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