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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)
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
3. Degrade proteins Proteinase K 4. Extract residual proteins phenol:chloroform extraction 5. Ethanol precipitation
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