1. Polymerase Chain Reaction PCR

2. PCR allows for amplification of a small piece of DNA. Some applications of PCR are in: –forensics (paternity testing, crimes) –identification of human remains Sometimes problems if DNA is degraded –Study human ancestry –Cloning – introduced amplified DNA into a vector

3. PCR PCR requires a thermostable DNA polymerase The most common type of DNA polymerase is Taq polymerase 2 Oligonucleotides called primers are also included in the PCR reaction (match and bind to the target sequence) 4 deoxynucleotides- The subunits of DNA that are incorporated into the new DNA copies

4. PCR Components Template DNA Nucleotides (dNTPS) PCR buffer 2Magnesium chloride (MgCl 2 ) Water Forward and Reverse primers (Taq) DNA polymerase

5. PCR machine The PCR mixture which contains DNA polymerase, buffer, deoxynucleotides, primers, and template, go through a cycle of varied temperatures 3 steps in a cycle -Denature -Anneal -Extend

6. Steps in PCR Denature -Temperature is raised to near boiling (92ºC) -Template DNA is separated into two strands Anneal -Temperature is lowered (50º-65ºC) -Allows the left and right primers to base pair to their complementary sequences -Primers purpose is to bracket the DNA region to be amplified

7. Steps in PCR Extend -Temperature is raised to 72ºC -Taq polymerase attaches at each priming site -Uses the nucleotides from the reaction mixture and attaches it to the growing strand; these nucleotides are complementary to the target sequence

8. PCR Animation http://www.maxanim.com/genetics/PCR /PCR.htmhttp://www.maxanim.com/genetics/PCR /PCR.htm

9. DNA replication in vivo Topoisomerase Helicase Primase Single stranded binding protein DNA polymerase DNA ligase

10. PCR Results These 3 steps of PCR result in exponential growth N =2 t x (No) of the DNA template in the mixture It’s typically repeated for 20-40 cycles But the product amount reaches a maximum after about 40 cycles due to depletion of the reaction components

11. PCR sample problem If you start with 30 copies of a target sequence and perform PCR for 45 cycles, then how many copies of the target sequence will you have at the end of the PCR amplification?

12. PCR- Problems Polymerase Errors –Taq polymerase lacks 3’ – 5’ exonulcease activity Unwanted amplification of products -Contamination -Nonspecific primer annealing

13. Electrophoresis follows PCR Used to determine if your PCR worked Determine if your sequence was present –If it was not present, the primers would not bind and you will not get a band.

14. Setting up a PCR Reaction Special small tubes or strips Have thin walls – efficiently transfer heat Try to prevent contamination –Tips with filters –Gloves –Often set up in hoods with UV lights Need to have negative controls Set up reactions on ice – don’t want nonspecific amplifications –For example, primers binding to themselves

15. Types of PCR Real time (quantitative PCR) Reverse transcription PCR

16. Real time (quantitative PCR) Conventional PCR –Not quantitative –PCR products analyzed at the end of the PCR Real time PCR –Quantitative –Amount of DNA measured at each cycle and input DNA is determined

17. Real-Time PCR This procedure uses a fluorescence dye that will bind the DNA. Can view the increase in the amount of DNA as it is amplified. As the PCR reaction progresses, more DNA is produced and more fluorescence is detected Determine how much produced by comparing to controls with a given amount of DNA

18. SYBR green is a fluorescence dye that binds DNA Adapted from Biorad.com

19. Sybr green can be first detected at the threshold cycle

20. Can also use Ethidium bromide Ethidium bromide intercalates into the DNA Take DNA after different cycles and add ethidium bromide Compare to known standards

21. Reverse transcription PCR Often used in combination with Real Time PCR Start with RNA and then make complementary DNA (cDNA) What enzyme is used?