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Welcome to Integrated Bioinformatics Friday, 30 September

Welcome to Integrated Bioinformatics Friday, 30 September. I. Intro to Scenario 3: Sequence alignment Bring up course web site Go to Scenario 3. Gdansk, Poland. Marbork Castle. Black Death. Scenario 3 Our Story. Samples:. Scenario 3: Our Story. You: Our first defense at CDC.

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Welcome to Integrated Bioinformatics Friday, 30 September

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  1. Welcome toIntegrated BioinformaticsFriday, 30 September • I. Intro to Scenario 3: Sequence alignment • Bring up course web site • Go to Scenario 3

  2. Gdansk, Poland

  3. MarborkCastle

  4. BlackDeath

  5. Scenario 3 Our Story

  6. Samples: Scenario 3: Our Story You: Our first defense at CDC Outbreak: . . . Anthrax? • Confirm agent • Identify strain

  7. Sample Toxin gene-specific primers (bacterial) DNA template Scenario 3: Our Story

  8. If DNA from bacterium with toxin gene If DNA NOT from bacterium with toxin gene? PCR Scenario 3: Our Story + DNA polymerase

  9. PCR (no product) Scenario 3: Our Story If DNA from bacterium with toxin gene If DNA NOT from bacterium with toxin gene? + DNA polymerase + DNA polymerase

  10. AATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATGAATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATG What’s the real story? >gi|16031490|emb|AJ413935.1|BAN413935 Bacillus anthracis partial lef gene, isolate Microsoft-6259 Length = 2417 Score = 155 bits (78), Expect = 2e-35 Identities = 138/158 (87%) Strand = Plus / Plus Query: 1 aatattgacgctttactacatcagtccatcggaagtacgttgtataataaaatatatctg 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1267 aatattgacgctttactacatcagtccatcggaagtacgttgtataataaaatatatctg 1326 Query: 61 tatgaaaacatgaatataaataacttaacagcaacgttaggtgccgatttagtagattcc 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1327 tatgaaaacatgaatataaataacctaacagcaacgttaggtgccgatttagtagattcc 1386 Query: 121 acagataatacaaaaattaatcgaggtatattcaatga 158 |||||||||||||||||||||||||||||||||||||| Sbjct: 1387 acagataatacaaaaattaatcgaggtatattcaatga 1424 Scenario 3: Our Story DG47

  11. PCR Toxin gene present Scenario 3: Our Story

  12. AATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATGAATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATG Do it! Scenario 3: Our Story DG47

  13. AATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATGAATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATG Toxin gene absent Scenario 3: Our Story DG47 Maybe it’s not from the toxin gene?? (Try a different approach)

  14. AATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATGAATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATG Translate NIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSIS Do it! Scenario 3: Our Story DG47

  15. DG47 nucleotide sequenceMatches nothing in GenBank DG47 amino acid sequence100% match to toxin gene Never mind Blast, What's the real answer?(Do it yourself)

  16. Do it! Scenario 3: Our Story Compare nucleotide sequences by hand DG47vslef

  17. Scenario 3: Our Story Compare nucleotide sequences by hand DG47      1  AATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTG          |||||||| |||||| ||||||| ||||| |||||||| ||||| |||||||| ||| || lef gene 1831  AATATTGATGCTTTATTACATCAATCCATTGGAAGTACCTTGTACAATAAAATTTATTTG DG47       61  TATGAAAACATGAATATAAATAACTTAACAGCAACGTTAGGTGCCGATTTAGTAGATTCC         |||||||| |||||||| |||||| | ||||||||  ||||||| |||||||| ||||||lef gene 1891  TATGAAAATATGAATATCAATAACCTTACAGCAACCCTAGGTGCGGATTTAGTTGATTCC DG47      121  ACAGATAATACAAAAATTAATCGAGGTATATTCAATGAGTTCAAAAAAAATTTCAAATAC            || |||||||| ||||||||| ||||||| |||||||| |||||||||||||||||||||lef gene 1951  ACTGATAATACTAAAATTAATAGAGGTATTTTCAATGAATTCAAAAAAAATTTCAAATAT DG47      181  AGTATTTCTA       |||||||||| lef gene 2011  AGTATTTCTA 89% identical! Now let Blast do it by hand

  18. AATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATGAATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATG Do it! Scenario 3: Our Story Compare nucleotide sequences by hand DG47 +lef gene

  19. AATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATGAATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTGTATG Sequence 1lcl|PCR Product DG47 Length190 Sequence 2lcl|M29081: Bacillus anthracis lethal factor (lef) gene, 1831-2020. Length190 No significant similarity was found Scenario 3: Our Story Compare nucleotide sequences by hand DG47 +lef gene

  20. DG47      1  AATATTGACGCTTTACTACATCAGTCCATCGGAAGTACGTTGTATAATAAAATATATCTG   |||||||| |||||| ||||||| ||||| |||||||| ||||| |||||||| ||| || lef gene 1831  AATATTGATGCTTTATTACATCAATCCATTGGAAGTACCTTGTACAATAAAATTTATTTG 89% identical! Sequence 1lcl|PCR Product DG47 Length190 Sequence 2lcl|M29081: Bacillus anthracis lethal factor (lef) gene, 1831-2020. Length190 No significant similarity was found Scenario 3: Our Story Why can’t Blast figure outwhat you can plainly see?

  21. Scenario 3: How does Blast work? • Theory behind nucleotide vs nucleotide Blast • Working BlastN program • Theory behind protein-protein Blast • How to get Blast to do what you want

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