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

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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)

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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 10 9 nucleotides (proof reading) -> in PCR (Taq polymerase) : 1 in 2x10 4 nucleotides High fidelity PCR -> Pfu,… (engineered polymerases) For Engineering purpose -> low fidelity -> introduction of mutations -Change of salt (Mg 2+ -> Mn 2+ ) 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

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22 Joining ends without ligation

23 RT-PCR

24 Real-time PCR

25 Detection of sex in prenatal cells

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27 DNA Sequencing 1.According to Maxam- Gilbert -> selective chemical degratation 2.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!!!

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31 Cycle Sequencing - PCR

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33 DNA sequencing by primer walking

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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

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41 Studying Transcription Primer Extension

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

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

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52 Homologous integration into chromosome Insertion on Bacillus subtilis chromosome

53 Principal factors in bacterial expression

54 Type of expression vectors

55 Fusion proteins increase production level facilitate purification (taq) detection of expression (GFP fusion) Redirection of proteins (secretion -> signal peptidases) Surface display (for screening of libraries) Tandem arrays (for small peptides, toxic proteins,..)

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