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Polymerase Chain Reaction (PCR) and Its Applications by Ayaz Najafov Boğaziçi University Department of Molecular Biology and Genetics What is PCR? PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro .

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polymerase chain reaction pcr and its applications

Polymerase Chain Reaction (PCR) and Its Applications

by

Ayaz Najafov

Boğaziçi University

Department of Molecular Biology and Genetics

what is pcr
What is PCR?

PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.

It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

what is pcr why polymerase
What is PCR? : Why “Polymerase”?

It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

what is pcr why chain
What is PCR? : Why “Chain”?

It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

what is pcr the reaction components
What is PCR? : The “Reaction” Components

1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - oligonucleotides that define the sequence

to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction

5) Mg++ ions - cofactor of the enzyme

6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

the reaction
The Reaction

PCR tube

THERMOCYCLER

slide7

Denature (heat to 95oC)

Lower temperature to 56oC Anneal with primers

Increase temperature to 72oC DNA polymerase + dNTPs

applications of pcr
Classification of organisms

Genotyping

Molecular archaeology

Mutagenesis

Mutation detection

Sequencing

Cancer research

Detection of pathogens

DNA fingerprinting

Drug discovery

Genetic matching

Genetic engineering

Pre-natal diagnosis

Applications of PCR
applications of pcr12
Applications of PCR

Basic Research

Applied Research

  • Mutation screening
  • Drug discovery
  • Classification of organisms
  • Genotyping
  • Molecular Archaeology
  • Molecular Epidemiology
  • Molecular Ecology
  • Bioinformatics
  • Genomic cloning
  • Site-directed mutagenesis
  • Gene expression studies
  • Genetic matching
  • Detection of pathogens
  • Pre-natal diagnosis
  • DNA fingerprinting
  • Gene therapy
applications of pcr13
Applications of PCR

Molecular Identification

Sequencing

Genetic Engineering

  • Bioinformatics
  • Genomic cloning
  • Human Genome Project
  • Site-directed mutagenesis
  • Gene expression studies
  • Molecular Archaeology
  • Molecular Epidemiology
  • Molecular Ecology
  • DNA fingerprinting
  • Classification of organisms
  • Genotyping
  • Pre-natal diagnosis
  • Mutation screening
  • Drug discovery
  • Genetic matching
  • Detection of pathogens
classification of organisms

Insufficient data

Molecular Identification:

Classification of Organisms

1) Relating to each other

2) Similarities

3) Differences

* Fossils

* Trace amounts

* Small organisms

! DNA !

detection of pathogens22

Molecular Identification:

Detection Of Pathogens

Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)

prenatal diagnosis

644 bp

440 bp

204 bp

Molecular Identification:

Prenatal Diagnosis
  • Chorionic Villus
  • Amniotic Fluid

Molecular analysis of a family with an autosomal recessive disease.

s equencing
SEQUENCING

Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP)

NO polymerisation after a dideoxynucleotide!

Fragments of DNA differing only by one nucleotide are generated

radioactive

Nucleotides are either or

fluorescent

summary
Summary

blood, chorionic villus, amniotic fluid, semen, hair root, saliva

68,719,476,736 copies

Gel Analysis, Restriction Digestion, Sequencing

conclusion
Conclusion

The speed and ease of use, sensitivity, specificity and

robustness of PCR has revolutionised molecular biology

and made PCR the most widely used and powerful

technique with great spectrum of research and

diagnostic applications.