1. POLYMERASE CHAIN REACTION (PCR) Prepared by: M. Mohsin Ali Dynamo

2. Introduction  You might have often strolled by clinics during your lounging out, and seen a small statement “PCR Lab” written on flex signs and boards. PCR is a technique to amplify the genetic information. In simpler terms, PCR is a method used to obtain large amounts of DNA in a laboratory test tube—large enough that analysis can be performed easily.  PCR was developed in 1983 by Kary Banks Mullis, an American Biochemist who won the Nobel Prize in Chemistry for it. You might be interested to note that while he got a Nobel prize in Chemistry, the PCR application is used mostly in biochemical analysis studied in biology.  PCR is entirely in vitro, i.e. it is performed in a completely artificial environment. PCR analysis is carried out in a PCR machine, formally called a thermocycler. We will get on to discuss the physics behind a thermocycler, but later. Copyrights Dynamo Study Guide 2013

3. What you should know about PCR Copyrights Dynamo Study Guide 2013

4. Specificity: PCR is very specific. The starting material for PCR amplification does not need to be purified DNA [DNA Purification is the extraction of DNA from cell debris, other nuclei acids and soluble proteins]. Only minute amounts of DNA are needed to carry out PCR analysis, and this DNA can be in a partially degraded state as well.

5. Copyrights Dynamo Study Guide 2013 Purpose: You might already know a bit about gene fingerprinting and genomic libraries. In a genomic library, there are clones of cells, each with a particular gene. To find a gene, we simply use a radioactive or a fluorescent tracer and off we go. Now, PCR can be used to amplify a gene prior to cloning. As a result, that gene becomes our most abundant DNA fragment, and it is easy for a tracer to spot it and the clone containing that gene.

6. Copyrights Dynamo Study Guide 2013

7. Note: Traditional cloning is still used whenever a large quantity of gene or protein product is needed.

8. Copyrights Dynamo Study Guide 2013

9. Primers: These are sequences of 20 bases complementary to bases on either side of the target DNA. In PCR, we use one forward and one reverse primer. Primers are used because DNA polymerase (an enzyme discussed below) cannot start a nucleotide chain; it can only add nucleotides to an already growing chain.

10. Copyrights Dynamo Study Guide 2013 Taq Polymerase: This is type of DNA polymerase enzyme, extracted from the thermophilic bacterium Thermus aquaticus. This bacterium inhabits hot springs, and the DNA polymerase extracted from it is thermostable (it can exist in its natural state even at high temperature. Other enzymes get denatured at high temperature). Ordinary DNA polymerase III denatures above 37°C, so it is not practical or efficient to use. See figure to visualize Thermus aquaticus.

13. Denaturation: The DNA is briefly heated at 94-96◦C to separate its strands.

14. Primer Annealing: Annealing means: “Hardening something by heat treatment.” In this step, the DNA is cooled to 50- 60◦C to allow the primers to bind by Hydrogen bonding to the ends of the target sequence, one primer on each strand. (Since DNA polymerase adds nucleotides from the 5’ to the 3’ end, therefore, technically, a forward primer and a reverse primer are used).

15. Copyrights Dynamo Study Guide 2013 Primer Extension: At about 72◦C, DNA Taq Polymerase adds nucleotides to the 3’ end of the primer using longer DNA strands as templates.

16. Copyrights Dynamo Study Guide 2013 The first cycle produces variable-length strands. After primer annealing in the second cycle of PCR, constant-length strands are produced.

17. Copyrights Dynamo Study Guide 2013

19. The doubling of the targeted DNA sequence takes a mere 5 minutes. After about 20 cycles, all product DNA molecules will consist of targeted sequences.

20. Copyrights Dynamo Study Guide 2013

21. To amplify molecules or sequences of RNA, it is initially converted to DNA using the enzyme reverse transcriptase. Then the converted DNA is amplified using the standard PCR procedure. This is called RT PCR.

22. Copyrights Dynamo Study Guide 2013

23. PCR has been used to amplify DNA from: 1. 40,000 years old frozen wooly mammoth. 2. Blood, tissue or semen for forensic analysis. 3. Single embryonic cell for rapid antenatal (before birth) diagnosis of genetic disorders. 4. Viral genes from cells infected with HIV.

24. Copyrights Dynamo Study Guide 2013