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Sequencing and Primer Design for Gene Characterization - A Helpful Guide

This guide provides step-by-step instructions for finalizing direct sequencing, editing and BLASTing contigs, primer walking, and designing primers for gene characterization. It also includes tips on redoing mitobim with alternative baiting and submitting to Genbank.

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Sequencing and Primer Design for Gene Characterization - A Helpful Guide

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  1. Today • HK • Finalize direct sequencing: 16S BLAST IDs(COI…) • *.ppt • Insert (plasmid) sequencing: edit, BLAST, contig • PRIMER WALKING, primer design

  2. Nolan et al., 2013 SO: redo mitobim with alternative baiting

  3. http://mitos.bioinf.uni-leipzig.de/result.py?hash=pWBtFmE1 Compare gene order, Find trnS2, check gene predictions Submit to Genbank

  4. DSEQ1 DSEQ2 x 3 x 4 201 202 203 x 205 206 208 x 209 x 211 212 213 214 215 216 217 218 219 220 5 7 x 8 x x x x 13 x 14 x X X X X 21 22 23 24

  5. Edit in chromas Ok sequence? 200-series: no for 203, 207, 210 Clear Ns Import in CodonCodes, assemble Edit more, Identify primers, blast ONLY experimental sequence

  6. >PE1_16S AGTCGGCAGATGAACTAAATTATTANTATTATTGTCGATATCTTCTGCCCGGTGTTTATAATAAACGGCCGCAGTACCTTGACTGTGTWAAGGTAGCATAATCAATTGGCTATTAATTGTAGTCTAGTATGAATGAAGTAATGAATAGTAACTATAATAAGAAATTTTATTTTAACTTAATGAACTGGTGAAAATACCAGTATTCAGAAAAAAGACGAGAAGACCCTTAGAGTTTTAAATAAAGATTGTTTAAGACTTTGATGTATCATCATCTTAAAGTACATTATACAACACACTTTTTTGTTGGGGCGACAAGAAAGCAATATAACCTTTCTATTTACGCTAAAAGTGGGTATTAGATCTGACGATATTATTATGAATGACTAAACTACCTGAGGGATAACAGCADAATGATAAATATTAGTTTGTGACCTCGATGTTGGACTAGGGACTTGTACACTAACCGTGATAAAAGCTTGTTCTGTTCGAACAATAAAGCC >PE2_16S AGCTTGAAGAATATATTTTAAGTTAATTCTGCCCGGTGCTTTAAGTTAACGGCCGCAGTACCTTGACTGTGCTAAGGTAGCATAATCAATTGGCTATTAATTGTAGTCTAGTATGAAAGAATAAATGATTAGGGGCTATAATAGGGGGTTTAGTTAAAACTTATTTATCAGGTGAAAATACCTGTAATAAGAAAAAAGACGAGAAGACCCTTAGAGTTTAAAAAATTACACTATATCTGATTAGTGGTGTTTTTTATTGGGGCGATAAGAAAACATTATTAAACTTTTCTATCAATGATTGTACAGGCCGAATTTATTATGGATGGCTAAACTACCTTAGGGATAACAGCATAATGATGTATTTTAGTTTGTGACCTCGATGTTGGACTAGGAACTTTAACACTAGCCSGTGGTAAAAGAATGTTCTGTTCGAACAGTTGTTTCC >LE4_16S AGCCCAAAGTTTTTTTTTGGGAAAATTCTGCCCAGCGTAATTTTTTAGCGGGCCGCAGTTCTTTGACTGTGCTAAGGTAGCATAATCAATTGGCCTTTAATTGTAGTCTGGAATGAAAGGACTAATAGGGGTTAGCTGTCTCTATACTATTATTTTGAATTTATTTATTAAGTGAAAATACTTATACAAAGAAAAAAGACGAGAAGACCCTTAGAATTTTAATAAAATATTTAATAATTTTTTTTTGTTGGGGCGACATTGAAACAATTAAACTTTCATTAACAATCAAGACATTTAGGTTTGAAAGAATAAATTACCTTAGGGATAACAGCATAATTAATTATTTAGTTTGTGACCTCGATGTTGGACTAGGAACTAGAAGATTAACCGTCTAAATAGATTGTTCTGTTCGAACAGAAATTCC >A2_16S AGTTCATTGAAATATATTTTGAATTTTTTCCTGCCCAGTGTATAAAAATAAATGGCCGCAGTACCTTGACTGTGCTAAGGTAGCATAATCAATTGGCTTTTAATTGAAGTCTTGGATGAAAGGATTTATGGGAATATTCTGTTTCAAAAGTATTATTTTTAATTTATTTATTAGGTGAAAATACCTTCATAATTATAAAAGACGAGAAGACCCTAAGAATTTTTAGGAAATTTTTTTTCATTATCCTTTTTGTTGGGGCGACAGATAAACAAAAAAACTTAATTTTTAGTATATATCGATTATTTTAGTAAAAATTAAATTACCTTAGGGATAACAGCATAGTTTTAAAAAGTTTGTGACCTCGATGTTGGACTAGGAACTTAATGGCTAGCAGTCAGTAAAGAGTGTTCTGTTCGAACAGTTATTGCC

  7. Snail 16S pp ID PE1 PE2 PE3 LE A2 Phylogeny to the rescue

  8. *.ppt

  9. MISPRIMING!! PRIMERS ARE ARTEFACTS

  10. Primer design 100 nt back from last good sequence 3’ GC clamp, make 3 of 6 3’ terminal residues G or C Recommend ~40% GC (not always possible) 18-24 nucleotides Annealing temp ≥ ~52C (higher Tm increases stringency, Tm must be similar for set of PCR primers) Count 2 for A/T, 4 for G/C Reverse complement for 5’-3’ of antisense primer Check with Netprimer (or alternative!): rating ≥80 E-mail your group’s forward and reverse primers for ordering and sequencing (provide in *txt format)

  11. http://www.premierbiosoft.com/netprimer/

  12. Exercise: Design a forward primer and a reverse primer for both sides of the insert Report primer sequences 5’->3’

  13. Primer Design Options • Literature • Sequence (also from gene discovery) • NO sequence from your organism • Align nt sequences from close relativeslook for conserved regions to design primers,use mixed bases if needed • Obtain protein sequencesback-translate (use correct genetic code)minimize degeneracy, consider inosine

  14. International Union of Pure and Applied Chemistry: The world authority on chemical nomenclature, terminology, standardized methods for measurement, atomic weights and other critically evaluated data http://www.bioinformatics.org/sms/iupac.html CGYGCWTATTTTACWGCYGCTAC

  15. International Union of Pure and Applied Chemistry: The world authority on chemical nomenclature, terminology, standardized methods for measurement, atomic weights and other critically evaluated data http://www.bioinformatics.org/sms/iupac.html CGYGCWTATTTTACWGCYGCTAC 2 x2 x2 x2 =16

  16. Peptide sequences from protein + Echinostoma paraensei Biomphalaria glabrata precipitate • MCDTTTDGGGWTIFQ • GKPDGAFXDNITVVE • LEIDLAQYVVDLTAR • FFTTFDKDNDDQQND MICRO- SEQUENCING 65 kDa TRANSLATE BACK AA TO NT SEQUENCE CHARACTERIZARION PCR PRIMERS

  17. International Union of Pure and Applied Chemistry: The world authority on chemical nomenclature, terminology, standardized methods for measurement, atomic weights and other critically evaluated data (puRines) (pYrimidines) http://www.bioinformatics.org/sms/iupac.html http://plato.stanford.edu/entries/information-biological/GeneticCode.png Met = ATG; Ser = TCN, AGY (WSN 2x2x4)

  18. M C D T T T D G GG W T I F Q ATG TCA GAT ACA ACA ACA GAC GGA GGA GGA TGG ACA ATA TTT CAT TCT GAC ACT ACT ACT GAT GGT GGT GGT ACT ATT TTC CAC TCC ACC ACC ACC GGC GGC GGC ACC ATC TCG ACG ACG ACG GGG GGG GGG ACG TGT TGC

  19. M C D T T T D G G G W T I F Q ATG TCA GAT ACA ACA ACA GAC GGA GGA GGA TGG ACA ATA TTT CAT TCT GAC ACT ACT ACT GAT GGT GGT GGT ACT ATT TTC CAC TCC ACC ACC ACC GGC GGC GGC ACC ATC TCG ACG ACG ACG GGG GGG GGG ACG TGT TGC ATG TCN GAY ACN ACN ACN GAY GGN GGN GGN TGG ACN ATH TTY CAR TGY 1 24 2 4 4 4 2 4 4 4 1 4 3 2 2

  20. M C D T T T D G G G W T I F Q ATG TCA GAT ACA ACA ACA GAC GGA GGA GGA TGG ACA ATA TTT CAT TCT GAC ACT ACT ACT GAT GGT GGT GGT ACT ATT TTC CAC TCC ACC ACC ACC GGC GGC GGC ACC ATC TCG ACG ACG ACG GGG GGG GGG ACG TGT TGC ATG TCN GAY ACN ACN ACN GAY GGN GGN GGN TGG ACN ATH TTY CAR TGY 1 24 2 4 4 4 2 4 4 4 1 4 3 2 2 GAY ACN ACN ACN GAY GGN G 2 x 4 x 4 x 4 x 2 x 4 = 1024 primers High primer degeneracy: allows for lots of miss-priming

  21. Reference(s) Ben-Dov, E., Shapiro, O.H., Siboni, N., Kushmaro, A. Advantage of Using Inosine at the 3’ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity. Appl. Environ. Microb. (2006), 72: 6902-6906. Loakes D. Survey and summary: The applications of universal DNA base analogues. Nucleic Acids Res. 2001 Jun 15;29(12):2437-47. Liu, H., Nichols, R. PCR amplification using deoxyinosine to replace entire codon and at ambiguous positions. Biotechniques. (1994), 16: 24-26. Oda, Y, Uesugi, S., Ikehara, M., Kawase, Y., Ohtsuka, E. NMR studies for identification of dI:dG mismatch base-pairing structure in DNA. Nucleic Acids Res. (1991), 19: 5263-5267. Ohtsuka, E., Matsuki, S., Ikehara, M., Takahashi, Y., Matsubara, K. An alternative approach to deoxynucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J. Biol. Chem. (1985), 260: 2605-2608. Martin, F.H., Castro, M.M., Aboul-ela, F., Tinoco, I. Base pairing involving deoxyinosine: implications for probe design. Nucleic Acids Res. (1985), 13: 8927-8938.

  22. M C D T T T D G G G W T I F Q ATG TCA GAT ACA ACA ACA GAC GGA GGA GGA TGG ACA ATA TTT CAT TCT GAC ACT ACT ACT GAT GGT GGT GGT ACT ATT TTC CAC TCC ACC ACC ACC GGC GGC GGC ACC ATC TCG ACG ACG ACG GGG GGG GGG ACG TGT TGC ATG TCN GAY ACN ACN ACN GAY GGN GGN GGN TGG ACN ATH TTY CAR TGY 1 24 2 4 4 4 2 4 4 4 1 4 3 2 2 GAY ACN ACN ACN GAY GGN G 2 x 4 x 4 x 4 x 2 x 4 = 1024 primers GAY ACI ACI ACI GAY GGN G 2 x 2 x 4 = 16 primers Universal base Inosine: 16-fold reduced primer degeneracy

  23. Other example: http://www-archive.thoracic.org/sections/research/additional-resources/research-methods/pcr-concepts-and-applications.html

  24. How to sequence full length? Primers where?

  25. Handout 446/546L Criteria for primer design 100 nt back from last good sequence 3’ GC clamp, make 3 of 6 3’ terminal residues G or C Recommend ~40% GC (not always possible) 18-24 nucleotides Annealing temp ≥ ~52C (higher Tm increases stringency, Tm must be similar for set of PCR primers) Count 2 for A/T, 4 for G/C Reverse complement for 5’-3’ of antisense primer Consider to Check with Netprimer (or alternative!): rating ≥80 Homework: E-mail your group’s forward and reverse primers for all three fragments (provide in *txt format by Friday morning 11.00 AM)Forward, Reverse + Forward, Reverse TARGET SEQUENCE 5’(GGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTT) GCGAATGGCTCATTAAATCAGCTATGGTTCCTTGGATCGTACATACTACATGGATAACTGTAGTAATTCTAGAGCTAATACATGCCACTATGCCCTGACCCGCAAGGGAACGGGTGGATTTATTAGAACAGAACCAACCGGGAGCGGCTTCGGTCGTTCCTGTTGCATTCTGTGATGACTCTGGATAACTTCACTGATCGCAGTCGGCCTTGTGTCGGCGACGGATCTTTCAAATGTCTGCCCTATCAATT Estimated ~400 bp gap CAGCGTGACTGGTGGGCTTGCCTGCTGGTCTGTTGGCATGCTTCTTGATGCCTTTAAACGGGTGTCGGAGGCGGACAGCACGTTTACTTTGAACAAATTTGAGTGCTCAAAGCAGGCCTTTGTGCCTGAAAATTCTTGCATGGAATAATGGAATAGGACTTCGGTTCTATTTTGTTGGTTTTCGGATCCGAAGTAATGGTTAAGAGGGACAGACGGGGGCATTTGTATGGCGGTGTTAGAGGTGAAATTCTTGGATCGCCGCCAGACAAACTACAGCGAAAGCATTTGCCAAGGATGTTTTCATTGATCTTGAGCG Estimated ~600 bp gap GCCTGAGCAGGTTGGGTAAACTGAATCATAACCGTCGTGACTGGGATCGGGGGCTTGCAATTGTTCCCCGTGAACGAGGAATTCCTGGTAAGTGCAAGTCATAAGCTTGCGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGTTTAGCAAGGTCCTCGGATTGGTGCCATTGTAGTGGCTTCGGCCGCTCGACCGGCGCTGAGAAGACGACCAAACTTGTTCATTTAGAGGAAGTAAA (NNNNNTCAAGGTTTCCGTAAGGGCGAAATCCAGCACACTGGCGGCCGTTACTAGTGGAT)3’

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