1. Today HK DNA samples (gel and spec) PCR background PCR targets – snail 16S and CO1 – parasite rDNA 18S and 28S Compose PCR reactions AmpliTaq Gold (ABI)

2. 15 minute powerpoint topics datetopicname 21-SepDiscovery of DNA structureJanette Mendoza 25-SepRestriction enzymesGabriela Perales 28-SepSouthern blottingCarlos GarciaG 2-OctCloningTimothy McBrideG 6-OctThe first sequenced geneConrad Greaves 9-Oct(q)PCR, specificity and sensitivityKrystal Charly 13-OctESTsIan Keller 16-OctBLAST and database searchesRyan HeimrothG 20-OctMicroarraysBianca Myers 23-OctForensicsJennifer Gutierrez 26-OctGenome sequencing, the $1000 genomeAyesha ArefinG 30-OctNext generation sequencingLeslie Janet LopezG 2-NovBioinformaticsAmalia Parra 6-NovEpigeneticsClyde Moya 9-Novnon-coding RNAHelen Nordquist 13-NovC-value paradoxKelsey CookG 16-NovPhylogenetic genomicsJennifer Cooksey 20-NovGenes associated with Type 1 diabetesKatie Kesler

3. http://sev.lternet.edu/about FIELDTRIP to Sevilleta LTER, Sample collection: Sunday 13 September (Sunday 20 September)

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/DNAzol Trizol TA cloning, B/W screening M13 sequencing Primer design, walking Qiagen plasmid extraction Restriction digests DNA RNA GenBank submission

5. http://www.jove.com/video/3923/agarose-gel- electrophoresis-for-the-separation-of-dna-fragments

6. Interpretation 1) Molecular weight marker, shows fragment size (bp) see website, staining intensity may provide reference for amount of experimental DNA. 2) Good genomic DNA, methods used yield fragments of 20-50 kbp, RNA may be visible as a smear low in the lane 3) A smear indicates degraded DNA NO signal does NOT mean no DNA! 1 2 3

7. 1-2 SP1;2 5-6 SP5;4 7-8 SP5;4 3 SP3 9-10 SP1;2 S1 S2 S3 S4 S5 P1 P2 P3 P4 P5

8. Polymerase Chain Reaction (PCR) Nobel prize Kary B. Mullis 1993 (developed 1984, patent 1985) Standard tool for molecular biology Pre PCR era and post PCR era Allows generation (amplification/detection) of DNA fragments from limited amounts of starting material (DNA or mRNA) Applications in gene characterization, forensics, diagnostics, phylogenetics, gene expression, ……

9. http://www.youtube.com/watch?v=-bF2QalUj1Y&feature=related

10. Key features of PCR High temperature denatures dsDNA to ss DNA Two primers hybridize ssDNA on opposite strands (NEED 2 PRIMERS) (5’ -> 3’)DNA polymerase makes new ds DNA downstream from ds to ss DNA junctions (5’ -> 3’) Thermostable DNA polymerases (like Taq polymerase from Thermophylus aquaticus) can do this repeatedly without losing activity. Exponential amplification of DNA between primer target sites

11. http://users.ugent.be/~avierstr/principles/pcr.html

12. http://www.dnalc.org/ddnalc/resources/pcr.html http://users.ugent.be/~avierstr/principles/pcrani.html Animations

13. http://www.ncbi.nlm.nih.gov/books

14. Polymerase Chain Reaction Molecular Biology of the Cell 4th ed. Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002Garland Science

15. PCR Theory: exponential target amplification x 2 30 (1,073,741,824) Reality: reagents limiting, routine PCR is NOT quantitative cycle number Amplification phases of PCR start-up exponential lag plateau

16. PCR needs DNA template (gDNA, PCR products, cDNA) DNA Polymerase Primers Enzyme cofactors (Mg) Buffer optimized for enzyme and primers dNTPs; deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP)

17. 1-2 SP1;2 5-6 SP5;4 7-8 SP5;4 3 SP3 9-10 SP1;2

18. Polymerases So many, not enough time to list Things to consider –Fidelity (proof reading, too many to list) –Template independent 3’ A addition –Hot start –Length of target sequence –HAVING TROUBLE? MAYBE TRY A DIFFERENT POLYMERASE! I routinely use AmpliTaq Gold (ABI, Life Sciences), and the Advantage polymerase for cDNA mix (Clontech) when things do not work. – For a list of available choices, go to Biocompare.com

19. Where do PCR primers come from? We choose or design them. (design defines optimum reaction conditions) Known targets: design from target DNA sequence Searching genes: design from conserved genes at DNA or protein level Random targets: design for common features or random More detail later (you will design some)

20. Enzyme cofactors (Mg) Buffer optimized for enzyme dNTPs http://www.diffen.com/difference/Image:Nucleotides.png

21. Thermo-cycling denature DNA 95C Anneal primer Tm Extend (make new DNA) 72C Repeat…….. Hot top PCR machines

22. Tm: melting temperature of primers: 50% of primers annealed to template Lower T, increased %, plus mismatches (ASPECIFIC) Higher T, reduced %, fewer/NO mismatches T (temperature) % primer bound to template 50 100 T melting Temperature gradient)

23. Anatomy of a good PCR product Correct size ds DNA (Primer 1) - amplified region – (primer 2) Checks/Controls: Positive (did the reagents work?) Tp1p2, Tp1-, T-p2, p1p2, T– (where T = template, p is primer)

24. Targets Mitochondrial rDNA and coding gene: 16S and CO1 Nuclear rDNA genes: 18S and 28S

25. TARGET 1 rDNA genes Repeated rDNA gene cassette Genes occur across phylogeny

26. Mol Biol Evol. 2002 Mar;19(3):289-301.

28. Nolan et al., 2013