1 characterization, amplification, expression screening of libraries amplification of dna (pcr)...

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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

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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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