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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

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DNA Sequencing

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  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

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