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Methods Seminar: Nucleic acids Ernesto Elorduy Vergara 19.11.2014 Outline • Biochemistry • Nucleic acid analy=cs – Isola=on & purifica=on – Polimerase chain reac=on – Restric=on analysis – BloDng (Southern, Northern) • Example: TALEN-‐produc=on in bacteria Outline • Biochemistry • Nucleic acid analy=cs – Isola=on & purifica=on – Polimerase chain reac=on – Restric=on analysis – BloDng (Southern, Northern) • Example: TALEN-‐produc=on in bacteria Biochemistry • Polymeric macromolecules made from monomers known as nucleo=des (discovered by Friedrich Miescher in 1869) • Include: DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) • Func=on: in encoding, transmiDng and expressing gene1c informa1on, catalysts (ribozymes) Building Blocks Structure of DNA solved by Watson and Crick 1953 Genomes • • • • • Viruses: DNA, RNA, single and double stranded Prokaryotes: DNA, mostly circular Mitochondria: circular DNA Chloroplasts: circular DNA Eukaryontes: Chromosomes with linear DNA Ouline • Biochemistry • Nucleic acid analy=cs – Isola=on & purifica=on – Polimerase chain reac=on – Restric=on analysis – BloDng (Southern, Northern) • Example: TALEN-‐produc=on in bacteria DNA isola=on • Eukaryote sample material: – =ssue – blood – cell culture Ø Chemical treatment: – Cell disrup=on: SDS (sodium dodecyl sulfate) – Protein degrada=on: protease K (histones!) Ø Mechanical treatment (homogenizator) – Flash freezing: liquid nitrogen – Pulveriza=on by shaking with glass/metal beads Phenol(-‐chloroform) extrac=on • Aim: purify samples of nucleic acids taken from cells • Phenol (also chloroform): non-‐polar compound which denatures proteins • DNA: Tris-‐HCl or TE (Tris-‐HCl/EDTA) pH 7,5 – 8 • RNA: RNase resistant to phenol!! „Sour phenol“ (denatura=on of DNA) Gelfiltra=on • Aim: purifica1on of NAs • Size exclusion chromatography – Sephadex G50, sephacel S300, Bio-‐Gel P-‐2 – Spin columns Ethanol precipita=on • Aim: increase concentra1on and further purifica1on of NAs • Working principle: NAs precipitates in EtOH in the presence of monovalent ions • DNA: sodium or ammonium acetate • RNA: lithium chloride Concentra=on measurement • • • • OD-‐measurement by absorp=on spectrometry UV-‐photometer, quartz cuveie UV-‐Vis Spectrometer NanoDrop 2000 OD 260 Protein contamina=on: OD280 absorption peak DNA/RNA: 260 nm Absorption peak proteins: 280 nm DNA: A260/280 ~1.8 RNA: A260/280 ~2 OD260= 1 (1 cm): 50 µg/ ml dsDNA 40 µg/ ml ssDNA/ RNA 20 µg/ml oligonucleotides Ø Lambert-‐Beer Law High-‐molecular DNA – Sensi=ve to shear forces Ø Careful mixing and pipeDng – Purifica=on: dialysis RNA – RNases!!! • very stable • no cofactors • ubiquitous Ø Inac=va=on: • solu=ons: DEPC (diethylpyrocarbonate) • glassware: 180-‐200°C; buyable materials and solu=ons • RNase-‐inhibitor (e.g. RNasin®, Promega) Outline • Biochemistry • Nucleic acid analy=cs – Isola=on & purifica=on – Polimerase chain reac=on – Restric=on analysis – BloDng (Southern, Northern) • Example: TALEN-‐produc=on in bacteria PCR • Invented by K. Mullis and co-‐workers in 1985 „Selec:ve amplifica:on of a specific DNA-‐sequence in a heterogenous mixture of DNA-‐sequences“ • Exponen=al amplifica=on (in vitro DNA-‐cloning) • Repeated hea=ng and cooling Ø Ethidiumbromide, sylver staining, radioac=vity marks, SYBR Green II, … PCR-‐components • Template DNA • DNA-‐Polymerase (5‘-‐3‘ polymeriza=on) – Taq-‐/Tth-‐DNA-‐Pol. (reverse transcrip1on ac=vity, Synthesis rate 2800 b/min, error rate 10-‐5) – Pwo-‐/Pfu-‐DNA-‐Pol. (5‘-‐3‘ exonuclease ac1vity, synthesis rate: 550/min, error rate: 10-‐6) • Primer • Buffer (Mg2+) • Nucleo=des (dATP,dGTP,dCTP,dTTP) • PCR-‐Cycler Primer design Primer design • Tm calcula=on: 2°C x (A+T) + 4°C x (G+C) • Avoid mismatches between the 3' end of the primer and the template • Avoid complementarity within primers and between the primer pair (-‐> primer dimers) • Avoid a T as ul=mate base at the 3' end • Ensure primer sequence is unique for the template sequence (mul=plex PCR!) • Use a concentra1on of 0.1–1.0 µM of each primer • Primer and Mg2+ concentra=on and annealing temperature need to be op=mized for each primer Temperature gradient PCR RT-‐PCR • Taq-‐/Tth-‐DNA-‐Pol. (reverse transcrip1on ac=vity) Ø Detec=on RNA expression • One-‐step/ two step PCR Semiquan=ta=ve RT-‐PCR Quan=ta=ve RT-‐PCR • Detec=on and quan%fica%on of a specific RNA within a RNA mixture • Detec=on of fluorophores during the PCR – dsDNA-‐bindende Fluorophore (SYBR-‐Green I) – Markierte Hybridisierungssonden (TaqMan) • LightCycler® -‐ Roche Life Science Qua=fica=on Sensi=vity (10-‐8) Specificity (primer design!!) Liile sample quan=ty needed Fast (30 – 120 min) RNA PCR kine=cs • Crossing Point CP: number of PCR cycles needed to reach a defined flourescence level (withing exp. Phase) Melt curve analysis Troubleshoo=ng • LiCle or no PCR product ₓ Poor quality of PCR templates, primers, or reagents → Template purifica=on • Poor PCR amplifica1on efficiency ₓ primer quality Ø accuracy of real-‐=me PCR!! • Primer dimer → increase the template amount • Non-‐specific amplicons ₓ Primer design, reac=on condi=ons, contamina=ons → gel electrophoresis, mel=ng curve analysis, sequencing → Nega=ve control Outline • Biochemistry • Nucleic acid analy=cs – Isola=on & purifica=on – Polimerase chain reac=on – Restric=on analysis – BloDng • Example: TALEN-‐produc=on in bacteria Restric=on enzymes • Restric1on endonuclease (REase) – cuts DNA at or near specific recogni=on nucleo=de sequences known as restric=on sites • 5 Types – Type II • recognize and cleave DNA at the same site • do not need ATP (but usually Mg2+) • Palindromic sequences • S=cky-‐/blunt ends Restric=on mapping Outline • Biochemistry • Nucleic acid analy=cs – Isola=on & purifica=on – Polimerase chain reac=on – Restric=on analysis – BloDng • Example: TALEN-‐produc=on in bacteria • Edwin Southern 1975 Ø Transfer of NAs to a membrane – Southern Blot: DNA – Northern Blot: RNA q Nitrocellulose q Nylon Outline • Biochemistry • Nucleic acid analy=cs – Isola=on & purifica=on – Polimerase chain reac=on – Restric=on analysis – BloDng • Example: TALEN-‐produc=on in bacteria TALEN-production in bacteria • Crea=on of a pair of plasmids, each encoding: – – – – – Sequence of TALEN that targets RAG1 in pig T7 promoter and terminator 6x His PTDTAT NLS Rag1-‐TALEN1 Rag1-‐TALEN2 pCAG-TALpCAG-TALlinker-Xtlinker-XtdSsR26Tal1 dSsRAG1Tal1 pTriEx-HTNC pCAG-TALpCAG-TALlinker-Xtlinker-XtdSsRAG1Tal2 dSsR26Tal2 pSL1180 PCR pTAL1-Rag1-pSL1180 with Designed Primers Take home message • It’s impossible do see biomolecular molecular processes with the naked eye • But there are TOOLS to explain them Ø Get acquainted with them • Don’t just passively follow established protocols Ø Stay curious! J Methyla=on-‐specific PCR à Detec=on of methyla=on status at CpG-‐islands (promoters) M: methylated-‐specific U: unmethylated-‐specific AFTER conversion!!! Transcription Activator-Like Effector Nucleases • Accurate and reliable genomic engineering tool • Chimeric DNA-‐binding proteins – TAL Effector-‐Like DNA-‐binding domain – DNA cleavage domain (FokI) – Linker • FokI is ac=ve only as dimer -‐> TALENs act in pairs • Posi=on specific introduc=on of DSB • Ac=va=on of DNA repair pathway NHEJ (or HR) -‐> INDELs Transcription Activator-Like Effector Virulence factors of the genus Xanthomonas Transloca=on signal -‐> bacterial type III secre=on system NLS -‐> transloca=on to the nucleus Transcrip=onal ac=va=on domain (AD) -‐> reprogram host gene expression • Central repeat domain -‐> DNA recogni=on • • • • – Polymorphisms at in posi=ons 12 and 13 -‐> base specificity = “repeat-‐variable diresidue” (RVD) – Each repeat (33-‐35 aa) acts in an independent modular way doi: 10.1146/annurev-‐phyto-‐080508-‐081936 Recombination Activating Gene 1 (RAG1) • Part of the soma=c V(D)J-‐recombina=on complex -‐> variety of an=bodies and lymphocyte receptors • V(D)J-‐recombina=on: a two step process – introduc=on of DSB within recombina=on signal sequences (RSS) – repair by non-‐ homologous end-‐joining (NHEJ) • Muta=ons in RAGs -‐> severe combined immunodeficiency (SCID) with T–B–NK+ genotype Adapted from doi: 10.1093/nar/29.7.1399
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