pcr polymerase chain reaction dauphin island graduate neurobiology
TRANSCRIPT
PCRPOLYMERASE 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?
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• 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)• 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
PCR: “THE MOTION PICTURE”
pcr.exe
Source : http://www.dnalc.org/resources/animations/pcr.html
• 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 receptorExpect PCR Product of 110 bases Annealing Temperature = 50 oC
• Tube 1 (Control) • 17 ul H20 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 H20 Blue tube unlabeled
• Total of 25 ul • Tube 2 (Test)• 17 ul H20 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 Machine1) Initial Denaturation 94oC 2 min2) Denature 94oC 30 sec3) Anneal 50oC 30 sec4) Extend (35 cycles) 72oC 30 sec5) Final 72oC 5 min6) Hold 10oChttps://www.youtube.com/watch?v=x5yPkxCLads&feature=kp
PCR: HANDS ON