BIOLOGY 207 - Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR) Required readings and problems: Reading: Open Genetics, Chapter 8.1 Problems: Chapter 8 Optional Griffiths (2008) 9 th Ed. Readings: pp 731-732 Problems: 9 th Ed.? Campbell (2008) 8 th Ed. Readings: Concept 20.1, pg. 404 Web pages: http://avery.rutgers.edu/wssp/studentscholars/project/archives/onions/pcr.html http://www.msu.edu/course/mmg/301/lec28.pdf http://users.ugent.be/~avierstr/principles/pcr.html Concepts: What is PCR and how can it be used in molecular genetics? 1. Polymerase chain reaction (PCR) is a method of DNA amplification that requires a template and some knowledge of the DNA sequence being amplified. 2. PCR has three basic steps (denaturation, annealing, and extension), which are repeated (or cycled) 20-40 times to produce a DNA product of a specific size. 3. PCR can be used for a variety of procedures that are used in DNA diagnostic, forensics, medical, and basic research. Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 1
The basics of a PCR reaction History Kary Mullis shared 1993 Nobel Prize in Chemistry Credited with the discovery of PCR See: http://www.nobel.se/chemistry/laureates/1993/ Reaction components: Buffer Nucleotides Template Primers Taq Polymerase Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 2
11/28/11 Thermocycler: Steps: Fig 8.6 OG 1. Denaturation 2. Annealing 3. Extension/Elongation Number of cycles: 1- Denaturation Heat to ~98-100 C for ~1 min 2- Annealing Temperature depends on the primers used, it ranges from ~40 to ~70 C for ~1 min. Usually estimated base on GC/AT content, and then confirmed empirically. 3- Extension/Elongation This is typically done at ~72 C for the polymerase to extend from the primer. The time depends on the length of product expected, with ~1 min/kb. See: http://www.youtube.com/watch?v=_ygxcj4n-kq http://www.youtube.com/watch?v=eecy9k_ksdi Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 3
Analysis of the PCR products Usually done by gel electrophoresis: Template DNA too few copies to see on a normal Agarose gel. Amplified Product see a band (many molecules of the same size) for each product produced. Detect deletion mutations: a b c d e f g h a b c f g h Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 4
Uses for PCR: Quantity -> Yes/No, how much? Very sensitive detection - Theoretically only one template molecule is needed to produce a PCR product - If itʼs there, it can be detected. - The amount of DNA product reaches a plateau at 20-40 cycles -> not directly correlated with the initial amount of target DNA. - Quantitative PCR - more reliable -> Real Time PCR (RT-PCR) - relies on fluorescence of the product measured over a fewer number of cycles - used to quantify nucleic acids (RNA & DNA) - relative quantification (fold differences) and absolute quantification (# molecules) Quality - PCR amplification product can determine distance between two primer locations -> detect insertions/deletions/rearrangements (see above) - Product can be sequenced to identify base pair changes in mutants - Products can reveal allelic differences between individuals, populations, or species. This relies on DNA sequence polymorphisms. (Lab Project #9) Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 5
Practical uses for PCR Detect trace contaminants - in air, food, water, tissue > presence/absence + type/species of contaminant Medical diagnostic tests - Detect pathogens in blood, tissues, body fluids. Forensic Genotyping - Identify trace human (and other species) tissue. -From DNA sample (any tissue) amplify up a fragment and determine its sequence directly - Use CODIS to obtain a unique genetic fingerprint of any person, or organism. - Do DNA fingerprinting RT PCR -> Reverse Transcriptase + PCR - Obtain tissue/organism -> extract RNA -> mrna - Reverse transcribe RNA to cdna - Do PCR on DNA sample to determine quantity and quality of mrna product - Quick assay for gene expression in widespread usage Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 6
Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 7 11/28/11