the polymerase chain reaction - norwell high school...polymerase chain reaction or pcr –...

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

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

Thermal Cyclers

▪ Water baths

▪ Thermal cyclers

Challenges of DNA Replication in a Tube

▪ Normal replication:

▪ Heat

▪ Heat-stable DNA polymerases

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

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

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

PCR Optimization

▪ Each reaction is very different

▪ Factors that must be taken into account vary with the purpose of the reaction

PCR Optimization

▪ Conditions and factors to optimize:

PCR Optimization

▪ Template quality

PCR Optimization

▪ Template concentration

PCR Optimization

▪ Primers – Primers bind to their complementary sequence on the target

PCR Optimization

▪ Primer concentration – The normal range: – Increasing the concentration can lead to

PCR Optimization

▪ Primer design – The sequence of the primers is crucial to the success of PCR

– Tm

– Annealing temperature

PCR Optimization

▪ Magnesium concentration – Magnesium

– Normal concentration

PCR Optimization

▪ Annealing temperature – Annealing temperature:

– Annealing temperatures are often adjusted

PCR Optimization

▪ Cycling temperatures and times – Amount of time that each step

– Only the annealing temperature is adjusted

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

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.

▪ PCR in medicine – Detection of disease-causing organisms

– Amplification of disease-causing organisms for quick diagnosis

– Amplification of genes of interest for cloning

▪ 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:

▪ 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:

▪ 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

▪ 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.

▪ PCR in forensics – Allele frequencies

– Combined DNA Index System (CODIS)

▪ PCR in paternity – Samples are amplified

using STR analysis

▪ PCR in human migration – Short interspersed

repetitive elements (SINEs) have been inserted randomly into our human genome over millions of years.

▪ PCR in wildlife conservation – Poaching endangers many animals in the world today

the polymerase chain reaction - norwell high school...polymerase chain reaction or pcr –...

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