Analysis of DNA Fragments with Gel Electrophoresis

1. Analysis of DNA Fragments with Gel Electrophoresis Pre-AP Biology

2. Bellwork • Explain how DNA fragments are created.

3. 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. Key Terminology • Agarose • Electrophoresis • Pore Spaces • Comb • Loading Wells

5. Overview of Gel Electrophoresis • 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

6. 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 http://ecx.images-amazon.com/images/I/51OWHEwM-fL._SL160_AA115_.jpg&imgrefurl http://www.bio-link.org/vlab/Graphics/Tools/DNAGel.jpg&imgrefurl Property of CTE Joint Venture

7. 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

8. 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

9. Pore Size Analogous to Textile Weave • Loose weave (fewer hydrogen bonds) • larger spaces between fibers • Tight weave (more hydrogen bonds) • smaller spaces between fibers g Loose Weave Tight Weave http://www.shutterstock.com/pic-1098831/ http://www.housebeautiful.com/cm/housebeautiful/images/fabric-17-xlg.jpg&imgrefurl Property of CTE Joint Venture

10. Think-Pair-Share What is the purpose of the agarose gel? 3 PROPERTY OF PIMA COUNTY JTED, 2010

11. 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 http://www.nslc.wustl.edu/courses/Bio2960/labs/07DNA/Gel/f21.gif Property of CTE Joint Venture

12. 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 http://www.connecticutvalleybiological.com/images/bt500.jpg&imgrefurl Property of CTE Joint Venture

13. 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

14. 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

15. Think-Pair-Share Explain how electrophoresis works IYOW. 3 PROPERTY OF PIMA COUNTY JTED, 2010

16. 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

17. Jumbled Sort Put the steps outlined on the next slide in order Be prepared to share 17 PROPERTY OF PIMA COUNTY JTED, 2010

18. 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

19. 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

22. 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

23. 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. 23 PROPERTY OF PIMA COUNTY JTED, 2010