devised by Kary Mullis c1983

POLYMERASE CHAIN REACTION - PCR

A 'licence' to do molecular biology
A key central technique that has revolutionised molecular and
consequently cell biology
POLYMERASE CHAIN REACTION - PCR
Kamchatka's thermaI hot springs

AmpIification via PCR

Multiple rounds of denaturation-
annealing-extension are
performed to create many copies
of the template DNA between
the two primer sequences.
Polymerase Chain Reaction (PCR)
PoIymerase Chain Reaction (PCR)
PCR Agarose gel electrophoresis
The final product UV visualisation
3-4 hours
-Cloning of genes or gene fragments
same species or homologous genes from different species
-Genetic diagnosis - Mutation detection
-Paternity testing
-Mutagenesis to investigate protein function
-Quantitate differences in gene expression
Reverse transcription (RT)-PCR
-Identify changes in expression of unknown genes
Differential display (DD)-PCR
-Forensic analysis at scene of crime
-Industrial quality control
-and so on...
APPLICATIONS OF PCR
Same principle different template
gDNA
- Maus / Mensch usw
- alte Quelle
pDNA
cDNA
Technische Voraussetzungen
Primer
Zyklenzahl
Polymerasen
Denaturation 93C - 95C
30 secs 1min
AnneaIing 37C - 65C
30 secs 1min depends on the duplex
Extension 72C
1min
(+ 30secs per 500bp DNA)
25-35 cycles
Final extension 2-10mins
CYCLING PARAMETERS
COMPONENT VOLUME Final
Concentration
10 X PCR Buffer
5 l
1X
10 X dNTPs (2mM)
5 l
200 M
Forward primer (10pmols/ l)
5 l
1M (50pmols/50l)
Reverse primer (10pmols/ l)
5 l
1M (50pmols/50l)
Genomic DNA template
2 l
1 g
Thermostable polymerase
(2U/ l)
0.5 l
1 unit
H
2
O (to 50 l Final volume)
27.5 l
25 or 50ls in a micro Eppendorf (0.5ml) tube
TYPICAL REACTION MIXTURE
PCR optimization - empiricaI technic
1.)GeneraIities
Components, PCR cycIe, viaIs
2.)Choosing PCR primers
How to design the primers
3.)Reaction voIumes
Do they infIuence the resuIts?
4.)Number of PCR products MuItipIexing. How ?
5.)Primer amount
How much primer?
6.)PCR buffers
Comparisons and concentration
7.)SaIt (KCI) concentration
EssentiaI optimization factor
8.)Designing PCR programs
Customize them for your purpose
9.)AnneaIing time and temperature
10.)Extension time and temperature
11.)DNA tempIate concentration
12.)Taq poIymerase(s)
Comparisons and concentration
13.)dNTP concentration
Interaction with magnesium
14.)MgCI2 concentration
Interaction with dNTP and saIt (PCR buffer)
15.)GeI eIectrophoresis
Agarose and poIyacryIamide (PAA)
16.)Adjuvants in PCR
DMSO, gIyceroI and BSA
NormaI concentrations of a PCR-reactions
NormaI concentrations of a PCR-reactions
TempIate:
1 - 100 ng genomic DNA
2 - 10 ng pIasmid DNA
Primer:
0,1 - 1 mM forward primer (mM = 1 pmoI I-1)
0,1 - 1 mM reverse primer (mM = 1 pmoI I-1)
NucIeotides:
0,2 mM dNTP (dATP, dTTP, dGTP, dCTP)
PCR-buffer:
1 x
MgCI2:
1,25 - 3 mM
Taq-poIymerase:
0,025 units I-1 (= ca. 0,5 U per reaction)
TotaI PCR-voIume:
20 - 100 I
PCR-buffers
PCR-buffers
1x PCR buffer simpIest:
50 mM KCI
10 mM Tris-HCI (pH 8.3)
1,5 mM MgCI2
1X ThermoPoI DF (Detergent-free) :
10 mM KCI
10 mM (NH4)2SO4
20 mM Tris-HCI
2 mM MgSO4
pH 8.8 @ 25C
1X ThermoPoI Reaction Buffer:
20 mM Tris-HCI
10 mM (NH4)2SO4
10 mM KCI
2 mM MgSO4
0.1 % Triton X-100
pH 8.8 @ 25C
pIus
0,1mg/mL BSA => Pfu buffer
PCR is a highly sensitive technique - contamination
with unwanted DNA can be a problem
-5 run NEGATIVE controls
Include a positive control if appropriate
Use dedicated filtered tips and positive displacement
pipettes
Dedicated areas?
Can use UV cabinets
ALWAYS REMEMBER!
RumIiche Trennung - "reine Werkbnke"
Depends on the PCR
Can be used where products are diffuse or absent
ADDITIVES?
DMSO 2-10% reduces Taq polymerase activity
dimethyl sulfoxide reduces secondary structure -GC rich templates
Betaine 1.0-1.7M use Betaine or Betaine (mono)hydrate and not Betaine HCl
N,N,N-trimethylglycine Q-Solution/Advantage-GC
Formamide 1-5% use as less as possible
Non-ionic detergents stabilise Taq polymerase and supress the formation of secondary structure
Triton X-100/Tween 20/Nonidet P-40 0.1-1% 0.1% increase yield but may also increase non-specific
0.5% Tween-20 or -40 will effectively neutralise this effect
TMAC 15-100mM
tetramethylammonium chloride
7-deaza-2'-deoxyguanosine facilitate amplification of templates with stable secondary structures
used in place of dGTP in a ratio of 3: 1- 7-deaza-2'-deoxyguanosine: dGTP
BSA amplify ancient DNA or templates which contain PCR inhibitors
bovine serum albumin
ThermocycIers and PCR viaIs
ThermocycIers and PCR viaIs
Variation in ampIification due to
-Iack of proper contact between the metaI bIock and some viaIs
Bottom Iine:
-use aIways same cycIer and viaIs
-or write down what you used
PCR-buffer / KCI conc
PrimerIength
optimaI primer design -> Rainer
optimaI Iength -> depends from aim
Longer primers (30-35 bp) seem to work in more simiIar cycIing conditions
compared with shorter primers, and can make muItipIexing easier.
Lanes A-F viaIs with identicaI reaction mixture - pIaced in different position
PCR ampIification of a pIymorphic Iocus in the presence of decreasing,
Iow amounts of genomic tempIate DNA and at an anneaIing temperature
10 C Iower than normaI.
AnneaIing temperature
Number of options available
Taq polymerase
Pfu polymerase
Tth polymerase
-How big is the product?
100bp 40-50kb
-What is end purpose of PCR?
Sequencing - mutation detection
Need high fidelity polymerase
integral 3 5' proofreading exonuclease activity
Cloning (TA cloning?)
CHOOSE YOUR POLYMERASE WITH CARE
USE MASTERMIXES WHERE POSSIBLE
If not.........troubleshoot
Qiagen PCR
methods
THE PERFECT RESULT