1 / 15

DNA Sequencing

DNA Sequencing. Sanger Di-deoxy method of Sequencing Manual versus Automatic Sequencing. Sequencing of DNA. Requires DNA amplification Labeling of DNA fragments Termination of extension Separation of fragments Detection of fragments. The Reaction Mixture. ddNTP (one) 5’-labeled primer

reece-bruce
Download Presentation

DNA Sequencing

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. DNA Sequencing Sanger Di-deoxy method of Sequencing Manual versus Automatic Sequencing

  2. Sequencing of DNA • Requires DNA amplification • Labeling of DNA fragments • Termination of extension • Separation of fragments • Detection of fragments

  3. The Reaction Mixture • ddNTP (one) • 5’-labeled primer • ATP, TTP, CTP, GTP • Terminated fragments • Followed by autoradiogram

  4. Dideoxy key point! • Sanger Method dideoxynucleotides: lack a 3’ OH • But have the 2’ and 3’ have hydrogen • NO DNA EXTENSION

  5. DNA PCR amplification is performed prior to gel electrophoresis Notice the reaction of this “one” reaction mixture!

  6. Separation followed by Detection

  7. Can you sequence without using radioactivity?

  8. ddGTP ddATP ddTTP ddCTP Orange Black Red Blue Example of Label the ddNTPs

  9. Automated Sequencing • Efficient • Does not require radioactivity

  10. The End • Onto DNA Mutations

More Related