Polymerase Chain Reaction (PCR) May 23, 2017

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1 Polymerase Chain Reaction (PCR) May 23, 2017

2 Outline History of PCR Uses of PCR How PCR works How to set up and run PCR

3 The structure of DNA

4 PCR Polymerase chain reaction Selective amplification of target DNA using specifically-designed ssdna primers Exponential amplification of DNA of interest in vitro, without the use of bacteria or other living systems Prior to PCR Very difficult to target specific regions SPECIFICITY Small amount of target to work with AMPLIFICATION

Specificity Pine: 68 billion bp Corn: 5.

7 thousand bp On a tape measure, the human genome (1 base

5 Specificity Pine: 68 billion bp Corn: 5.0 billion bp Soybean: 1.1 billion bp Human: 3.4 billion bp Housefly: 900 million bp Rice: 400 million bp E. coli: 4.6 million bp HIV: 9.7 thousand bp On a tape measure, the human genome (1 base per mark) would stretch 5,366 miles! Finding a 500 bp section would be like trying to find a 4-foot section Courtesy BioRad

6 Discovery of PCR Kerry Mullis, 1983 Cetus Corporation commercialized Mullis made nothing But, he won the Nobel Prize in 1993 Revolutionized molecular biology Recognized as one of the greatest achievements in science in 20th century

7 Uses of PCR Forensics Amplify DNA from biological sample Match with suspect based on patterns of amplification First publicized example was OJ Simpson case

8 Uses of PCR GMO detection GMO content is regulated in US exports PCR amplifies markers of genetic modification

9 Uses of PCR Paternity testing Used in courts and commercially PCR amplifies markers of parents and progeny to identify matches (or mismatches)

10 Uses of PCR Disease diagnoses Sometimes a pathogen is not culturable PCR can detect the nucleic acid trace from the pathogen Example: influenza

11 Uses of PCR Basic research PCR is a core tool of nearly every lab in the biological sciences field Not only amplifies, but also quantifies DNA and RNA (quantitative PCR)

12 PCR PCR by itself is not a very informative technique for molecular epidemiology PCR, coupled with other techniques, forms the basis of many highly informative methods PCR of organism-specific DNA or RNA sequences can also be a powerful diagnostic test Sensitivity and specificity issues become complex, however.

13 How does it work? pt8w y/biotech-dna-technology/dna-sequencingpcr-electrophoresis/v/the-polymerase-chainreaction-pcr

14 Components of a PCR reaction Water Buffer stabilizes everything DNA template contains what you want to amplify Primers targets what you want to amplify Nucleotides the letters (A, T, G, C) Magnesium cofactor required for enzyme Polymerase enzyme that does the work

Primer REV DNA Polymerase DNA Template Total Volume 38.

15 Setting up a PCR reaction Sterile Water 10X PCR Buffer MgCl2 (50mM) dntp s (10mM each) Primer FWD (25 pmol/ul) Primer REV DNA Polymerase DNA Template Total Volume 38.0 ul 5.0 ul 2.5 ul 1.0 ul 1.0 ul 1.0 ul 0.5 ul 1.0 ul 50.0 ul

16 Setting up a master mix Component 1X 20X Sterile Water 38.0 ul 760 ul 10X PCR Buffer 5.0 ul 100 ul MgCl2 (50mM) 2.5 ul 50 ul dntp s (10mM each) 1.0 ul 20 ul PrimerFWD (25 pmol/ul) 1.0 ul 20 ul PrimerREV 1.0 ul 20 ul DNA Polymerase 0.5 ul 10 ul DNA Template 1.0 ul -- Total Volume 50.0 ul 980 ul Aliquot 49 ul Add DNA as last step

17 The thermal cycler 1. Initial Denaturation 95 C 5 min 2. DNA Denaturation 95 C 30 sec 3. Primer Annealing 60 C 30 sec 4. Primer Extension 72 C 1 min 5. Go to step #2, repeat 25x-30x 6. End

18 Analyzing the PCR reaction otech-dna-technology/dna-sequencing-pcrelectrophoresis/v/gel-electrophoresis-dna

19 Multiplex PCR Instead of amplifying one target (gene), you are trying to amplify >1 target

20 Salmonella multiplex PCR Purpose: to differentiate/identify specific common serotypes of Salmonella

21 Salmonella multiplex PCR

22 Salmonella multiplex PCR Master mix is already prepared You have unknown Salmonella isolates You will add DNA and run the PCR

23 Salmonella multiplex PCR Thermal cycling program for Salmonella multiplex PCR

24 Salmonella multiplex PCR Interpreting the results

The Polymerase Chain Reaction. Chapter 6: Background

The Polymerase Chain Reaction. Chapter 6: Background The Polymerase Chain Reaction Chapter 6: Background PCR Amplify= Polymerase Chain Reaction (PCR) Invented in 1984 Applications Invention of PCR Kary Mullis Mile marker 46.58 in April of 1983 Pulled off