Characterization, Amplification, Expression

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

13. Chromosome Walking

14. 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)

17. 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

18. 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,…

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

22. Joining ends without ligation

23. RT-PCR

24. Real-time PCR

25. Detection of sex in prenatal cells

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

28. DNA Sequencing – Sanger method

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

31. Cycle Sequencing - PCR

33. DNA sequencing by primer walking

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

36. 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

37. Northern Blot -> to study transcription level

38. Studying Transcription Microarray technique – DNA chips

39. Studying Transcription Microarray technique – DNA chips

41. Studying Transcription Primer Extension

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

43. Promoter studies by using reporter genes

44. Analyzing Translation – Western Blot

45. 2 D Electrophoresis

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

47. Comparison of expression systems

48. Prokaryotic Expression vector

49. Eukaryotic Expression vector