logging in or signing up A new method to generate full-length HIV molecular clones Shityakov Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 21 Category: Science & Tech.. License: All Rights Reserved Like it (0) Dislike it (0) Added: January 16, 2012 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript A new method to generate full-length HIV molecular clones (advantages and perspectives): A new method to generate full-length HIV molecular clones (advantages and perspectives) MD/PhD student Sergey W. Shityakov IRTG 2 nd International Symposium Kloster Banz , 2010Aim of the project:: Aim of the project: Establishment of HIV molecular infectious clones that can be used in drug-resistance study, viral pathogenesis research, lentiviral vectors construction for gene therapy etc.Strategies to create HIV infectious clones: Strategies to create HIV infectious clones PCR-cloning Lambda phage-cloning Homologous recombination-mediated cloning ( recombineering )PCR-cloning: PCR-cloning Preparation of DNA from cultured PBMC PCR amplification and subcloning of subgenomic fragments Restriction and ligation of the DNA fragments to recover a complete full-length clone Labor intensive High number of defective clonesPowerPoint Presentation: Rodriguez et al., 2006 PCR-cloningPhage-based cloning: Phage-based cloning Preparation of DNA from cultured PBMC Clone into vector Screen E. coli colonies for positive clones ( more than 1000) with specific probes Labor intensive High amount of clones Lack of precision (swallows up whole DNA)PowerPoint Presentation: Phage-based cloning http://web-books.com/MoBio/PowerPoint Presentation: http://web-books.com/MoBio/Recombination assay: Recombination assay Precise cloning of DNA Relatively fast, in comparison to other methods High yield of recombinant clones No need for PCR to amplify viral DNA fragments (mutations) No need for subcloning (in absence of LTRs) Full-length viral genome cloningBackground of homologous pKD20-based recombination assay: λ Red (γ, β, Exo) recombinase-flippase (FLP) : Background of homologous pKD20-based recombination assay: λ Red ( γ , β , Exo) recombinase-flippase (FLP) Datsenko et al., 2000PowerPoint Presentation: Human DNA with HIV provirus HIV genome pKD20-based recombination assay (modified)Thank you for your attention: Thank you for your attention You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
A new method to generate full-length HIV molecular clones Shityakov Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 21 Category: Science & Tech.. License: All Rights Reserved Like it (0) Dislike it (0) Added: January 16, 2012 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript A new method to generate full-length HIV molecular clones (advantages and perspectives): A new method to generate full-length HIV molecular clones (advantages and perspectives) MD/PhD student Sergey W. Shityakov IRTG 2 nd International Symposium Kloster Banz , 2010Aim of the project:: Aim of the project: Establishment of HIV molecular infectious clones that can be used in drug-resistance study, viral pathogenesis research, lentiviral vectors construction for gene therapy etc.Strategies to create HIV infectious clones: Strategies to create HIV infectious clones PCR-cloning Lambda phage-cloning Homologous recombination-mediated cloning ( recombineering )PCR-cloning: PCR-cloning Preparation of DNA from cultured PBMC PCR amplification and subcloning of subgenomic fragments Restriction and ligation of the DNA fragments to recover a complete full-length clone Labor intensive High number of defective clonesPowerPoint Presentation: Rodriguez et al., 2006 PCR-cloningPhage-based cloning: Phage-based cloning Preparation of DNA from cultured PBMC Clone into vector Screen E. coli colonies for positive clones ( more than 1000) with specific probes Labor intensive High amount of clones Lack of precision (swallows up whole DNA)PowerPoint Presentation: Phage-based cloning http://web-books.com/MoBio/PowerPoint Presentation: http://web-books.com/MoBio/Recombination assay: Recombination assay Precise cloning of DNA Relatively fast, in comparison to other methods High yield of recombinant clones No need for PCR to amplify viral DNA fragments (mutations) No need for subcloning (in absence of LTRs) Full-length viral genome cloningBackground of homologous pKD20-based recombination assay: λ Red (γ, β, Exo) recombinase-flippase (FLP) : Background of homologous pKD20-based recombination assay: λ Red ( γ , β , Exo) recombinase-flippase (FLP) Datsenko et al., 2000PowerPoint Presentation: Human DNA with HIV provirus HIV genome pKD20-based recombination assay (modified)Thank you for your attention: Thank you for your attention