PCR Optimization: Challenges and Successes May 8, 2009 DNA Facility Seminar Series Outline Components of the PCR reaction Cycling Conditions Variations on basic PCR.
Download
Report
Transcript PCR Optimization: Challenges and Successes May 8, 2009 DNA Facility Seminar Series Outline Components of the PCR reaction Cycling Conditions Variations on basic PCR.
PCR Optimization:
Challenges and Successes
May 8, 2009
DNA Facility Seminar Series
Outline
Components of the PCR reaction
Cycling Conditions
Variations on basic PCR
PCR: History
PCR Invention: 1987 Kary Mullis
PCR is essentially DNA replication in a tube.
Series of repetitive steps enabling amplification
of target DNA from a complex mixture of DNA
Starting Thoughts
Think about purpose of PCR and downstream
applications for your PCR product
Think about “Carry over effect”
Set up area keeping in mind PCR has the potential
sensitivity to amplify a single molecule
Basics
Target
dNTP’s
Buffer
Primers
DNA Taq polymerase
Denature- 920C-950C
(940C)
Anneal- 500C-720C
Aim for 50C below
calculated Tm
(520C-580C generally best)
Extension - 680C-800C
(720C)
highest efficiency 700C-800C
Template
Plasmid
cDNA (RT-PCR)
Genomic DNA
P
C
Plasmid
P
Purified (P)
Crude Lysate (C)
C
Genomic
40ng 10ng 1ng
dNTPs
Mixture of dATP, dCTP, dGTP, dTTP or dUTP
Purity- chemical or enzymatic synthesis
Stability- concentration – Li or Na salt form
dNTPs
Purity can effect PCR
Buffer
All 10x Buffers are not the same
Salt
10-50 mM Tris pH 8.3
Monovalent cation
100-150 mM KCl or NaCl
Divalent cation
Mg2+, Mn2+
1.5uM or > MgCl2+
Additives
Detergent, Glycerol, Gelatin
Buffer Systems
Modifications:
Mg
pH
Ionic strength
Additives
Mg2+ g
Ionic strength g
Buffer Additives
Q-solution-Betaine
DMSO
BSA
Glycerol
Gelatin
PEG
GC-melt
Formamide
Detergents
Q
D
B
G
P Q/D F
D
Primers
Pair complementary to opposite strands
5’g3’ sense primer
3’g5’ anti-sense primer
Features
18-26 nucleotides
Equal mix GC to AT bases
Match Tm of primers
Tm oC= 2(A/T) + 4(G/C)
3’ Stability
GG or GC clamps
Additional Considerations
Secondary structure- avoid hairpins, self-dimers, crosshomology
Avoid di-nucleotide repeats that occur consecutivelyATATATAT
Avoid long runs of single bases- ACGGGGGGAT
Avoid cross-homology- BLAST Test
Primer Variation Example
PCR 1st Round vary primer pairs
Sets A-F
A= Primer 1F
B= Primer 2F
C= Primer 3F
D= Primer 1F
E= Primer 2F
F= Primer 3F
Forward primers
Primer 1: GAGGGCAGATTCGGGAATG
Primer 2: TCGGGAGAGGCCCTTCCC
Primer 3: CAGTTTCCCGGGTTCGGC
Reverse primers
Primer 1: AGCCTAATCAAGTCACTATCAAG
Primer 2: GCAAGTGAGAAAATGGGGAG
Tm=600c
Tm=620c
Tm=600c
Tm=620C
Tm=600C
Primer 1R
Primer 1R
Primer 1R
Primer 2R
Primer 2R
Primer 2R
DNA Taq Polymerases
Considerations: Aim of experiment
Thermal stability
Processivity
Fidelity
DNA Taq Polymerases
Standard polymerase
Standard polymerase with loading dye
Hot Start polymerase
works for most applications
Polymerase blends or cocktails
combine polymerases for
fidelity with speed
Taq blend
Standard Taq
aids in higher through-put
inhibits non-specific primer
extension
Hot Start Taq
Fidelity
PCR
PCR
product sequence
product T/A cloned
Individual isolates sequenced
PCR Cycling
Modified PCR Methods
Hot Start PCR
Manual Hot Start
Physical Barrier
Modified Taq DNA polymerase
Oligo Inhibitors
Modified dNTP’s
Semi-Nested or Nested PCR
Touch down PCR
Semi-Nested or Nested-PCR
Specificity
g g
-----------------------
-------------------f f
Sensitivity
Additional PCR Methods
Allele-specific PCR
Assembly PCR (PCA)
Breakpoint PCR
Intersequence-specific PCR (ISSR)
Inverse-PCR (IPCR or RE-PCR)
Ligation Mediated PCR (LM-PCR)
Long distance PCR
Multiplex-PCR
Methylation Specific PCR
Mini-primer PCR
Quantitative PCR or Real-time PCR
Reverse Transcriptase PCR (RT-PCR)
RT-PCR
Quality
Reverse
of RNA
Transcriptase-QC
oligo dT
random hexamers
gene specific primers
f
Multiplex-PCR
Increase
throughput
Increase data with limited material
1
Exon 7 and 8
Exon 9
Exon 3
Exon 5
Exon 1
Exon 2
Exon 6
Exon 4
2
3
4
5
6
7 8
9
Long-PCR
Analyze
Tool
large area in single reaction
to analyze inserts and breakpoints
14kb
3kb
20kb
1.6kb
Breakpoint-PCR
Isolate low frequency event
Inverse-PCR and RE-Inverse PCR
Isolate unknown flanking region
Digest with restriction enzyme
Ligate with T4 DNA ligase
Real-Time PCR or Q-PCR
Increased Sensitivity
Increased Specificity
Increased Throughput
Questions