Polymerase Chain Reaction

1. Polymerase Chain Reaction

2. What is Polymerase chain reaction? (PCR) PCR is a technique that is used to amplify one sample of DNA thousands of times over to create a large enough DNA sample for extensive analysis. i.e. each time a PCR cycle is performed the total amount of DNA is doubled. It is In Vitro amplification of DNA.

3. What are some uses of PCR? PCR can be used for many applications • Paternity Tests – The child’s tandem repeats are compared to the mother’s and the father’s. • Detecting mutations- Comparing one persons DNA to the DNA of the person with the mutation

4. The use of PCR in forensics To create enough DNA from a small sample to create a DNA profile.

5. What is needed? • The DNA fragment to be copied • DNA polymerase – for PCR it is taken from bacteria that live in hot springs so it is tolerent to heat • Primers – short pieces of DNA that bind to the start of each end of fragment – also help to prevent both ends from joining back up

6. Primers in excessThe annealing reaction isvery efficient because the primers are "in excess" in the reaction. In a typical PCR reaction, 10,000 molecules of a template may be used, which is 1.6 x 10-20 moles (0.016 attomoles). On the other hand, 5 picomoles of each primer may be used (5 x 10-12 moles) -- that is a 3 x 108 fold excess.

7. What is needed? • The DNA fragment to be copied • DNA polymerase – for PCR it is taken from bacteria that live in hot springs so it is tolerent to heat • Primers – short pieces of DNA that bind to the start of each end of fragment – also help to prevent both ends from joining back up • Nucleotides • Thermocycler – a computer controlled machine that varies temperatures precisely for set time periods

9. Biorad – PCR song (find on youtube)

10. The process of PCR Step 1: Denature the DNA “Denature” means to separate the DNA strands into 2 separate strands This involves heating the DNA sample up to 96 degrees!

11. The process of PCR Step 2: Anneal the DNA - “annealing” means to add In this step 2 primers are added to the 2 separated DNA strands. ( 1 on each strand) The temperature is “cooled” to 65 degrees, this help the primers bind to the DNA. The primer is an attachment that signals to a polymerase where to start synthesising (making) new DNA

12. The process of PCR Step 3: “Extension” of DNA- New DNA Created 2 Polymerase molecules attach to the 2 Primers on the 2 DNA strands and move along the strand. As they move along they create new “complementary” DNA. Temperature goes up to 72 degrees

13. The process of PCR Done! By heating the DNA to separate it into 2 strands (Denaturation) Cooling the DNA to add 1 primer to each of the 2 DNA strands (Annealing) And by Adding 1 polymerase to each strand to synthesise DNA from where the primer attached (Extension) You just doubled the mount of DNA present.

15. Animation • http://www.dnalc.org/resources/animations/pcr.html

16. Making more DNA Every time you do a Cycle of PCR you double the DNA. You can do this process as many times as needed to create millions of strands of DNA.

17. Now complete these questions • When you finish you can use the computer room to run through a simulation PCR simulation http://learn.genetics.utah.edu/content/labs/pcr/

18. PCR simulation http://learn.genetics.utah.edu/content/labs/pcr/