Download 5 PCR - UNM Biology

Today
• HK, CARC/GALAXY
• DNA samples (gel and spec)
• PCR background
• PCR targets
– snail 16S and CO1
– parasite rDNA 18S and 28S
• Compose PCR reactions
SNAIL AND PARASITES BIOLOGY
DNA
“identity, possibilities”
phylogenetics
RNA
“intentions”
transcriptomics
CTAB/DNAzol
Trizol
gel electrophoresis
nanodrop spec
Illumina (full)
genome sequencing
Bioanalyzer
DNA-free,
PCR
rDNA/mito
TA cloning, B/W screening
electrophoresis
Qiagen plasmid extraction
Restriction digests
Qubit Fluorometry
Covaris fragmentation
Ampure (fragment collection)
Kapa DNA library preparation kit
Pippin size selection
QC Bioanalyzer, Qubit, qPCR
Illumina run
direct sequencing
M13 sequencing
Sequence ID (BLAST)
editing
Primer design, walking
Phylogenetics
GenBank
submission
Galaxy
QC Data file
(MT) genome assembly
Mitos, manual annotation
Gene annotation
RT-PCR
gel
http://www.jove.com/video/3923/agarose-gelelectrophoresis-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
1Lsc1, 1Sc1; 2Lsc1, 2Sc1
7PSt, 7St;
8PSt, 8St
3Lsc2, 3Sc3; 4Lsc2, 4Sc21
5PE, 5E;
9HDau, 10HDau, 9Dau, 10Dau
6PE, 6E
1Lsc1, 1Sc1; 2Lsc1, 2Sc1
7PSt, 7St;
8PSt, 8St
3Lsc2, 3Sc3; 4Lsc2, 4Sc21
5PE, 5E;
9HDau, 10HDau, 9Dau, 10Dau
6PE, 6E
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 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 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
Animations
http://users.ugent.be/~avierstr/principles/pcrani.html
http://www.dnalc.org/ddnalc/resources/pcr.html
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)
Amplification
phases of PCR
start-up
exponential
lag
plateau
cycle number
• Reality: reagents limiting,
routine PCR is NOT quantitative
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)
1Lsc1, 1Sc1; 2Lsc1, 2Sc1
7PSt, 7St;
3Lsc2, 3Sc3; 4Lsc2, 4Sc21
8PSt, 8St
5PE, 5E;
9HDau, 10HDau, 9Dau, 10Dau
6PE, 6E
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
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 template
Lower T, increased %, plus mismatches (ASPECIFIC)
Higher T, reduced %, fewer/NO mismatches
% primer
bound to template
100
50
T (temperature)
T melting
Temperature 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 1
rDNA genes
Repeated rDNA
gene cassette
Genes occur
across phylogeny
Mol Biol Evol. 2002 Mar;19(3):289-301.
Nolan et al., 2013