Navigating DNA Analysis Techniques: From Gel Electrophoresis to Genome Sequencing

1. Today • HK • DNA samples (gel and spec) • PCR background • *.ppt • Compose PCR reactions • PCR targets • snail 16S and CO1 • parasite rDNA 18S and 28S

2. SNAILAND PARASITES BIOLOGY DNA “identity, possibilities” phylogenetics CTAB/DNAzol CTAB/DNAzol Illumina (full) genome sequencing gel electrophoresis nanodrop spec Qubit Fluorometry Covaris fragmentation Ampure (fragment collection) Kapa DNA library preparation kit Pippin size selection QC Bioanalyzer, Qubit, qPCR Illumina run PCRrDNA/mito TA cloning, B/W screening electrophoresis Qiagen plasmid extraction Restriction digests direct sequencing M13 sequencing Sequence ID (BLAST) editing Galaxy QC Data file (MT) genome assembly Mitos, manual annotation Gene annotation Primer design, walking Phylogenetics GenBank submission

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

4. 1 2 3 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, standard extraction methods yield fragments of 20-50 kbp, RNA may be visible as banding or a smear low in the lane 3) A smear indicates degraded DNA NO signal does NOT mean no DNA! NEVER THROW A SAMPLE AWAY BEFORE PCR TEST

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7. FLUORESCENT STAINING ng 1000 500 250 100 50 10 Ethidiumbromide drop method (Quantitative binding DNA-EthBr) known amounts DNA compare intensity of sample “dynamic range” is limited Fluorometry “Hoechst dyes” reference sample of known amount sensitive but expensive Qubit Fluorometry 600 ng (Black box)

8. Spectrometry Chemicals in solution absorb light Depending on the chemical, Some wavelengths are absorbed more than others The amount of light absorbed depends on the concentration,transmission is reduced with increasing concentration

10. Transmittance and Absorbance I T = ---- = 10 –al = 10 –elc I0 a = absorbance coefficient (molar absorbance x concentration) I A = - log10 T = - log10(----) = - log10(10 –elc ) I0 A = elc The Beer-Lambert Law: A = εlc where A is absorbance, ε is the extinction coefficient (units: M-1cm-1), l is the path length that the light has travelled (units: cm) and c is concentration (units: M). http://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law

11. Spectrophotometry of DNA Compound max absorbance Nucleic acids 260 nm Proteins 280 nm Organic solvents 230 nm diluant light (x nm) DNA A 260nm x 50 x dilution = ng/l double stranded DNA A 260nm x 40 x dilution = ng/l single stranded RNA 260/280 ratio of 1.8-2.0 indicates good DNA sample (low protein) watch the A 230nm for organic contamination Half the concentration, half the absorbance

12. UV spectrophotometric measurement of DNA concentration and purity DNA itself, and most of the common contaminants found in DNA preps, have absorbances in the region 230nm to 320nm so measurement of the absorbances in this region allows measurement of the DNA concentration and provides information about the contaminant levels. The most important wavelengths to note are: 230nm: Guanidium salts (used to facilitate DNA binding to silica columns) and phenol (used in phenol/chloroform extractions) absorb strongly at 230nm, therefore high absorbances at this wavelength can be indicative of carry-over of either of these compounds into the sample. 260nm: DNA absorbs light most strongly at 260nm so the absorbance value at this wavelength (called A260) can be used to estimate the DNA concentration using the equation Concentration (µg/ml) = (A260 reading) × 50 , which is derived from Beer’s Law . 280nm: Since tyrosine and tryptophan residues absorb strongly at this wavelength, the absorbance at 280nm is used as an indicator of protein contamination. 320nm: A320 provides a general measurement of the turbidity of the sample and is normally subtracted from the A260 value as a background reading for the calculation of DNA concentration, but excessive values may indicate non-specific contamination. A good quality DNA sample should have a A260/A280 ratio of 1.7–2.0 and an A260/A230 ratio of greater than 1.5, but since the sensitivity of different techniques to these contaminants varies, these values should only be taken as a guide to the purity of your sample. http://bitesizebio.com/2007/08/22/dna-concentration-purity/

13. NANODROP: great for SMALLVOLUMES

14. SO, Do you have good/useful DNA samples? Gel results: Spec results: Never throw away samples until you tried PCR

15. *.PPT

16. 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, ……

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

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

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

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

21. 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; c2002

22. phases of PCR start-up exponential lag plateau Amplification cycle number PCR • Theory: exponential target amplification x 230 (1,073,741,824) • Reality: reagents limiting, routine PCR is NOT quantitative

23. 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)

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25. 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 start with AmpliTaq Gold (ABI, Life Sciences), and use Advantage polymerase for cDNA mix (Clontech) if things do not work. • For a list of available choices, go to Biocompare.com

26. Where do PCR primers come from? We choose or design them.(design defines optimum reaction conditions) Known targets: literature 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)

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

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

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

31. 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)

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

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

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

36. Nolan et al., 2013