Restriction Enzymes By: Nicole Carroll
Restriction Enzyme presentation overview • Definition • Bacteria • Sticky vs. Blunt ends • Uses and purpose • Nobel Prize • My lab experience
Definition of a Restriction Enzyme • An enzyme (endodeoxyribonucleases) that recognizes and cleaves a specific nucleotide sequence within a double stranded DNA. This cleavage results in particular ends of the DNA that allows the characterization of DNA in a repeated matter and makes recombinant bioengineering possible.
Bacteria • Defense against bacteriophages: prevents the insertion of the phages DNA within it’s own genome (host) by using it’s restriction enzymes. -Bacteria cells achieve this without cleaving their own DNA. • Three types of restriction enzymes: -Type I: cuts DNA at random locations for 1000+ bp from site. -Type III: cuts DNA at random locations for about 25 bp from site. -Type II: cuts DNA at that specific sequence only. -does not require ATP like type I and III and is more efficient. -named after the bacterial species it was isolated from. -Example: EcoRI is isolated from E. Coli.
Sticky vs. Blunt ends EcoRISmaI Recognition sequence: G/AATTC 5’ overhang Recognition sequence: CCC/GGG Inverted palindrome recognition site
Uses and purposes • Nucleotide sequences analysis • DNA reconstruction (recombinant DNA) • Virus protection and identification -Lambda DNA/HindIII Marker • Forensics -DNA Fingerprinting • Ordering restriction enzymes: New England BioLabs • Other important tools (mini prep, DNA ligase, and cloning)
History-1978 Nobel Prize in Medicine Werner Arber- Suggested the idea of bacteria being able to protect themselves from foreign DNA through some catalyzed enzymatic reaction. Found two methods: restriction and modification (methylation) Hamilton Smith- Verified Arbers hypothesis and proved that these restriction enzymes cleaves only the foreign DNA and not the bacterium's (host) DNA. Lastly, he found that they cleave at a specific sequence of 6 base pairs. Dan Nathans-Researched the simian virus (SV40).By using multiple restriction enzymes simultaneously, he constructed the first genetic map of SV40 (with this chemical method) and the start of genetic engineering.
My lab experience Plasmid map of restriction sites and sizes
Sources: • A. Pray, L. (2018). Restriction Enzymes | Learn Science at Scitable. [online] Nature.com. Available at: https://www.nature.com/scitable/topicpage/restriction-enzymes-545 [Accessed 12 Sep. 2018]. • Nathans, D., and Smith, H. O., 1975. RESTRICTION ENDONUCLEASES IN THE ANALYSIS AND RESTRUCTURING OF DNA MOLECULES. • https://www-annualreviewsorg.libproxy.unm.edu/doi/pdf/10.1146/annurev.bi.44.070175.001421 • Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002. Section 9.3, Restriction Enzymes: Performing Highly Specific DNA-Cleavage Reactions. Available from: https://www.ncbi.nlm.nih.gov/books/NBK22528/ • https://www.neb.com/ • http://www.nobelprize.org/nobel_prizes/medicine/laureates/1978/press.html • Pingoud A, Jeltsch A. Structure and function of type II restriction endonucleases. Nucleic Acids Research. 2001;29(18):3705-3727.