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Pyromania

USAFSAM “EPI Lab”. Pyromania. MSgt Stephen Christian, USAF, MSgt, MT (ASCP) Lucinda Sinclair, GS-11. Distribution Statement A: Approved for Public release; distribution is unlimited. 311 ABG/PA No. 10-080, 12 Mar 2010. Overview. Introduction to Influenza WhyWhat to Sequence

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Pyromania

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  1. USAFSAM “EPI Lab” Pyromania MSgt Stephen Christian, USAF, MSgt, MT (ASCP) Lucinda Sinclair, GS-11 Distribution Statement A: Approved for Public release; distribution is unlimited. 311 ABG/PA No. 10-080, 12 Mar 2010

  2. Overview • Introduction to Influenza • Why\What to Sequence • Sequencing (Pyro versus Sanger) • Pyrosequencing for influenza antiviral resistance markers (Tamiflu resistance in 2009 A (H1/N1)

  3. Influenza http://www.ncbi.nlm.nih.gov/genomes/FLU/

  4. Types of influenza 1918 = H1N1 50,000,000 deaths 1957 = H2N2 1,000,000 deaths 1968 = H3N2 500,000 deaths Annual flu 250,000 deaths H5N1 annual 25 - 75 A,B, and C (Epidemics = A &B) Pandemics = A only 2009 A/H1N1

  5. Influenza Genome PB2 PB1 PA HA NP NA M NS

  6. Why/What to Sequence HA1 region of HA gives the most bang for the buck. PB2 PB1 PA HA NP NA M NS 13,588 bp’s Tamiflu Amantadine Presence/Absence of Antiviral resistance markers

  7. Sequencing (Pyro versus Sanger) Nucleic Acid Extraction Traditional RT PCR Sequencing

  8. Sanger Sequencing • Sequencing by Chain Termination • Extract Nucleic Acid • Produce amplicon • Clean Amplicon • Big Dye Rxn (Chain Termination) • Clean Big Dye Rxn • Electrophoresis of Big Dye (Sequencer) • Analyze Sequence Data

  9. Produce Amplicon (HA1) Template (Amplicon)

  10. Template (Amplicon) Template (Amplicon) Clean Amplicon (HA1) (dATP’s) (dGTP’s) (dCTP’s) (dTTP’s) Polymerase Salts Buffers Clean PCR Product Amplicon

  11. Primer Primer Primer Primer Primer Primer Big Dye Reaction Template (Amplicon) Primer dNTP’s A (dATP’s) G(dGTP’s) C (dCTP’s) T (dTTP’s) ddNTP’s A G C T

  12. Big Dye Reaction Template (Amplicon)

  13. Clean Big Dye Reaction

  14. Electrophoresis

  15. Results from Electrophoresis

  16. Sequence Analysis • Build Single Contig • Analyze constructed nucleotide sequence (Protein) • Relate to current vaccine strain on phylogenetic tree

  17. Pyrosequencing • Sequencing by Addition • Extract Nucleic Acid • Produce amplicon • Pyrosequencer

  18. Pyrosequencing

  19. Simple Sample Preparation 1. Amplify relevant region by PCR (100 - 300 bp), using 1 biotinylated primer 2. Immobilize biotinylated PCR products onto streptavidin-coated beads 3. Separate strands by denaturation in NaOH 4. Wash /neutralize the immobilized strand 5. Anneal sequencing primer 96 samples are processed in parallel

  20. Pyrosequencing 1.) Hybridize sequence primer along with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosuflate (APS) and luciferin. luciferin luciferase Apyrase APS sulfurylase Polymerase Polymerase

  21. Pyrosequencing 2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi. luciferin luciferase Apyrase APS sulfurylase dNTP’s added one at a time Polymerase

  22. Pyrosequencing 2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi. luciferin luciferase Apyrase APS sulfurylase Polymerase

  23. Pyrosequencing 2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi. luciferin luciferase APS sulfurylase Apyrase PPi Polymerase

  24. Pyrosequencing ATP sulfurylase converts PPi and APS to ATP. Luciferase converts luciferin and ATP to oxyluciferin which generates light read by a camera. sulfurylase luciferin luciferase ATP APS + PPi = Apyrase Polymerase

  25. Pyrosequencing ATP sulfurylase converts PPi and APS to ATP. Luciferase converts luciferin and ATP to oxyluciferin which generates light read by a camera. sulfurylase luciferase ATP APS oxyluciferin + PPi = luciferin Apyrase Polymerase

  26. Pyrosequencing Hybridize sequence primer, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosuflate (APS) and luciferin. Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi. ATP sulfurylase converts PPi and APS to ATP. Luciferase converts luciferin and ATP to oxyluciferin which generates light read by a camera. Apyrase degrades dNTP’s and ATP, when complete next dNTP is added. As process continues, the correct DNA sequence is built up and displayed.

  27. Sanger sequencing steps • VS. 2 Pyrosequencing steps

  28. Neuraminidase Pyrosequencing

  29. Questions

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