate of thereaction or to differences in the intracellular conditions remainsto be elucidated.

To assess the functionality of the Phelp-driven expression ofluxABCDE in gram-negative bacteria in vivo, three of the la-beled strains were tested in murine models of infection in 6- to8-week-old conventional female BALB/c mice and comparedto their wild-type strains. C. rodentium, S. enterica serovarTyphimurium, and P. aeruginosa were chosen as models fornoninvasive intestinal, invasive intestinal, and invasive pulmo-nary pathogens, respectively. Animals were kept in a conven-tional animal colony, and all experiments were approved by theanimal ethics committee of University College Cork. Animalswere infected by gavage (C. rodentium and S. enterica serovarTyphimurium) or intranasally (P. aeruginosa) with bacteriafrom overnight cultures washed with phosphate-buffered saline(PBS). Doses used were 2 $ 109 CFU/animal in 100 %l of PBSfor C. rodentium, 1 $ 107 CFU/animal in 100 %l PBS for S.enterica serovar Typhimurium, and 1 $ 106 CFU/animal in 10%l PBS for P. aeruginosa. At various time points during infec-tion (Fig. 2), animals were anesthetized with isoflurane and

FIG. 1. (A) Plasmid map of p16Slux with relevant restriction sites and arrangement of the E. coli DH10B 16S sequence (blue), the Phelp promoterregion (green arrow), and luxABCDE (red arrows). (B) Gram-negative strains tagged by chromosomal integration of p16Slux (i, E. coli DH10B::p16Slux;ii, C. rodentium ICC169::p16Slux; iii, S. enterica serovar Typhimurium UK-1::p16Slux; iv, P. aeruginosa PAO1::p16Slux; v, E. sakazakii DPC6440::p16Slux;vi, S. flexneri 2a ATCC 700930::p16Slux; vii, Y. enterocolitica NCTC13174::p16Slux). The color bar indicates bioluminescence signal intensity (in photonss"1 cm"2). Strains were grown on LB agar plates containing erythromycin under nonpermissive conditions and imaged using the Xenogen IVIS100imaging system. min, minimum; max, maximum. (C) Growth (symbols) and luminescence (bars) of wild-type (wt) and lux-tagged strains of C. rodentiumICC169 (C. rod) and S. enterica serovar Typhimurium UK-1 (S. typh) on LB medium. Data for luminescence are presented as mean RLU & standarddeviations of results for four wells, and the results from one representative of three independent experiments are shown. The top panel showsrepresentative wells containing the indicated strain in LB broth at the indicated time points.

VOL. 73, 2007 BIOLUMINESCENT IMAGING OF GRAM-NEGATIVE BACTERIA 7093

+ + + +

− − − −1. 2. 3.

1.   XhoI2.   PstI

3.   XhoI+PstI

10221bp

3660bp

5831bp

4390bp

730bp





RestrikPonsendonukleasenwerdeninderMolekularbiologievorAllemeingesetztfür:

2.   Klonierungszwecke,dabeiwerdenFragmentezunächstingewünschterWeisegeschnikenundanschließendwiederzusammengefügt(➜rekombinanteDNA)

genA%

Resistenzgen)

Replika.onsursprung)

Polylinker)

Promotor)genA

Of:AmplifikaPonderZiel-sequenzdurchPCRmitPrimern,diediegewünschtenSchnikstellenenthalten

genA

PCR

genA



ModifikaPonvonDNA:LigaPon



WeitereArbeitsschrike,diefürdieKlonierungvonDNAwichPgsind:  VerbindenvonFragmenten

Ligase=molekularerKleberfür(kompaPble)DNA-Enden


  Schneiden(RestrikPonsverdau


DNA-Ligase

AuchhierbedienenwirunsbeidenEnzymen,diefürdieDNA-ReplikaPonbenöPgtwerden:

BrockMikrobiologie,Abb.7.16BrockMikrobiologie,Abb.7.14


DNA-Ligase

FürmolekularbiologischeArbeitenmeistensT4Ligase(ausT4Phagen)

1.   Freie,kompaPbleDNAEndenfindensichundlagernsichloseaneinenader(H-Brücken)

2.   Ligaseknüpf1.Esterbindung

3.   Ligaseknüpf2.Esterbindung

4.   (ehemalsfreie)DNA-EndenanbeideSträngekovalentbebunden(ligiert)


DNA-Ligase

ZwischenproduktvorLigaPonstabilisiertdurchH-Brücken

BenöPgtfreie3‘-OH-UND5‘-Phosphat-Gruppen!!!!

3‘

5‘

5‘


-OH

-PO4-

3‘

5‘

5‘


OH-

PO4--

Ligase

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G


DNA-Ligase

LigaseverbindetauchEndenohneÜberhang(Blunt)

3‘

5‘

PO4-

OHG T T: : : C A A

HpaI

OH 5‘

3‘A A C: : : T T G

PO4-

Ligase

3‘

5‘

5‘

3‘G T T A A C: : : : : :C A A T T G

AllerdingsistdieLigaPonvonBlunt-EndensehrvielineffizienterKeinZwischenprodukt,beidemdieBindungdurchH-Brückenstabilisiertwird, 3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G



ModifikaPonvonDNA:weitere




  RestriPonsverdau  LigaPon

ModifikaPonfreierEnden

ModifikaPonfreierEnden


1.Dephosphorylierung

Phosphatasen:EnzymezurunspezifischenEn�ernungvonendständigenPhosphatrestenvonbiologischenMakromolekülen(DNA,RNA,Proteinen...)inderMolekularbiologiewerdenverschiedenePhosphataseeingesetzt,hauptsächlichzurDephosphorylierungvonfreienDNA-Enden  CalfintesPnalphosphatase(CIP)

  Shrimpalkalinephosphatase(SAP)  AntarkPcphosphatase

3‘

5‘

5‘


-OH

-PO4-

3‘

5‘

5‘


OH-

PO4--

CIP

3‘

5‘

5‘


-OH

3‘

5‘

5‘


OH-


1.Dephosphorylierung

Grund:VerhinderungderReligaPonvonz.B.KlonierungsvektorennachSchnikmiteinemRestrikPonsenzym

3‘

5‘

5‘


-OH

3‘

5‘

5‘


OH-

Ligase

3‘

5‘

5‘

3‘G A A T T C: : : : : :C T T A A G

genA%

LigaPonohneCIPmit

PO42--

-PO4-


2.Phosphorylierung

(T4)PolynukleoPdkinaseÜberträgteinenPhosphatrestvonATPauffreie,unphosphorylierte5‘-EndenvonDNAZweck:VorbereitungvonunphosphoryliertenDNA-FragmentenzurLigaPon

3‘

5‘

5‘


-OH

3‘

5‘

5‘


OH-

T4Kinase

3‘

5‘

5‘


-OH

3‘

5‘

5‘


OH-

PO4--

-PO4-


2.Phosphorylierung

Warumbrauchtmandas?➜PCR-Primersindam5‘-EndemeistnichtphosphoryliertundkönntensonstnichtdirektohneSchnikligiertwerdenz.B.Pfu-PolymeraseproduziertBlunt-EndenohnefreiePO4

--Enden,nachPhosphorylierungkönnendiesedirektmitdem(dephosphorylierten)Zielvektorligiertwerdenkönnen

A" T" C" A" G" C" T" A" C" C" T" T" C" A" G" G" A" C" A" T" A" C" G" G" T" A"T" A" G" T"

3‘"

5‘"

5‘"

3‘"Pfu"

A" T" C" A" G" C" T" A" C" C" T" T" C" A" G" G" A" C" A" T" A" C" G" G" T" A"T" A" G" T" C" G" A" T" G" G" A" A" G" T" C" C" T" G" T" A" T" G" C" C" A" T"

3‘"

5‘"

5‘"

3‘"Pfu-PCR

A" T" C" A" G" C" T" A" C" C" T" T" C" A" G" G" A" C" A" T" A" C" G" G" T" A"T" A" G" T" C" G" A" T" G" G" A" A" G" T" C" C" T" G" T" A" T" G" C" C" A" T"

3‘"

PO4++"

+PO4+"

3‘"

Polynukleotidkinase

OH# 5‘#

3‘#A A C!: : : !T T G !

3‘#

5‘# OH#G T T!: : : !C A A !


3.AuffüllenvonÜberhängen

Wirdverwendet,wennVektorundzuklonierendesFragmentnicht-kompaPbleEndenhaben

OH# 5‘#

3‘#A A C!: : : !T T G !

3‘#

5‘# OH#G T T!: : : !C A A !

EcoRI KpnI

G" C" T" A" C" C" T" T" C" A" G" G" A" C" A" T" A" C"A" A" T" T" C" G" A" T" G" G" A" A" G" T" C" C" T" G" T" A" T" G" G" T" A" C"

3‘"

5‘"

5‘"

3‘"


3.AuffüllenvonÜberhängen

Wirdverwendet,wennVektorundzuklonierendesFragmentnicht-kompaPbleEndenhaben

Enzym:Klenow-Fragment=dasgrößerederbeidenProteinfragmentederDNA-PolymeraseIausE.colinachenzymaPscherSpaltungmitSubPlisin

Besitzt2enzymaPscheAkPvitäten:

1.   5‘→3'Polymerase-AkPvität2.   3‘→5'Exonuklease-AkPvität(proofreading)

UrsprünglichesEnzymzurAmplifikaPonvonDNAdurchPCR(vorVerwendungvonTaq)

2.   3‘→5'Exonuklease-AkPvität

G C T A C C T T C A G G A C A T A CA A T T C G A T G G A A G T C C T G T A T G G T A C

3‘

5‘

5‘

3‘

AT AT

3‘

3‘

1.   5‘→3'Polymerase-AkPvität

PhosphorylierungundLigaPon

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Methoden der Molekularbiologie Nachweis von DNA - Agarose … · Seite 1 Riedel | Molekularbiologie, Biotechnologie, Gentechnik 02 | WS2015/16 Methoden der Molekularbiologie Nachweis