Investigation strategies and methods

Polymerase Chain Reaction

- Devansh Gupta (11712)
 Learning 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

 History of PCR

Invented and patented in 1983

Revolutionary technique
 PRC overview

Enzymatic DNA amplification

Need two short sequences on the DNA

Repetition of 30-35 cycles of three steps

 Technical 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
 Background

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……..
 What 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)

 Denaturation
• The double stranded DNA is heated to 96 degrees to
break down the strand into two pieces.

• The template DNA and other core ingredients are added

into a solution with temperatures around 96 degrees.

• The high temperature causes hydrogen bonds between

the bases in the template to break down.

• Two single strands that are obtained now act as template

for the replication.

• Usually takes 15-30 seconds, but don’t raise the

temperature!
 Annealing
• When the temperatures are lowered, the DNA primers attach to the template
DNA

• The temperature is lowered to 50 to 65 degrees.

• The primers attach to the specific location of the single strand DNA by the
way of Hydrogen Bonding.

• Primers are single strands of DNA/RNA with length around 20 to 30

bases.

• Primers are designed to be complementary in sequence to short sections of

DNA on each end of the sequence to be copied.

• Primers act as starting point for DNA synthesis. The polymerase can only add
DNA bases to double strand of DNA.

• The two separated strands of DNA are complementary, and run in opposite
directions. There are two types of Primers - Forward and Reverse.
 Extending

• When the temperature is raised, a new strand of DNA

is made by the Taq Polymerase enzyme.

• The heat is slowly raised to 72 degrees to enable the

new DNA to be made by a special Taq DNA
polymerase enzyme which adds DNA bases.

• Taq DNA Polymerase is an enzyme from a bacteria -

Thermus Aquaticus

• The temperature is chosen as it is the optimum

temperature to build a complementary strand. It
attaches to the primer and then adds DNA bases to
the single strand.
 What 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)

 What you need for PCR

Put into one reaction tube:

• Sample (+/- target DNA)

• Primers for the specific detection

• Nucleotides

• Enzyme
 Performing 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

Logaritmic multiplication
Logaritmic multiplication
Logaritmic multiplication
Logaritmic multiplication
Logaritmic multiplication
Logaritmic multiplication
Logaritmic multiplication
 Advantages of PCR

Quick

Reliable

Sensitive

Relatively easy

Specific
 Disadvantage 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
 Applications 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
 Restrictions 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