BIOLOGY Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR)

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1 BIOLOGY 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 Problems: 9 th Ed.? Campbell (2008) 8 th Ed. Readings: Concept 20.1, pg. 404 Web pages: 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) 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

2 The basics of a PCR reaction History Kary Mullis shared 1993 Nobel Prize in Chemistry Credited with the discovery of PCR See: 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.

Temperature depends on the primers used, it ranges from ~40 to ~70 C for ~1 min.

3- Extension/Elongation This is typically done at ~72 C for the polymerase to extend from the primer.

3 11/28/11 Thermocycler: Steps: Fig 8.6 OG 1. Denaturation 2. Annealing 3. Extension/Elongation Number of cycles: 1- Denaturation Heat to ~ 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: Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 3

Amplified Product see a band (many molecules of the same size) for each product

4 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

5 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 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

6 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

7 Biol207 Dr. Locke section Lecture#27 Fall ʻ09 page 7 11/28/11

INTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist

INTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist Objective of PCR To provide a solution to one of the most pressing