Recombinant DNA Technology………..

1. Recombinant DNA Technology……….. BTEC3301

2. Polymerase Chain Reaction (PCR) Introduction • The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying small quantities of DNA. • Kary Mullins won the Nobel Prize in 1993 for development of this novel technique.

3. Polymerase Chain Reaction (PCR) Introduction • The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus auquaticus , discovered in hot springs.

4. Polymerase Chain Reaction (PCR) Introduction • The use of thermostable DNA polymerases was an important breakthrough since this enzyme does not denature at the temperatures( 950C) used to cause denaturation of the DNA.

5. Polymerase Chain Reaction (PCR) Introduction • The most well-known of these thermostable DNA polymerases is Taq. • This enzyme has a molecular size of 94kD and an optimum reaction temperature of 75-80oC.

6. Polymerase Chain Reaction (PCR) Introduction • Amplification means making multiple identical copies (replicates) of a DNA sequence. • PCR method of DNA amplification has proved very important in recombinant DNA technology and is used in a range of applications in medicine and forensic science .

7. Polymerase Chain Reaction (PCR) What is PCR? • It is used to amplify a specific DNA (target) sequence lying between known positions (flanks) on a double-stranded DNA molecule. • The amplification process is mediated by oligonucleotide primers that, typically, are 20-30 nucleotides long.

8. Polymerase Chain Reaction (PCR) What is PCR? • The primers are single-stranded DNA. • Primers anneal to the flanking regions by complementary-base pairing (G=C and A=T) using hydrogen bonding.

9. Polymerase Chain Reaction (PCR) • The amplified product is known as an amplicon. • Generally, PCR amplifies smallish DNA targets 100-1000 base pairs (bp) long.

10. Polymerase Chain Reaction (PCR) Material required in PCR process: • Thermal cycler (PCR machine, available in different specificity and models).

11. Polymerase Chain Reaction (PCR) : • PCR mix for a reaction has the following: • sample DNA with a target sequence • thermostable DNA polymerase (Taq is commonly used) • two oligonucleotide primers which are complementary to the sequence flanking the target sequence.

12. Polymerase Chain Reaction (PCR) • deoxynucleotide triphosphates (dNTPs) • reaction buffer containing magnesium ions and other components. Homework:Function of Magnesium for Quiz!!!

13. Polymerase Chain Reaction (PCR) Steps in PCR reactions: • 1.    Heat denaturation • A DNA molecule carrying a target sequence is denatured by heat at 90-95oC. The two strands separate due to breakage of the hydrogen bonds holding them together.

14. Polymerase Chain Reaction (PCR) • 2.   Primer annealing • Primers are added to the dissociated target strands and incubated together first at a temperature that allows the primers to hybridize to the target strands (usually somewhere between 40 and 55 oC).

15. Polymerase Chain Reaction (PCR) • 3. Primer extension • DNA polymerase is then added and complementary strands are synthesized at a temperature of 60-75oC. The polymerase causes synthesis of new material in the 5' to 3' direction away from each of the primers.

16. Polymerase Chain Reaction (PCR) • This allows a first round of synthesis to occur on each of the DNA templates. • Following primer extension, the mixture is heated (again at 90-95oC) to denature the molecules and separate the strands and the cycle repeated

17. Polymerase Chain Reaction (PCR) • Each new strand then acts as a template for the next cycle of synthesis. • Amplification (replication) proceeds at an exponential (logarithmic) rate (amount of DNA produced doubles at each cycle).

18. Polymerase Chain Reaction (PCR) • A thermal cycler (a machine that automatically changes the temperature at the correct time for each of the stages and can be programmed to carry out a set number of cycles) is used for a PCR reaction.

19. Polymerase Chain Reaction (PCR) • The amplified product can be detected using gel electrophoresis to view the band containing DNA fragments of a particular size containing the gene of interest in the original starter DNA sample.

20. Diagrammatic steps in PCR process (Read summary): • Three major steps in PCR: 1. Template denaturation 2. Primer annealing 3. Primer extension

21. Home work for next class!! Summarize the steps in PCR process • The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template. • This results in the synthesis of new DNA strands which are complementary to the parent template strands. • After each cycle the newly synthesized DNA strands can serve as templates in the next cycle.

22. Polymerase Chain Reaction (PCR) Diagrammatic steps in PCR process • PCR amplification is achieved by using oligonucleotide primers.

23. Polymerase Chain Reaction (PCR) Diagrammatic steps in PCR process •

24. Fig. 3.8 The Polymerase Chain Reaction

25. Go to Animation courtesy of the following websites:References • http://faculty.plattsburgh.edu/donald.slish/PCRmov.html (Animation) • http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html • http://www.people.virginia.edu/~rjh9u/pcranim.html ( PCR Animation) • http://www.escience.ws/b572/L3/L3.htm (PCR Animation) • http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect4.html • http://en.wikipedia.org/wiki/Polymerase_chain_reaction • http://www.escience.ws/b572/L3/L3.htm • http://allserv.rug.ac.be/~avierstr/principles/pcrani.html