PCR POLYMERASE CHAIN REACTION Dauphin Island Graduate Neurobiology.

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Research Techniques Made Simple: Polymerase Chain Reaction

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PCR POLYMERASE CHAIN REACTION Dauphin Island Graduate Neurobiology

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:

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

IN CASE YOU DON’T KNOW THE MAURY SHOW…

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

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

PCR: “THE MOTION PICTURE” Source :

Gel Electrophoresis DNA sequencing PCR RESULTS VERIFICATION

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

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 PCR: HANDS ON