Polymerase Chain Reaction (PCR) and its Applications

Lesson Plan 1

•PCR • What does it do? • What do Scientists use it for? • The reaction sample in detail

•How to use micropipettes

•Practical – Set up your own PCR reactions and amplify them in the Thermal Cycler

•Gel electrophoresis – How to visualise the DNA that has been amplified in your PCR reactions

Lesson Plan 2

•Practical – load your amplified PCR samples onto a gel and apply electric current to the gel to separate the DNA fragments according to their size

•Using PCR to detect faulty BRCA2 genes and how these genes are involved in breast cancer

•Practical – visualise the DNA fragments that have been separated by gel electrophoresis

•Interpret the results

Amplifies a small, specific region

of DNA

PCR

-Alzheimer’s Disease -Osteoarthritis -Cardiovascular disease -Pancreatitis-Breast cancer

Conservation of endangeredspecies

Applications of PCR

…….CAGTCGCTAAGTTCTAACGTCC……

ATTCAAGATT

PCR: The Steps

1. Find out the sequence of the piece of DNA that you would like to amplify

2. Design & add a short piece of DNA (a PRIMER) which is perfectly complimentary to that sequence

• Primers – short single-stranded pieces of DNA which are chosen to PERFECTLY MATCH a portion of the DNA segment to be copied. There is a FORWARD (F) and a REVERSE (R) primer.

DNA

F

R

…….CAGTCGCTAAGTTCTAACGTCC……

ATTCAAGATTGG GGGG AA

CCGGCCAA GGGG

PCR: The Steps3. This then allows a whole new complimentary strand to be produced

4. The process can be repeated many times to produce lots of new copies of the original DNA

• Split the double-stranded DNA into 2 single strands (this is called DENATURATION)

• Join on short pieces of DNA (primers) which perfectly match the denatured DNA (this is called ANNEALING)

• Extend the DNA to make a perfect copy of the single strand (EXTENSION)

PCR

Temperature

PCR only works if the temperature is correct and accurate for each step

•Denaturation: 95ºC

•Annealing: 50 – 65ºC

•Extension: 72ºC

Temperature - Thermal Cycler

• Accurate temperatures• Fast changes

In the Reaction Tube

• Buffer – a chemical that enables the PCR reaction to take place. Has optimal pH and salt components

• Source of DNA• Primers• MgCl – Magnesium chloride is a salt which is

crucial for the DNA polymerase enzyme to work

In the Reaction Tube

• Nucleotides – free nucleotides (A, T, C & G) are needed to EXTEND the DNA chain on each cycle

• Taq DNA Polymerase – enzyme used to read the original DNA segment and add on new nucleotides to make a complimentary copy of that sequence

Taq DNA Polymerase

The Taq (Thermus aquaticus) DNA polymerase has to be a special heat-stable enzyme which is able to survive at high temperature

Hot Springs at Yellowstone National Park, USA.

Image by Billy Gast. Used under licence.

How to avoid the DNA polymerase being broken down in the thermal cycler

DNA Polymerase

• Taq DNA polymerase is an example of a big improvement

• Before its discovery, normal DNA polymerase was used and had to be added freshly after each amplification cycle (since it was degraded at high temperatures)

• This has made the procedure less labour intensive and cheaper

Improvements