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Characterization, Amplification, Expression

Characterization, Amplification, Expression. Screening of libraries Amplification of DNA (PCR) Analysis of DNA (Sequencing) Chemical Synthesis of DNA Expression Studies General Considerations of Gene Expression in Prokaryotes + Eukaryotes. Screening of Libraries.

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Characterization, Amplification, Expression

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  1. Characterization, Amplification, Expression • Screening of libraries • Amplification of DNA (PCR) • Analysis of DNA (Sequencing) • Chemical Synthesis of DNA • Expression Studies • General Considerations of Gene Expression in Prokaryotes + Eukaryotes

  2. Screening of Libraries 1. Screening libraries with gene probes: -> Hybridisation: - Colony Hybridisation - Plaque Hybridisation 2. Screening Expression libraries: -> Activity screening (-> HTS of Directed Evolution Libraries) -> with Antibodies

  3. Screening of Libraries 1. Hybridisation:

  4. Gene Probes • Homologous gene probes (DNA from the same gene, same organism) • -> if you have already an incomplete clone of the gene • -> if you want to clone neighboring regulatory elements (promoters) • -> if you have cDNA clone but want the genomic clone as well • -> genetic variations between individuals (mutation causing diseases) • Heterologous gene probe (DNA from the same gene, different organism) • -> if you have already the gene from the same gene family but different organism (insulin from rat in order to screen human library) • - Probe generated by back translation -> degenerated oligonucleotide probe

  5. A degenerate oligonucleotide probe.

  6. Colony Hybridisation

  7. Plaque Hybridisation

  8. Screening of Expression Libraries with Antibodies Primary Antibody: against protein of interest (specific) Secondary Antibody: against proteins (antibodies) produced in rabbit, mouse, bird,… (unspecific but labeled)

  9. Characterization of gene products • Restriction analysis • Southern blot hybridisation • PCR • DNA sequencing • Chromosome walking - Characterization of large fragments -> make ordered libraries) - Identify genes (clone genes)

  10. Characterization of Nucleotide sequences and protein sequences - Blots • Blots -> Transfer of target molecules to filters -> analysis of target molecules on filters • Southern Blot: • -> Hybridisation of DNA (target) with DNA or RNA (Probe) • used for detection and characterization of gene fragments • 2. Northern Blot: • -> Hybrisation of RNA (target) with DNA or RNA (probe) • used for detection of transcrition level (mRNA) of expressed genes (can also be done by real-time PCR) -> analysis of gene expression • used for detection of size of transcript (length of mRNA) -> analysis of alternative splicing • 3. Western Blot: • -> Interaction of Antigen with Antibody • used for detection and localization of proteins

  11. Detection of DNAs containing specific base sequences by the Southern blot technique. Page 111

  12. Chromosome Walking

  13. PCR – Polymerase Chain Reaction 1993 Kary B Mullis received the Nobel Prize in Chemistry 1. Step -> Denaturation (94-96º C) 2. Step -> Annealing (variable Temp.) T -> 2-4 C below melting T 3. Step -> Extension (68-72º C)

  14. PCR Reaction mix: • Primers (15 – 30 bp) -> GC at 3’ end • Nucleotides (A,T, G,C) • Buffer -> Mg 2+ • Target DNA (around 10 ng) • Taq Polymerase (from Thermus aquaticus -> thermostable) Fidelity: -> rate of misincorporation -> in DNA replication : 1 in 109 nucleotides (proof reading) -> in PCR (Taq polymerase) : 1 in 2x104 nucleotides High fidelity PCR -> Pfu,… (engineered polymerases) For Engineering purpose -> low fidelity -> introduction of mutations • Change of salt (Mg 2+ -> Mn2+) and salt concentration • increase concentration of polymerase

  15. PCR Applications • Amplification of DNA • Modification of ends for cloning (RACE) • Analysis of PCR products (nested primers) • Cloning of genes (amplification from genome or library) • Introduction of site-specific mutations • Joining ends (religation of different DNA molecules) without ligation • Invitro splicing • Reverse Transcriptase (RT)-PCR • Real-time PCR -> Diagnostics • Asymmetric PCR -> ssDNA -> sequencing • Detection of Infections (bacterial, viral) -> Diagnostics • Detection of sex in prenatal cells • Fingerprinting -> forensic medicine • PCR on a Chip -> Detection of human pathogen organisms • In situ PCR -> studying disease states, mapping chromosomes,…

  16. Adding of restriction sites for cloning of a PCR product

  17. Joining ends without ligation

  18. RT-PCR

  19. Real-time PCR

  20. Detection of sex in prenatal cells

  21. DNA Sequencing • According to Maxam- Gilbert -> selective chemical degratation • According to Sanger -> polymerase reaction with nucleotide analog

  22. DNA Sequencing – Sanger method

  23. DNA Sequencing – Sanger method Polymerase Reaction: 5’-> 3’ -> incorporation of ddNTP -> 3’ end has NO OH group -> Polymerase reaction stops!!!

  24. Cycle Sequencing - PCR

  25. DNA sequencing by primer walking

  26. Chemical synthesis of DNA Chain grows: 3’-> 5’

  27. Gene Expression Expression studies: 1. Analyzing Transcription - Northern blot - Micro array - real-time PCR - Primer extension 2. Promoter studies Use of report genes to study regulatory elements 3. Analyzing Translation - Western blot - immuno assays - 2D electrophoresis - proteomics

  28. Northern Blot -> to study transcription level

  29. Studying Transcription Microarray technique – DNA chips

  30. Studying Transcription Microarray technique – DNA chips

  31. Studying Transcription Primer Extension

  32. Promoter Studies Use of green fluorescent protein (GFP) as a reporter gene. • Used reporter genes: • Lac Z • GFP • Luciferase Promoter

  33. Promoter studies by using reporter genes

  34. Analyzing Translation – Western Blot

  35. 2 D Electrophoresis

  36. General consideration about Gene Expression • Expression Host -> Expression System • Promoter system -> expression vector • Properties of product -> stability • Production level

  37. Comparison of expression systems

  38. Prokaryotic Expression vector

  39. Eukaryotic Expression vector

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