Restriction Enzyme Digestion of DNA

1. Restriction Enzyme Digestion of DNA

2. WHAT ARE RESTRICTION ENZYMES? • Restriction Enyzmes – molecular scissors able to cut DNA

3. Discovering Restriction Enzymes Werner Arber Daniel Nathans Hamilton Smith Werner Arber – discovered restriction enzymes Daniel Nathans - pioneered the application of restriction for the construction of genetic maps Hamilton Smith - showed that restriction enzyme cuts DNA in the middle of a specific sequence

4. WHERE DO RE’S COME FROM? • Bacteria • “Immune system” to protect against bacteriophages (bacteria-infecting viruses like Lambda).

5. Biological Role • Most bacteria use Restriction Enzymes as a defence against bacteriophages. • Restriction enzymes prevent the replication of the phage by cleaving its DNA at specific sites. • The host DNA is protected by Methylases which add methyl groups to adenine or cytosine bases within the recognition site thereby modifying the site and protecting the DNA.

7. HOW DO RESTRICTION ENZYMES WORK? • Usually cut DNA at a “palindrome” such as GAATTC. • Palindrome – word or phrase when spelled backwords, spells the same word or phrase 5’ 3’ “Restriction site” or “Recognition Sequence” GAATTC | | | | | | CTTAAG 3’ 5’

8. HOW DO RESTRICTION ENZYMES WORK? • RE’s cut DNA’s phosphodiester bonds and hydrogen bonds.

9. Restriction fragments can be blunt ended or sticky ended 5’ G A A T T C 3’ 5’ G A T A T C 3’ 3’ C T T A A G 5’ 3’ C T A T A G 5’ Sticky Ends Blunt Ends Sticky ends or blunt ends can be used to join DNA fragments. Sticky ends are more cohesive compared to blunt ends.

10. Example of RE that produce sticky end cutters:

11. HaeIII Some restriction enzymes cut DNA at opposite base. They leave blunt ended DNA fragments These are called blunt end cutters

12. Isoschizomers and Neochischizomers • Restriction enzymes that have the same recognition sequence but cleave the DNA at a different site within that sequence are Neochizomers. SmaIand XmaI • Restriction enzymes that have the same recognition sequence as well as the same cleavage site are Isoschizomers. C CC G GG C CC G GG G GG C CC G GG C CC Xma I Sma I

14. Restriction Maps

15. Tools used for Restriction studies. Site Address NEB Cutter http://tools.neb.com/NEBcutter2/index.php Restriction/Mapper http://arbl.cvmbs.colostate.edu/molkit/mapper/index.html http://www.restrictionmapper.org/ Webcutterhttp://www.ccsi.com/firstmarket/cutter/cut2.html In Silico Restriction http://insilico.ehu.es/restriction/ WatCuthttp://watcut.uwaterloo.ca/watcut/watcut/ EnzymeXhttp://mekentosj.com/enzymex/ Silent http://bioweb.pasteur.fr/seqanal/interfaces/silent.html

16. cloning vectors • A cloning vector is a small piece of DNA, taken from a virus, Bacteria or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. • Multiple Cloning Sites • Origin of Replication • Antibiotic Resistance Genes

18. Plasmid vectors • Plasmids are circular DNA molecules present in the cytoplasm of the bacteria • Capable of autonomous replication • Can transfer genes from one cell to other • Act as vectors in genetic engineering. • Can also present in Yeasts

19. pBR322 ori

20. Bacteriophage vectors • Advantages: • Useful for cloning large DNA fragments (10 - 23 kbp) • Disadvantages: • Less easy to handle

21. l vectors • Left arm: • head & tail proteins • Right arm: • DNA synthesis • regulation • host lysis • Deleted central region: • integration

22. ori 21.5 kb TetR cos Cos site is the only requirement for packaging into phage particle EcoRI Cosmids • Hybrid vectors: plasmids that contain bacteriophage lambda cos sites • DNA (~ 33-48 kb) cloned into restriction site, the cosmid packaged into viral particles and these phages used to infect E.coliSomewhatunstable, difficult to maintain

24. Other Vectors • BACs (Bacterial artificial chromosomes) • Useful for sequencing genomes, because insert size 100 - 300kb • YAC (Yeast Artificial Chromosome) • Can accept 200 kb -1000 kb; useful for sequencing • Ti plasmids; to introduce genes into plants

25. Gene Cloning • Gene cloning is a commonly used molecular biological technique in which a gene of interest is fused into a self-replicating genetic element called a plasmid, which when introduced into a suitable host (usually bacteria), self-replicates and generates a large number of identical copies of the particular gene.

26. Gene cloning involves using bacteria to make multiple copies of a gene • Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell • Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA • This results in the production of multiple copies of a single gene

27. Five steps of cloning: • Cutting the DNA to be cloned from the chromosomal using sequence specific Restriction Endonuclease. • Selecting a cloning vector (a small molecule capable of self-replicating inside host cells), and cutting the cloning vector with the same restriction endonuclease producing the cohessive ends. • Incubating the vector and subject DNA togather to aneal and then joining them using DNA ligase. The resultant DNA is called recombinant DNA. • Transferring the reconbinant DNA to an appropriate host such as bacteria, virus or yeast which will provide necessorybiomachinary for DNA replication. • Identifying the host cells that contain the recombinant DNA.

31. Blotting Techniques • Concept: hybridization of nucleic acids to identify sequence of interest. • Southern Blotting is named after its inventor, the British biologist Edwin Southern (1975) • Other blotting methods (i.e. western blot, WB, northern blot, NB) that employ similar principles, but using protein or RNA, have later been named in reference to Edwin Southern's name.

32. In Molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length (usually 100–1000 bases long) which can be radioactively labeled. It can then be used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe

33. Southern Blot

34. Northern Blot mRNA is... RNA

35. Western Blot mRNA is... RNA

36. Applications • Applied to detect Restriction Fragment Length Polymorphism (RFLP) and Variable Number of Tandem Repeat (VNTR) Polymorphism. The latter is the basis of DNA fingerprinting. • To check gene expressions • To find Antigens in blood