This document provides an overview of polymerase chain reaction (PCR) for field epidemiologists. It describes the history and invention of PCR in 1983. It explains that PCR enzymatically amplifies DNA using two primers and 30-35 cycles to exponentially replicate the target DNA sequence. The document outlines the process of PCR including denaturing, annealing and extending DNA strands. It discusses the advantages of PCR being quick, reliable and sensitive for detecting specific pathogens or genes. However, it also notes disadvantages like the need for equipment and risks of contamination producing false results. Examples of PCR applications include detecting viruses, bacteria, and analyzing antibiotic resistance genes.

1. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Polymerase Chain Reaction
Investigation strategies and methods
May 2007

2. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Learning objectivesLearning objectives
At the end of the presentation, participants should know:
• History of polymerase chain reaction (PCR)
• Definition and short technical overview of PCR
• Applications of PCR
• Restrictions of PCR
• Examples for diagnostics with PCR

3. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
History of PCRHistory of PCR
Invented and patented in 1983
Revolutionary technique

4. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
PRC overviewPRC overview
Enzymatic DNA amplification
Need two short sequences on the DNA
Repetition of 30-35 cycles of three steps

5. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Technical overviewTechnical overview
DNA consists of four elements: A, C, G and T
DNA molecule
• Double stranded DNA strands
• Bound together by chemical forces
– Exception: single stranded DNA/RNA viruses

6. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
BackgroundBackground
Double stranded DNA:
…….A T G G C A T A T C G……..
…….T A C C G T A T A G C……..

7. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
What you need for PCRWhat you need for PCR
Two short DNA fragment that stick specifically to each
of the DNA strands at some distance of each other
Primers
• Can be specific for:
– A certain bacterium
– Bacterial species
– Genes (e.g., toxin gene)

8. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
What you need for PCRWhat you need for PCR
Apparatus to perform about 35 cycles of a three
temperature procedure
• 95 °C (denaturation of DNA)
• 50-60 °C (annealing of primers)
• 72 °C (extension of the primers)

9. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
What you need for PCRWhat you need for PCR
Put into one reaction tube:
• Sample (+/- target DNA)
• Primers for the specific detection
• Nucleotides
• Enzyme

10. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Performing PCRPerforming PCR
1. Put your tube in the apparatus
2. Let the program run (35 cycles)
3. If primers fit, there is amplification of target DNA
4. If primers do not fit, no amplification product
=> the DNA (micro-organism) was not in the sample
5. Detect if there is PCR product

11. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Logaritmic multiplicationLogaritmic multiplication

12. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Logaritmic multiplicationLogaritmic multiplication

13. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Logaritmic multiplicationLogaritmic multiplication

14. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Logaritmic multiplicationLogaritmic multiplication

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Logaritmic multiplicationLogaritmic multiplication

16. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Logaritmic multiplicationLogaritmic multiplication

17. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Logaritmic multiplicationLogaritmic multiplication

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19. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Advantages of PCRAdvantages of PCR
Quick
Reliable
Sensitive
Relatively easy
Specific

20. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Disadvantage of PCRDisadvantage of PCR
Need for equipment
Taq polymerase is expensive
Contamination
False reactions
Internal control
Cross-reaction
Enrichment steps in (contaminated) samples
Capacity building needed
Unspecific amplification

21. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Applications of PCRApplications of PCR
Detection of specific genome
• Classical with a primer pair
• Nested – amplification of larger area then specific detection
in multiplied genome part (more sensitive)
• Real time PCR to quantify the amount of genome in sample
• Detection of RNA with reverse transcriptase
Screening specific genes for unknown mutations
Genotyping using short primers or primer pairs that are
often repeated in the genome

22. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Restrictions of PCRRestrictions of PCR
Contamination of reagents or lab results in false
positive results
Failure due to a mistake in the protocol
Different materials/parts of the sample can inhibit the
PRC process

23. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
PRC diagnosticsPRC diagnostics
Viruses
• HIV, SARS, H5N1
Bacteria
• meningococcus, legionellosis
Analysis for resistant genes
• MRSA, VRE

24. E P I D E M I C A L E R T A N D R E S P O N S ELaboratory Training for Field Epidemiologists
Developed by the Department of Epidemic and
Pandemic Alert and Response of the World Health
Organization with assistance from:
European Program for Intervention
Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
Investigation strategies and methods