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 [...]... introduction of mutations - Change of salt (Mg 2+ -> Mn2+) and salt concentration - increase concentration of polymerase - Not equal amount of nucleotides or dITP 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... 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 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 ->