1. POLYMERASE CHAIN REACTION (PCR) Bridges 2014

2. Polymerase Chain Reaction  Simple reaction  Produces many copies of a specific fragment of DNA  Live replication takes much longer  Only requires a small amount of DNA (ng)  Many types of PCR

3. What goes into a PCR?  Mostly water  Autoclaved MilliQ  Forward Primer  Front end of target DNA sequence  Reverse Primer  Back end of target DNA sequence  Template DNA

4. What goes into PCR?  PCR Buffer  Balances pH levels  MgCl 2  Helps target specificity and stabilizes DNA  dNTPs  Nucleotides (A, G, C, T)  Polymerase  Polymerase used to “build” target sequence

5. PCR is SENSITIVE!  Need specific amounts of each reagent  Too much or too little can RUIN a PCR  Highly subject to contamination  Use sterile technique!  Needs specific thermocycling program

6. Thermocycler

7. Thermocycler program  Denature  Anneal  Extend

8. Biological Response to Thermocycler

9. Exponential growth of template DNA

10. 16S rRNA  Ribosomal Sequence  Ribosomes make proteins  All bacteria have 16S  Has conserved and hypervariable regions  Conserved regions- primer binding site  Hypervaribale regions are for species identification