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Restriction Enzyme Digestion

Restriction Enzyme Digestion. http://www.chemsoc.org/timeline/graphic/1984_dnaf.jpg. Definition. http://previews5.nvtech.com/75/tf05310/NVTech_vc063467.jpg. http://www.esemag.com/0105/dna2.jpg. Restriction enzyme:

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Restriction Enzyme Digestion

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  1. Restriction Enzyme Digestion http://www.chemsoc.org/timeline/graphic/1984_dnaf.jpg

  2. Definition http://previews5.nvtech.com/75/tf05310/NVTech_vc063467.jpg http://www.esemag.com/0105/dna2.jpg • Restriction enzyme: A protein that recognizes a particular sequence of DNA and cuts the DNA at that site (the restriction site) • Digestion: the act of breaking down into pieces

  3. Restriction enzymes • Recognize short specific sequences, often palindromes • Isolated from bacteria as endogenous “restrictors” of bacterial pathogens http://arbl.cvmbs.colostate.edu/

  4. Different enzymes…different sites…different cuts http://www.phschool.com/science/ http://www.phschool.com/science/ http://www.phschool.com/science/ Eco RI Pst I Sma I

  5. Animations Cutting and pasting 3D animation

  6. Quick Quiz A restriction enzyme cuts: • Single-stranded DNA • RNA • Proteins • Double-stranded DNA

  7. Quick Quiz A restriction enzyme works by identifying a specific DNA sequence and cleaving: • The sugar-phosphate backbone of one strand • The sugar-phosphate backbone of both strands • The nitrogenous bases from one strand • The nitrogenous bases from both strands

  8. Examples • Restriction enzyme digestion is used for… • Identifying individuals (DNA fingerprinting) • Identifying species (e.g. Mytilus) • Cloning (moving genes in and out of plasmids) http://www.nottingham.ac.uk/biology http://www.bio.davidson.edu/courses/genomics/method/inducepromoter.gif

  9. Quick Quiz ITS PCR products must be cut by restriction digestion to identify the different Mytilus species because: • Both mussel species have an ITS PCR product of the same size • Both mussel species have the same restriction sites • ITS PCR products can vary in size • Neither mussel species gives good ITS PCR bands

  10. Non-Examples Restriction enzyme digestion is NOT used for… • Cutting RNA • Sequencing DNA

  11. Digestion Procedure Prepare Master Mix Add buffer and enzyme in correct proportions. Add restriction digest master mix to DNA Mix thoroughly by flicking tube. Incubate Temperature and time depend on enzyme. View result by gel electrophoresis

  12. Downstream Applications • Ligation and….. • Bacterial transformation http://www.kochi-u.ac.jp/~tatataa/tech2/gene/ligation.jpg http://hthbiotech.sandiegostc.org/tr-2a-b.html

  13. END

  14. Resources http://www.phschool.com/science/biology_place/biocoach/red/intro.html http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html http://web.indstate.edu/thcme/mwking/molecular-medicine.html#restriction http://www.fermentas.com

  15. California State Chemistry Standards Grades 9-12 2. Properties of matter result from the ability of atoms to form bonds… 6.Solutions are homogenous mixtures of two or more substances 10b. Students know the bonding characteristics of carbon…

  16. California State Biology Standards Grades 9-12 5d. Students know the general structure and function of DNA

  17. California State Investigation and ExperimentationStandards Grade 7 a. Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display data Grades 9-12 c. Identify possible reasons for inconsistent results, such as sources of error or uncontrolled conditions d. Formulate explanations by using logic and evidence k. Recognize the cumulative nature of scientific evidence l. Analyze situations and solve problems that require combining and applying concepts from more than one area of science

  18. National Standards Grades 6-12 Content Standard A: Science as Inquiry Content Standard C: Life Science Content Standard E: Science and Technology

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