Polymerase Chain Reaction (PCR) and its Applications

1. Polymerase Chain Reaction (PCR) and its Applications

2. 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

3. 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

4. PCR Amplifies a small, specific region of DNA

5. Applications of PCR Forensic science Medical science -Alzheimer’s Disease -Osteoarthritis -Cardiovascular disease -Pancreatitis -Breast cancer Conservation of endangered species Molecular ecology

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

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

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

9. ATTCAAGATT PCR: The Steps …….CAGTCGCTAAGTTCTAACGTCC……

10. ATTCAAGATT A G C G G PCR: The Steps G G G A C …….CAGTCGCTAAGTTCTAACGTCC……

11. PCR • 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)

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

13. 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. F DNA R

14. 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

15. Taq Polymerase The Taq (Thermusaquaticus) 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.

16. DNA Polymerase

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

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

19. Thermal Cycler

20. Improvements • 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