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Molecular Biology

Molecular Biology. Dr. Chaim Wachtel May 28, 2015. GENE EXPRESSION. Topics. Enzymes: Reverse Transcriptase, DNA polymerase, RNA polymerase. Microarray Deep Sequencing PCR Real-Time Digital Nanostring. Microarray in theory. Microarray in practice. Common Applications.

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Molecular Biology

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  1. Molecular Biology Dr. Chaim Wachtel May 28, 2015

  2. GENE EXPRESSION Topics Enzymes: Reverse Transcriptase, DNA polymerase, RNA polymerase • Microarray • Deep Sequencing • PCR • Real-Time • Digital • Nanostring

  3. Microarray in theory

  4. Microarray in practice

  5. Common Applications • Gene expression profiling • Chromatin immunoprecipitation on Chip (ChIP) • SNP detection • Alternative splicing detection

  6. Microarray • Affymetrix • Agilent • Same idea different implementation • Pros and cons to each method

  7. The Chips Agilent Affymetrix

  8. Printing of Affymetrix Chips

  9. Printing of Agilent Chips

  10. Printing of Agilent Chips Probes are 60 nucleotides in length vs 25 for Affymetrix

  11. Affymetrix workflow

  12. Agilent workflow

  13. Analysis “home-made”=low-density • Feature Extraction • Bioinformatics Agilent array

  14. Pros and Cons • Cost • Dual color vs single • Specificity

  15. Deep Sequencing Next Generation Sequencing (NGS) High Throughput Sequencing (HTS)

  16. Timeline and Comparison of Commercial HTS Instruments High-Throughput Sequencing Technologies, Molecular Cell, 58, 586–597 ( May, 2015) Jason A. Reuter1, Damek V. Spacek1, Michael P. Snyder

  17. Overview of Selected HTS Applications High-Throughput Sequencing Technologies, Molecular Cell, 58, 586–597 ( May, 2015) Jason A. Reuter1, Damek V. Spacek1, Michael P. Snyder

  18. Who is this? And what is he holding??? In 1958, he was awarded a Nobel Prize in chemistry "for his work on the structure of proteins, especially that of insulin". In 1980,  he shared with Walter Gilbert half of the chemistry prize "for their contributions concerning the determination of base sequences in nucleic acids". The other half was awarded to Paul Berg "for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA"

  19. Sanger Sequencing Sanger program

  20. Capillary Sequencing Machines have 1-96 capillaries

  21. Multiple Platforms • Illumina • 454 • Solid • Nanopore/Oxford • Ion Torrent • Pacific Biosciences

  22. Different companies different methods

  23. Workflow for Sequencing

  24. Workflow for Sequencing • Depends on project- RNA or DNA • Protocols are ~90% the same for all systems

  25. Illumina

  26. Workflow for Sequencing • Bridge PCR • Emulsion PCR

  27. Sequencing with Illumina

  28. Ilumina Machines

  29. Ilumina Machines

  30. Sequencing with Solid

  31. The Ion Torrent

  32. Scalability

  33. The Chip

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