PCR POLYMERASE CHAIN REACTION Dauphin Island Graduate Neurobiology

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?

Image Source: http://betastuffs.blogspot.com

• 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