Today HK DNA samples (gel and spec) PCR background PCR targets – snail 16S and CO1 – parasite...

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)

15 minute powerpoint topics date topic name

21-Sep Discovery of DNA structure Janette Mendoza

25-Sep Restriction enzymes Gabriela Perales

28-Sep Southern blotting Carlos Garcia G

2-Oct Cloning Timothy McBride G

6-Oct The first sequenced gene Conrad Greaves

9-Oct (q)PCR, specificity and sensitivity Krystal Charly

13-Oct ESTs Ian Keller

16-Oct BLAST and database searches Ryan Heimroth G

20-Oct Microarrays Bianca Myers

23-Oct Forensics Jennifer Gutierrez

26-OctGenome sequencing , the $1000

genomeAyesha Arefin G

30-Oct Next generation sequencing Leslie Janet Lopez G

2-Nov Bioinformatics Amalia Parra

6-Nov Epigenetics Clyde Moya

9-Nov non-coding RNA Helen Nordquist

13-Nov C-value paradox Kelsey Cook G

16-Nov Phylogenetic genomics Jennifer Cooksey

20-Nov Genes associated with Type 1 diabetes Katie Kesler

http://sev.lternet.edu/about

FIELDTRIP to Sevilleta LTER, Sample collection:

Sunday 13 September

(Sunday 20 September)

PARASITES AND SNAILPARASITES AND SNAIL BIOLOGY BIOLOGY

“identity, possibilities”phylogenetics

“intentions”transcriptomics

PCRrDNA/mito

BioanalyzerDNA-free,

direct sequencing

gel electrophoresisnanodrop spec

Sequence ID (BLAST)editing

Phylogenetics

electrophoresisRT-PCR

gel

CTAB/DNAzol

Trizol

TA cloning, B/W screening

M13 sequencing

Primer design, walking

Qiagen plasmid extraction Restriction digests

DNA

RNA

GenBank submission

http://www.jove.com/video/3923/agarose-gel-electrophoresis-

for-the-separation-of-dna-fragments

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

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

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, ……

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

Key features of PCR• High temperature denatures dsDNA to ss DNA • 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’)(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

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

http://www.dnalc.org/ddnalc/resources/pcr.html

http://users.ugent.be/~avierstr/principles/pcrani.html

Animations

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

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

PCR• Theory: exponential target amplification

x 230 (1,073,741,824)

• Reality: reagents limiting, routine PCR is NOT quantitativecycle number

Am

plif

icat

ion

phases of PCRstart-upexponentiallagplateau

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)

1-2 SP1;2

5-6 SP5;4 7-8 SP5;4

3 SP3

9-10 SP1;2

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

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)

Enzyme cofactors (Mg) Buffer optimized for enzyme dNTPs

http://www.diffen.com/difference/Image:Nucleotides.png

Thermo-cycling• denature DNA 95C

• Anneal primer Tm

• Extend (make new DNA) 72C

• Repeat……..

• Hot top PCR machines

Tm: melting temperature of primers:50% of primers annealed to templateLower T, increased %, plus mismatches (ASPECIFIC)Higher T, reduced %, fewer/NO mismatches

T (temperature)

% p

rim

erbo

und

to te

mpl

ate

50

100

T meltingTemperature gradient)

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)

Targets

• Mitochondrial rDNA and coding gene:16S and CO1

• Nuclear rDNA genes: 18S and 28S

TARGET 1rDNA genes

Repeated rDNAgene cassette

Genes occuracross phylogeny

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

Nolan et al., 2013