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

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

Screening of Libraries 1. Hybridisation:

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

A degenerate oligonucleotide probe.

Colony Hybridisation

Plaque Hybridisation

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)

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)

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

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

Chromosome Walking

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)

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

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,…

Adding of restriction sites for cloning of a PCR product

Joining ends without ligation

RT-PCR

Real-time PCR

Detection of sex in prenatal cells

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

DNA Sequencing – Sanger method

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

Cycle Sequencing - PCR

DNA sequencing by primer walking

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

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

Comparison of expression systems

Use of Lac promoter (pLac) for expression of foreign protein

Prokaryotic Expression vector

Prokaryotic Expression vector PstI T7/lac phoA cutinase NarI SalI HindIII BamHI phoA-cut NdeI S/D Term pFCEX1

Eukaryotic Expression vector

Promoters

Control of transcription of the lac operon. Page 95

Terminators

Northern Blot -> to study transcription level

Expression studies by microarray technique