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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
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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
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Screening of Libraries
1. Hybridisation:
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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
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A degenerate oligonucleotide probe.
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Colony Hybridisation
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Plaque Hybridisation
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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)
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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)
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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
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Detection of DNAs containing specific base sequences by the Southern blot technique.
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Chromosome Walking
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PCR – Polymerase Chain Reaction1993 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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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
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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,…
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Adding of restriction sites for cloning of a PCR product
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Joining ends without ligation
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RT-PCR
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Real-time PCR
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Detection of sex in prenatal cells
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DNA Sequencing
1. According to Maxam- Gilbert -> selective chemical degratation
2. According to Sanger -> polymerase reaction with nucleotide analog
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DNA Sequencing – Sanger method
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DNA Sequencing – Sanger method
Polymerase Reaction:
5’-> 3’
-> incorporation of ddNTP -> 3’ end has NO OH group -> Polymerase reaction stops!!!
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Cycle Sequencing - PCR
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DNA sequencing by primer walking
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Chemical synthesis of DNA
Chain grows: 3’-> 5’
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General consideration about Gene Expression
• Expression Host -> Expression System
• Promoter system -> expression vector
• Properties of product -> stability
• Production level
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Comparison of expression systems
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Use of Lac promoter (pLac) for expression of foreign protein
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Prokaryotic Expression vector
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Prokaryotic Expression vector
PstI
T7/lac
phoA cutinase
NarISalI HindIII
BamHI
phoA-cut
NdeI
S/D Term
pFCEX1
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Eukaryotic Expression vector
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Promoters
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Control of transcription of the lac operon.P
age
95
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Terminators
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Northern Blot
-> to study transcription level
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Expression studies by microarray technique
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