Polymerase Chain Reaction (PCR) May 23, 2017
Outline History of PCR Uses of PCR How PCR works How to set up and run PCR
The structure of DNA
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: 5.0 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
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
Uses of PCR Forensics Amplify DNA from biological sample Match with suspect based on patterns of amplification First publicized example was OJ Simpson case
Uses of PCR GMO detection GMO content is regulated in US exports PCR amplifies markers of genetic modification
Uses of PCR Paternity testing Used in courts and commercially PCR amplifies markers of parents and progeny to identify matches (or mismatches)
Uses of PCR Disease diagnoses Sometimes a pathogen is not culturable PCR can detect the nucleic acid trace from the pathogen Example: influenza
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)
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.
How does it work? https://www.youtube.com/watch?v=x2juqhs pt8w https://www.khanacademy.org/science/biolog y/biotech-dna-technology/dna-sequencingpcr-electrophoresis/v/the-polymerase-chainreaction-pcr
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
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
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
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
Analyzing the PCR reaction https://www.khanacademy.org/science/biology/bi otech-dna-technology/dna-sequencing-pcrelectrophoresis/v/gel-electrophoresis-dna
Multiplex PCR Instead of amplifying one target (gene), you are trying to amplify >1 target
Salmonella multiplex PCR Purpose: to differentiate/identify specific common serotypes of Salmonella
Salmonella multiplex PCR
Salmonella multiplex PCR Master mix is already prepared You have unknown Salmonella isolates You will add DNA and run the PCR
Salmonella multiplex PCR Thermal cycling program for Salmonella multiplex PCR
Salmonella multiplex PCR Interpreting the results