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