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Polymerase Chain Reaction (PCR)
Fundamentals
DNA Extraction
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Introduction to PCR
DNA Genetic information for every animal, plant andmicroorganism
Unique variations in DNA allow us to track it back to the
organism it originated from with precision
Comparative genomics, forensics, fingerprinting oftenrequire significant amounts of DNA
PCR can synthesize, characterize and analyze any specificpiece of DNA
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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
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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
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PCR Applications
Food Science:
Detection or molecular confirmation of specificmicroorganisms 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
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PCR Applications
PCR detection particularly useful when
Classical detection too time-consuming
Differentiation from closely related non-pathogenicorganisms is difficult
Listeria monocytogenes
Only species in Listeria genera that is pathogenic tohumans
PCR assay targeted to detect hemolysin (hlyA) gene candetect presence and differentiate L. monocytogenesfrom other Listeria spp.
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PCR Reaction
High temperature melts double strand DNAhelix into single strand DNA
Two synthetic sequences of single stranded DNA
(18-24 bases) known as primers target a region ofgenome
Forward primer and Reverse primer flank theregion of interest (usually
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PCR Reaction Components
1 DNA template
2 DNA polymerase
3 Primers (oligonucleotides)
4 Deoxynucleotidetriphosphate bases (dNTPs)
5 MgCl26 Buffer
7 Sterile ultrapure water
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PCR Reaction Set-up
1 - DNA Template
Theoretically, PCR can detect as little as one DNAmolecule
Template DNA should be present in small amounts (