Lecture 32 PCR & DNA Extraction

8/13/2019 Lecture 32 PCR & DNA Extraction

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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 (

Lecture 32 PCR & DNA Extraction

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