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 C onservation of endangered species Applications of PCR

Decreasing size of DNA fragments Gel electrophoresis is used to visualise the amplified DNA fragments

Reminder: DNA DNA is made of up of two long strands –helix- made up of simpler units called nucleotides

Double- stranded helix Nucleotides pair up Adenine always pairs with Thymine Guanine always pairs with Cytosine Nucleotide DNA Structure

…….CAGTCGCTAAGTTCTAACGTCC…… ATTCAAGATT PCR: The Steps 1. Find out the sequence of the piece of DNA that you would like to amplify 2. Design a short piece of DNA (a PRIMER) which is perfectly complimentary to that sequence

…….CAGTCGCTAAGTTCTAACGTCC…… ATTCAAGATTG G G A CG C A G G PCR: The Steps 3. 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 which perfectly match the denatured DNA (this is called ANNEALING) Extend the DNA to make a perfect copy of the single strand (EXTENSION) PCR

In the Reaction Tube Buffer – a chemical that enables the PCR reaction to take place. Has optimal pH and salt components Source of DNA

In the Reaction Tube 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

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.

DNA Polymerase

In the Reaction Tube MgCl – Magnesium chloride is a salt which is crucial for the DNA polymerase enzyme to work

Temperature PCR only works if the temperature is correct for each step Denaturation:95ºC Annealing:50 – 65ºC Extension: 72ºC

Thermal Cycler

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 This has made the procedure less labour intensive and cheaper Improvements