1. 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

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.
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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)

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

37. Comparison of expression systems

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

39. Prokaryotic Expression vector

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

41. Eukaryotic Expression vector

45. Promoters

47. Control of transcription of the lac operon.
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52. Terminators

56. Northern Blot
-> to study transcription level

57. Expression studies by microarray technique