1. PCR POLYMERASE CHAIN REACTION Dauphin Island Graduate Neurobiology

2. It is a revolutionary method developed by Dr. Kary Mullis in 1983, that enables the amplification of a gene of interest. WHAT IS PCR? Image Source: http://betastuffs.blogspot.com

3. Obtain more copies of a gene. Detection and diagnosis of diseases. Functional analysis of genes. Identification of genetic fingerprints: – Paternity Testing (e.g. Maury Show) – Forensics (e.g. CSI) PCR APPLICATIONS

4. IN CASE YOU DON’T KNOW THE MAURY SHOW…

5. DNA template DNA polymerase (DNAp) Primers Nucleotides (dNTPs or deoxynucleotide triphosphates) PCR REQUIRES:

6. Initial Denature (95 °C for 2 mins) 30-40 times: – Denature (95 °C for 30 s) – Anneal (50-70 °C for 30 s) – Extend (68-72°C for 30-60 s) Final Extend (68-72°C for 5 mins) Infinite Hold (4-10 °C for∞ ) PCR STEPS

7. PCR: “THE MOTION PICTURE” Source : http://www.dnalc.org/resources/animations/pcr.html

8. Gel Electrophoresis DNA sequencing PCR RESULTS VERIFICATION

9. RT-PCR (Reverse Transcription PCR) qPCR (Quantitative PCR also known as Real Time PCR) PCR : FAMILY

10. DNA- mouse retina cDNA Primers to ryanodine receptor Expect PCR Product of 110 bases Annealing Temperature = 50 o C Tube 1 (Control) 17 ul H 2 0 Blue tube unlabeled 2 ul Forward Primer (5 uM stock) Green tube labeled F 2 ul Reverse Primer (5 uM stock) Yellow tube labeled R 4 ul H 2 0 Blue tube unlabeled Total of 25 ul Tube 2 (Test) 17 ul H 2 0 Blue tube unlabeled 2 ul Forward Primer (5 uM stock) Green tube labeled F 2 ul Reverse Primer (5 uM stock) Yellow tube labelled R 4 ul DNA Template (500 ng/ul) Orange tube labeled DNA Total of 25 ul PCR Reaction in PCR Machine 1)Initial Denaturation94 o C2 min 2)Denature94 o C30 sec 3)Anneal50 o C30 sec 4)Extend (35 cycles)72 o C30 sec 5)Final 72 o C5 min 6)Hold 10 o C https://www.youtube.com/watch?v=x5yPkxCLads&feature=kp PCR: HANDS ON