1 / 56

Characterization, Amplification, Expression

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. Screening of Libraries.

nansen
Download Presentation

Characterization, Amplification, Expression

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

  12. Chromosome Walking

  13. 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)

  14. 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

  15. 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,…

  16. Adding of restriction sites for cloning of a PCR product

  17. Joining ends without ligation

  18. RT-PCR

  19. Real-time PCR

  20. Detection of sex in prenatal cells

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

  22. DNA Sequencing – Sanger method

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

  24. Cycle Sequencing - PCR

  25. DNA sequencing by primer walking

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

  27. 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

  28. Northern Blot -> to study transcription level

  29. Studying Transcription Microarray technique – DNA chips

  30. Studying Transcription Microarray technique – DNA chips

  31. Studying Transcription Primer Extension

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

  33. Promoter studies by using reporter genes

  34. Analyzing Translation – Western Blot

  35. 2 D Electrophoresis

  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. Prokaryotic Expression vector

  39. Eukaryotic Expression vector

More Related