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The Polymerase Chain Reaction
Chapter 6: Background
Polymerase Chain Reaction(PCR)
• PCR • Amplify=
• Invented in 1984
• Applications
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com‹#›
Invention of PCR
▪ Kary Mullis – Mile marker 46.58 in April of 1983
– Pulled off the road and outlined a way to conduct DNA replication in a tube
DNA Replication vs. PCR
• PCR
• The laboratory version
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What is PCR?
▪ Polymerase chain reaction or PCR – Simplified version
– Copies a specific sequence of DNA
– Target is
– Annealing
PCR: the in vitro version of DNA Replication
The following components are needed to perform PCR in the laboratory:
1) Template DNA 2) DNA Polymerase 3) Four 4) Buffers 5) Magnesium chloride (MgCl2) 6) Primers 7) Tubes 8) Thermal cycler
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Thermal Cyclers
▪ Water baths
▪ Thermal cyclers
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Challenges of DNA Replication in a Tube
▪ Normal replication:
▪ Heat
▪ Heat-stable DNA polymerases
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Heat-stable DNA Polymerase
▪ PCR involves very high temperatures
▪ DNA polymerases would denature
▪ Taq DNA polymerase
▪ Taq
The three main steps of PCR• Basis of PCR:
• In a PCR reaction:
• Step 1: Denature DNA
Step 2: Primers Anneal
Step 3: DNA polymerase Extends the DNA chain
Denaturation of DNA
Step 2 Annealing or Primers Binding
Forward Primer
Reverse Primer
Step 3 Extension or Primer Extension
extension
extension
The Size of the DNA Fragment Produced in PCR is Dependent on the Primers
• The PCR reaction
Forward primer
Reverse primer
The DNA of interest is amplified by a power of 2 for each PCR cycle
Example:
Overview of PCR
1. Temperature Cycling Denaturation Annealing Extension
2. Every cycle DNA between primers is duplicated
PCR Amplification
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Types of PCR
▪ There are many variations of traditional PCR – Real-time or quantitative PCR (qPCR) – Reverse transcription PCR (RT-PCR) – Multiplex PCR – Degenerate PCR – Nested PCR – Random amplification of polymorphic DNA (RAPD) – Fast PCR
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PCR Optimization
▪ Each reaction is very different
▪ Factors that must be taken into account vary with the purpose of the reaction
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com‹#›
PCR Optimization
▪ Conditions and factors to optimize:
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com‹#›
PCR Optimization
▪ Template quality
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PCR Optimization
▪ Template concentration
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com‹#›
PCR Optimization
▪ Primers – Primers bind to their complementary sequence on the target
DNA
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com‹#›
PCR Optimization
▪ Primer concentration – The normal range: – Increasing the concentration can lead to
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com‹#›
PCR Optimization
▪ Primer design – The sequence of the primers is crucial to the success of PCR
– Tm
– Annealing temperature
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PCR Optimization
▪ Magnesium concentration – Magnesium
– Normal concentration
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PCR Optimization
▪ Annealing temperature – Annealing temperature:
– Annealing temperatures are often adjusted
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PCR Optimization
▪ Cycling temperatures and times – Amount of time that each step
– Only the annealing temperature is adjusted
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PCR Optimization
PCR has become a very powerful tool in molecular biology
• Start with a single sperm cell or stand of hair
• Can amplify fragments of interest
• Can use the selectivity of the primers
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Other Applications of PCR
▪ PCR in medicine
PCR and prenatal diagnosis
• For prenatal diagnosis, PCR used to amplify DNA
• Single base changes then detected by: - -
• Combination of two methods provides confirmation.
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Other Applications of PCR
▪ PCR in medicine – Detection of disease-causing organisms
– Amplification of disease-causing organisms for quick diagnosis
– Amplification of genes of interest for cloning
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Other Applications of PCR
▪ PCR and Disease – Primers can be created that will only bind and amplify certain
alleles of genes or mutations of genes
– Some diseases that can be diagnosed with the help of PCR:
Huntington’s Disease (HD)
• HD
• Caused by: • In individuals with HD, the HD gene is “expanded”
– In non-HD individuals:
– In HD individuals:
• PCR can be performed on an individual’s DNA to determine whether the individual has HD.
Cystic Fibrosis (CF)
• CF
• Caused by:
• In non-CF individuals:
• In CF individuals,
• The presence of CTFR mutations in a individual can be detected by performing PCR and sequencing on that individual’s DNA.
Human Immunodeficiency Virus (HIV)
• HIV
• HIV tests
• PCR product = • No PCR product = • Before the appearance of HIV antibodies
• PCR can detect:
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Other Applications of PCR
▪ PCR in agriculture – GMO – Compliance with reporting
requirements
PCR in Forensics
Crucial forensic evidence may be present in very small quantities. • Too little material
• PCR also possible on extensively degraded DNA.
Other advantages of PCR in forensic science are:
Major legal problems with PCR:
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Other Applications of PCR
▪ PCR in forensics – Short Tandem Repeat (STR) analysis
is commonly used in forensics – PCR used:
– Regions contain
– Primers are chosen that will amplify these repeated areas
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Other Applications of PCR
▪ PCR in forensics – Short tandem repeats
(STR) analysis • Short tandem repeats are
amplified and used as a method of identification
Using STR to compare forensic and suspect samples
Individuals A & C are excluded by this analysis. The samples from individual B will be subjected to further tests.
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Other Applications of PCR
▪ PCR in forensics – Allele frequencies
– Combined DNA Index System (CODIS)
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Other Applications of PCR
▪ PCR in paternity – Samples are amplified
using STR analysis
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Other Applications of PCR
▪ PCR in human migration – Short interspersed
repetitive elements (SINEs) have been inserted randomly into our human genome over millions of years.
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Other Applications of PCR
▪ PCR in wildlife conservation – Poaching endangers many animals in the world today