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ABE Workshop 2005July 15, 2006 Polymerase Chain Reaction (PCR) Kabi Neupane, Ph.D. Leeward Community College.

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Presentation on theme: "ABE Workshop 2005July 15, 2006 Polymerase Chain Reaction (PCR) Kabi Neupane, Ph.D. Leeward Community College."— Presentation transcript:

1 ABE Workshop 2005July 15, 2006 Polymerase Chain Reaction (PCR) Kabi Neupane, Ph.D. Leeward Community College

2 Polymerase Chain Reaction Polymerase: DNA polymerase – – DNA polymerase duplicates DNA – –Before a cell divides, its DNA must be duplicated – –Chain Reaction: The product of a reaction is used to amplify the same reaction – –Results in rapid increase in the product

3 ABE Workshop 2005July 15, 2006 Polymerase Chain Reaction (PCR) PCR performs the chemistry of DNA duplication in vitro PCR performs the chemistry of DNA duplication in vitro Numerous PCR applications make this process a staple in most biology laboratories Numerous PCR applications make this process a staple in most biology laboratories Understanding properties of DNA polymerases helps understanding PCR Understanding properties of DNA polymerases helps understanding PCR

4 Discovery PCR was discovered by Kary Mullis PCR was discovered by Kary Mullis –On a long motorcycle drive –Mentally visualized the process Nobel Prize in Chemistry Nobel Prize in Chemistry –1993

5 DNA polymerase Duplicates DNA Duplicates DNA Necessary for reproduction of new cells Necessary for reproduction of new cells More than one DNA polymerases exist in different organisms More than one DNA polymerases exist in different organisms

6 Properties of DNA polymearse 3’ 5’ 3’ Needs a pre-existing DNA to duplicate Needs a pre-existing DNA to duplicate –Cannot assemble a new strand from components –Called template DNA Can only extend an existing piece of DNA Can only extend an existing piece of DNA –Called primers

7 Properties of DNA polymearse DNA polymerase needs Mg ++ as cofactor DNA polymerase needs Mg ++ as cofactor Each DNA polymerase works best under optimal temperature, pH and salt concentration Each DNA polymerase works best under optimal temperature, pH and salt concentration PCR buffer provides optimal pH and salt condition PCR buffer provides optimal pH and salt condition

8 Properties of DNA polymearse DNA strands are anti-parallel DNA strands are anti-parallel –One strand goes in 5’  3’ –The complementary strand is opposite DNA polymerase always moves in one direction (from 5’  3’) DNA polymerase always moves in one direction (from 5’  3’) 3’ 5’ 3’

9 Properties of DNA polymearse DNA polymerase incorporates the four nucleotides (A, T, G, C) to the growing chain DNA polymerase incorporates the four nucleotides (A, T, G, C) to the growing chain dNTP follow standard base pairing rule dNTP follow standard base pairing rule 3’ 5’ 3’ dCTP dTTP dCTP dGTP dATP dGTP dCTP dTTP dATP dGTP dCTP dTTP dATP dGTPdCTPdTTPdATPdGTP dATP dGTP dTTP dATP dCTP dTTP

10 Properties of DNA polymearse The newly generated DNA strands serve as template DNA for the next cycle The newly generated DNA strands serve as template DNA for the next cycle PCR is very sensitive PCR is very sensitive Widely used Widely used

11 Setting up a PCR Reaction Add template DNA and primers Add template DNA and primers 3’ 5’ 3’ dCTP dTTP dCTP dGTP dATP dGTP dCTP dTTP dATP dGTP dCTP dTTP dATP dGTPdCTPdTTPdATPdGTP dATP dGTP dTTP dATP dCTP dTTP Add dNTPs Add dNTPs Add DNA polymerase Add DNA polymerase

12 Taq DNA polymerase Derived from Thermus aquaticus Derived from Thermus aquaticus Heat stable DNA polymerase Heat stable DNA polymerase Ideal temperature 72C Ideal temperature 72C

13 Thermal Cycling A PCR machine controls temperature A PCR machine controls temperature Typical PCR go through three steps Typical PCR go through three steps –Denaturation –Annealing –Extension

14 Denaturation Heating separates the double stranded DNA Heating separates the double stranded DNA –Denaturation Slow cooling anneals the two strands Slow cooling anneals the two strands –Renaturation Heat Cool

15 Annealing Two primers are supplied in molar excess Two primers are supplied in molar excess They bind to the complementary region They bind to the complementary region As the DNA cools, they wedge between two template strands As the DNA cools, they wedge between two template strands Optimal temperature varies based on primer length etc. Optimal temperature varies based on primer length etc. Typical temperature from 40 to 60 C

16 Extension DNA polymerase duplicats DNA DNA polymerase duplicats DNA Optimal temperature 72C Optimal temperature 72C

17 PCR Amplification Exponential Amplification of template DNA

18 Typical PCR mix In a thin wall Eppendorf tube assemble the following PCR components Amount Template DNA (5-200 ng) 1 mM dNTPs (200 uM final) 10 X PCR buffer 25 mM MgCl2 (1.5 mM final) 20 uM forward primer (20 pmoles final) 20 uM reverse primer (20 pmoles final) 5 units/uL Taq DNA polymerase (1.5 units) Water Final Volume variable 10 uL 5 uL 3 uL 1 uL 0.3 uL Variable 50 uL

19 Applications Ubiquitous applications Ubiquitous applications Revolutionized how we study biology Revolutionized how we study biology –Research –Diagnostics –Forensics


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