Library Screening, Characterization, and Amplification

1. Library Screening, Characterization, and Amplification • Screening of libraries • Amplification of DNA (PCR) • Analysis of DNA (Sequencing) • Chemical Synthesis of DNA

2. Screening of Libraries 1. Screening libraries with gene probes (DNA level): -> Hybridisation: - Colony Hybridisation - Plaque Hybridisation 2. Screening Expression libraries (Protein level): -> 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 1. 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. Restriction fragment length polymorphism or RFLP analysis is used to identify a change in the genetic sequence that occurs at a site where a restriction enzyme cuts. RFLPs can be used to trace inhertitance patterns, identify specific mutations, and for other molecular genetic techniques.

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) Normally around 30-35 cycles -> around 1 mill copies

15. PCR Reaction mix: • Primers (15 – 30 bp) -> GC at 3’ end, not too high Tm (40-70˚C), ho hairpine • 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 • Not equal amount of nucleotides or dITP

16. 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,…

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

19. Joining ends without ligation

20. RT-PCR – Reverse Transcriptase PCR

21. Real-time PCR

22. Detection of sex in prenatal cells

23. PCR fingerprinting: for identification of bacteria, forensic purpose (to assist in the identification of individuals on the basis of their respective DNA profiles

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

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

27. Cycle Sequencing - PCR

29. DNA sequencing by primer walking

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