Polymerase Chain Reaction (PCR)

1. Polymerase Chain Reaction (PCR) By: Yat Chum Kristen Fung Alicia Kassee

2. WHAT IS PCR? • Polymerase Chain Reaction (PCR) • A process that directly amplifies (makes more of) a desired DNA sequence (target DNA) by repeating cycles of strand separation and replication.

3. History • Problem: It was impossible to make multiple copies of the desired gene without placing it into a plasmid • Reason: Due to restriction endonucleases, DNA ligase, and plasmids, the researcher would have to extract the plasmid DNA from the bacteria, then excise the fragment needed. • Kary Mullis first proposed the process of PCR in 1987. • Mullis was awarded the Nobel Prize in Chemistry in 1992

4. PCR PROCESS Help You Understand • Mimics DNA replication • Does not replicate 100% of original strand Important Players • Primers • DNA fragments complimentary to target strand • TAQ DNA Polymerase • Heat resistant enzyme • Synthesizes new DNA strand • Deoxynucleotide Triphosphates (dNTPs) • Building blocks of new strand

5. PCR PROCESS (cont.) • STEP 1: • Temperature raised to 95°C to break H bonds between complementary bases TIP: Think of DNA helicase in DNA replication

6. STEP 2: • Temperature is dropped to 60°C • DNA Primers (5’-3’) anneal to the target strand (3’-5’). One is the forward primer and the other is the reverse primer • work oppositely, towards one another Unlike DNA Replication which uses RNA primers, DNA primers are used for PCR because they are easy to obtain

7. STEP 3: • Temperature raised to 72°C (Taq DNA Polymerase works most efficiently at this temperature) • TaqPolymerase synthesizes the complementary strand from the primer (adding onto the primer’s 3’ end) using dNTPs as building blocks TIP: To remember Taq polymerase’s job, think of DNA polymerase III DNA Polymerase denatures at 37°C. Taq polymerase can withstand high temperatures.

8. Process repeats again times because there are variable length strands (unequal, replicated strands of DNA) • By the 3rd repetition, there are constant length strands (equal replicated strands of DNA) • Then target strands of DNA increase exponentially

9. After 25 Cycles… there are 33, 554, 432 strands of the target DNA

10. HOW CAN WE USE PCR? • PCR is used for improving medical diagnosis • Identifying sexually transmitted diseases • Identifying and eliminating viruses that are found in the body which can be faster and safer than traditional methods. • PCR is used for forensics investigation • Since little genetic information is needed, it is perfect for forensic application due to the somewhat limiting DNA in a crime scene. • PCR is used to identify ancient fossil remains • PCR can identify and help scientist distinguish between different species.

11. To Help Your Understanding • Watch this video :D! • http://www.sumanasinc.com/webcontent/animations/content/pcr.html