Yesterday…

P to the C to the R PCR

Biotechnology Tools Restriction Endonucleases / enzymes Methylases DNA ligase Gel Electrophoresis Plasmids Transformation 7. PCR 8. RFLPs 9. DNA sequencing

Who dun it? Pretend you are Gil Grissom You are at a murder crime scene of a celebrity After completely scouring the scene for days you are only able to find a TINY drop of blood How can you use such a small amount of DNA to find the culprit? Wouldn’t you run out after one test?

Enter… PCR Polymerized Chain Reaction Technique used to amplify DNA generating thousands or millions of copies from one sample.

PCR Developed in 1983 by Kary Mullis –An American biotechnologist When driving home with his girlfriend one night, he had an incredible ‘genetic idea’ Why not use TWO primers on a gene? –You’ll get two copies of the gene THEN, use two primers on those two copies –Then use two primers on those copies…. Etc etc

PCR In 1993, he received the Nobel Prize for his work –Sadly, his gf broke up with him for it all came together “I was sagging as I walked out to my little silver Honda Civic. Neither [assistant] Fred, empty Beck's bottles, nor the sweet smell of the dawn of the age of PCR could replace Jenny. I was lonesome."

So how does PCR work? Closely related to DNA replication –Two DNA strands are separated using the enzymes DNA helicase and DNA gyrase In PCR, the strands are separated by heat –94-96 oC –Breaking the H-bonds between bases of the two strands

PCR Once separated, the two strands can be used as templates –creating new complementary strands In replication, RNA primers are used to start DNA polymerase III In PCR, DNA primers are used –As they are easily made in the laboratory

PCR Primers Primers must be complementary to the target area being copied: –One primer is complementary to one end of the target DNA –The other primer is complementary of the opposite end of the opposite strand Bind to 3’ end

PCR Once the primers are added, –Temperature is lowered to oC –Allowing the DNA primers to anneal Next, a different DNA polymerase builds complementary strands from DNA primers –Taq Polymerase –This occurs at 72 oC

Oh, that crazy Taq Polymerase Taq Polymerase was isolated from the bacteria Thermus Aquaticus –Lives in hot springs –Thus, has enzymes that will not denature at 72 oC –Can replicate 1000 base pairs in 10 seconds DNA polymerase III would denature above 37 oC –Thus, not overly useful in this process

Finally… The four strands separate, and are free to bind to four more primers

PCR Each subsequent cycle doubles the amount of DS target DNA –Exponential growth After 30 cycles, more than 1 billion copies of target DNA exist –2 30

PCR & HIV Can’t confirm presence of HIV HIV can’t be confirmed immediately by looking for specific antigens on infected cells –Mutations occur which makes identification hard With PCR, Primers can be designed to bind to specific regions of HIV DNA –DNA can then be amplified to test for the presence of the HIV genome