Polymerase Chain Reaction (PCR) Fundamentals DNA Extraction
Introduction to PCR
DNA Genetic information for every animal, plant and microorganism Unique variations in DNA allow us to track it back to the organism it originated from with precision Comparative genomics, forensics, fingerprinting often require significant amounts of DNA PCR can synthesize, characterize and analyze any specific piece of DNA
What is PCR ?
Polymerase Chain Reaction:
in vitro (DNA synthesis in a tube) Yields million of copies of target DNA sequence Repeated cycling action Involving DNA polymerase enzyme
PCR Principles
Conceptualized by Kary Mullis in 1983 DNA amplification in vitro using the following components:
Two synthetic oligonucleotides (primers) complementary to each end of targeted DNA sequence Single nucleotide bases as substrate DNA polymerase; a naturally occurring enzyme responsible for in vivo DNA replication and repair
PCR Applications
Food Science:
Detection or molecular confirmation of specific microorganisms present in foods Molecular subtyping of isolates
Molecular Biology:
Mutagenesis, cloning or sequencing
Evolutionary Biology:
Re-create the evolutionary history of a group of taxa
PCR Applications
PCR detection particularly useful when
Classical detection too time-consuming Differentiation from closely related non-pathogenic organisms is difficult
Listeria monocytogenes
Only species in Listeria genera that is pathogenic to humans PCR assay targeted to detect hemolysin (hlyA) gene can detect presence and differentiate L. monocytogenes from other Listeria spp.
PCR Reaction
High temperature melts double strand DNA helix into single strand DNA Two synthetic sequences of single stranded DNA (18-24 bases) known as primers target a region of genome Forward primer and Reverse primer flank the region of interest (usually <1000 base pair)
PCR Reaction Components
1 DNA template 2 DNA polymerase 3 Primers (oligonucleotides) 4 Deoxynucleotidetriphosphate bases (dNTPs) 5 MgCl2 6 Buffer 7 Sterile ultrapure water
PCR Reaction Set-up
1 - DNA Template
Theoretically, PCR can detect as little as one DNA molecule Template DNA should be present in small amounts (< 106 target molecules; 1 ng of E. coli DNA = 3x105) Bacterial cells need to be lysed to make their DNA accessible for PCR
PCR Reaction Set-up
2 Primers
Should be in great excess to template DNA to ensure that most strands anneal to a primer and not each other Generally between 0.1 and 0.5 M optimal concentration Higher primer concentrations may cause nonspecific products
PCR Reaction Set-up
3 - Deoxynucleotide Triphosphates (dNTPs)
Building blocks of DNA As, Ts, Gs, & Cs Essential to have enough dNTPs to make desired number of target sequence copies Final concentration of EACH dATP, dTTP, dCTP and dGTP should be 0.1 mM
PCR Reaction Set-up
4 - DNA Polymerase Enzyme:
Original PCR performed with E. coli DNA polymerase, but high temperature (94-95oC) needed to denature double stranded DNA also denatured this polymerase Problem solved using thermostable Taq DNA polymerase Concentration should be around 0.5 to 5 units/100l reaction volume
PCR Reaction Set-up
5 - MgCl2
Essential for optimum Taq activity and for proper primer annealing Excessive concentrations cause non-specific products Standard PCR reaction contains between 1.5 and 5.0 mM MgCl2
PCR Reaction Set-up
6 - PCR buffer
Buffer keeps the PCR reaction at the proper pH (6.8-7.8) for enzyme activity Contains 10-50mM Tris-HCl and KCl or NaCl (50 mM) Salt also helps to stabilize hybridization of primer to target DNA
PCR Reaction Set-up
7 High quality water
Dilutes template, buffers etc.
PCR Demo Cycling Action
http://www.dnalc.org/files/swfs/animationlib /pcr.exe
Standard Thermal Cycling Conditions
25-40 cycles 94-96C 94-96C 2-10 min 1-2 min
*Initial denaturation *Denaturation *Annealing *Extension
50-55C 1-2 min
72C 1-2 min
72C 5-7 min
*Final extension
4 C
Newly synthesized extension product of one primer serves as template for annealing of the second primer in subsequent cycles Each reaction cycle doubles the number of DNA copies or PCR products
Amplification Cycles
Initial denaturation at 94-96oC for 2 min Standard PCR protocol has 20-45 cycles of the following time/temperature combinations 1-2 min @ 94-96oC 1-2 min @ 50-55oC 1-2 min @ 72oC
Denature DNA Primers hybridize by forming hydrogen bonds to complementary sequence DNA polymerase binds and extends a complementary strand from each hybridized primer
One final hold at 72oC for 5-7 min optional for higher product yield
Exponential Amplification
Newly synthesized extension product of one primer serves as template for annealing of the second primer in subsequent cycles Each reaction cycle doubles the number of DNA copies or PCR products
Important PCR Innovations
Acquisition of heat stable DNA polymerase (Taq) from Thermus acquaticus which inhabits hot springs
Remains active during repeated denaturation cycles
Thermal cycler or thermocycler
Computer controlled heating block for repetitive temperature change cycles required for PCR reaction
Basics of DNA Extraction
Goal Gain access to DNA 1) Classic DNA Extractions methods 2) Silica membrane (commercial kit) 3) Microwave (yep, just nuke the sample)
Classic DNA Prep
1. Disrupt the cell wall
Mechanically Beadbeating, sonication, French press Enzymatically Lysozyme
2. Remove membrane lipid
Triton-X, Sodium dodecyl sulfate (SDS)Detergents
3. Degrade proteins Proteinase K 4. Extract residual proteins phenol:chloroform extraction 5. Ethanol precipitation
Commercial Silica Column DNA Purification
1.Enzymatic lysis & protein degradation 2.Apply supernatant to silica membrane in a column 3.DNA is negatively charged; silica membrane is negatively charged Na+
Commercial Silica Column DNA Purification , cont.
4.High salt and Ethanol washes rinse degraded protein through the column 5. Elution Water with low salt concentrations disrupt the DNA-Na-Silica interaction releasing the DNA from the column
Microwave
Colony PCR or dirty lysates 1. Select an isolated colony from an agar plate 2. Transfer a portion to a PCR tube (0.2 mL tube) 3. Microwave Time dependent! 4. Add water 5. Add PCR reagents & thermal cycle
