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Analysis of DNA Fragments with Gel Electrophoresis
Pre-AP Biology
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Bellwork Explain how DNA fragments are created.
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Objectives Define electrophoresis
Explain the relationship between agarose concentration and pore space Explain the role of the buffer in electrophoresis Explain how DNA is detected in agarose gels Property of CTE Joint Venture 4/23/2017
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Key Terminology Agarose Electrophoresis Pore Spaces Comb Loading Wells
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Overview of Gel Electrophoresis
Uses electricity to separate molecules Used to analyze biological molecules Uses a gel to separate molecules based on charge, shape and/or size Biological molecules most commonly analyzed by gel electrophoresis include DNA RNA Proteins Property of CTE Joint Venture 4/23/2017
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Two Types of Gel Electrophoresis
Classified based on orientation of gels Horizontal gel lays flat commonly used for separating DNA fragments Vertical gel is upright commonly used for separating proteins Property of CTE Joint Venture 4/23/2017
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Gel commonly used to analyze DNA
Agarose (carbohydrate derived from seaweed) Separates DNA fragments ranging in size between 500 and 25,000 base pairs (bp) Separates DNA fragments by acting as a molecular strainer Smaller fragments move through the pores easier than large ones Property of CTE Joint Venture 4/23/2017
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Pore Size created in Agarose
The number of hydrogen bonds between and within the carbohydrate chains of agarose create the pore size The greater number of hydrogen bonds the smaller the pore size 4/23/2017
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Pore Size Analogous to Textile Weave
Loose weave (fewer hydrogen bonds) larger spaces between fibers Tight weave (more hydrogen bonds) smaller spaces between fibers Loose Weave Tight Weave Property of CTE Joint Venture 4/23/2017
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PROPERTY OF PIMA COUNTY JTED, 2010
Think-Pair-Share What is the purpose of the agarose gel? 3. Think-pair-share teacher presents a question teacher gives wait time for student to form answer teacher instructs students to share their answer with a partner teacher calls on non-volunteers to share with the class PROPERTY OF PIMA COUNTY JTED, 2010 3
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Agarose Gel Preparation:
Dissolve powdered agarose into electrophoresis buffer Boil to dissolve the agarose Pour into gel trays and insert comb to form loading wells Allow to harden at room temperature Remove comb to reveal loading wells Property of CTE Joint Venture 4/23/2017
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Electrophoresis Apparatus
Place solidified gel in a gel box and cover with electrophoresis buffer Electrophoresis Buffer commonly used is TAE Buffer pH 8 Purpose of the buffer Stabilizes the pH Maintains shape of DNA molecule Conducts electricity Property of CTE Joint Venture 4/23/2017
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Electrophoresis Apparatus
The gel box has electrodes at each end of the box anode (negative electrode, black) cathode (positive electrode, red) The power supply that generates the electrical field is attached to the electrodes of the gel box
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Movement of DNA in an Electric Field
DNA molecules are negatively charged because of the phosphate groups Under an electric field they will move towards the positive electrode (cathode) The smaller DNA fragments will move farther down the gel because they more easily move through the pore spaces Property of CTE Joint Venture 4/23/2017
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PROPERTY OF PIMA COUNTY JTED, 2010
Think-Pair-Share Explain how electrophoresis works IYOW. 3. Think-pair-share teacher presents a question teacher gives wait time for student to form answer teacher instructs students to share their answer with a partner teacher calls on non-volunteers to share with the class PROPERTY OF PIMA COUNTY JTED, 2010 3
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Detection of DNA in Agarose Gels
DNA can be detected by using a stain: Methylene Blue Works by bonding with the DNA and turning it dark blue
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Jumbled Sort Put the steps outlined on the next slide in order
Be prepared to share 13. Jumbled sort teacher supplies each student or group of students with random strips of instructions, key words, safety procedures, etc, and asks them to put the strips in order teacher calls on non-volunteers to contribute their sorted material 17 PROPERTY OF PIMA COUNTY JTED, 2010
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Steps of gel electrophoresis for analyzing DNA fragments
Pour agarose gel Isolate DNA Insert electrodes into power supply Load agarose gel with DNA fragments Cover agarose gel with electrophoresis buffer Load DNA samples in agarose gel Stain gel with methylene blue Cut DNA with restriction fragments
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DNA Fingerprints DNA Fingerprints Way of identifying individuals
By analyzing sections of DNA that vary widely between individuals DNA fingerprints are created by cutting the DNA with restriction enzymes separating those fragments on agarose gels 19
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DNA Fingerprints important for
Forensics for identifying who is responsible for a crime Paternity/maternity suits for identifying who is the father/mother of a child Identification of bodies, for example after 9/11 22
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PROPERTY OF PIMA COUNTY JTED, 2010
2 Minute Paper You have two minutes to write down how agarose gel electrophoresis would be useful in identifying an individual’s DNA fingerprint. Be prepared to share. 29. Minute paper or Quick Write teacher provides a prompt from the lesson students have one minute to write down a summary of the key points students compare with a partner teacher uses a strategy to check all PROPERTY OF PIMA COUNTY JTED, 2010 23 23
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