1. Today House Keeping –CARC –MIDTERMs Exosap, sequencing fungal amplicons Ppt presentation Primer design

3. PCR amplicon clean-up, sequencing EXOSAP http://media.affymetrix.com/support/technical/usb/brochure/ExoSAP-IT_Brochure.pdf

4. PARASITES AND SNAIL BIOLOGY “identity, possibilities” phylogenetics “intentions” transcriptomics PCR rDNA/mito Bioanalyzer DNA-free, direct sequencing gel electrophoresis nanodrop spec Sequence ID (BLAST) editing Phylogenetics electrophoresis RT-PCR gel CTAB Trizol TA cloning, B/W screening M13 sequencing Primer design, walking Qiagen plasmid extraction Restriction digests DNA RNA GenBank submission + Sevilleta snails

8. How to make primers? Posthodiplostomum sp. NSMT:Pl_5926 genes for 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA, partial and complete sequence

9. Primer Design Options -Literature -Sequence (also from gene discovery) -NO sequence from your organism

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

13. CGYGCWTATTTTACWGCYGCTAC http://www.bioinformatics.org/sms/iupac.html 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

14. CGYGCWTATTTTACWGCYGCTAC 2 x2 x2 x2 =16 http://www.bioinformatics.org/sms/iupac.html 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

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

18. Met = ATG; Ser = TCN, AGY (WSN 2x2x4) http://plato.stanford.edu/entries/information-biological/GeneticCode.png http://www.bioinformatics.org/sms/iupac.html (pYrimidines) (puRines) 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

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

21. 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

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 High primer degeneracy: allows for lots of miss-priming

23. 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.

24. 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

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

26. 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)

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

29. 1 80 GNNNNNNNNN NNNNGGCGAT TGGGCCCTCT AGATGCATGC TCGAGCGGCN NNNNNTGTGA TGGATATCTG CAGAATTCGC CNNTAGGTCG ACCCGCTGAA TTTAAGCATA TCACTAAGCG GAGGAAAAGA AACTAACCAG GATTCCCCTA GTAACGGCGA GTGAACAGGG ATCAGCCCAG CACCGAAGCC TGTGGCCGTT TGGCTGCTAG GCAATGTGGT GTTTAGGTCG TCTCTTGGCA TTACTGCTCC ATCCTAAGTC CAGCAYTGAG TATGGCATCT GGAGTGGCCC ATTGAGGGTG AAAGGCCCGT GGGGGTGGAG ATCAAGTCGG CCAGTATTGC CCTGAGTAGA CCTTGGAGTC GGGTTGTTTG TGAATGCAGC CCAAAGTGGG TGGTAAACTC CATCCAAGGC TAAAATACTA GCACGAGTCC NATAGCGAAA NNNNTACCGT GAGGGGAAAN NTNNAAAAGT ACTTTGAANA NAAGAGTAAA ACAGTGCGTG AAANCCCTTC NNAGGNAAAC CNNGGNGGAN TTTGAANNTG NAANCTCCGG GGAATTCNAN NGGGGNNNNG NGNCNTGGGC TTTGGCCNCT TTTTGGCCNG GCNCNNNNAN NNCCNNNTCN NTGNCGGGCC NTGNTCCTTT CCGGANNNNA NNNGGNNNNN NNNTTNCNCT AGNGANNGNG NNCCCTGGNC GGNANNNTGC NNGCTGGNCN NNNNNNTTTN TTTNNCNGAN NGNCNCCCCC AGGCNNCNNT NNNCCTCNNN GCNTNNNNCA ACCCGNCNGN TTGNNNNNNG NNNNNNTTNN NNNGNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNTNNNNN NNNNNNNNNN NNNNNNNNTN NNNNNNNNNNNNNN Exercise: Design a forward primer and a reverse primer Report primer sequences 5’->3’