Molecular Techniques

1. Molecular Techniques Reminder: All molecular techniques are based on the chemical “personality” (or chemical properties) of the DNA molecule (or nucleic acids)

2. Studies of cell • Fractionation • Purification/ Identification • Structure/ Function Proteins Carbohydrates Lipids Nucleic acids Organelle level • Cell fractionation • Nucleus • Mitochondria • Ceell membrane • Cytosol Cellular level Microscope Molecular level: Macromolecules Atomic level C, H, O, N, S, P

3. - - - - Negatively-charged phosphate-sugar backbone Various lengths Specificity of nucleotides Hydrogen bonds Nucleic Acids

4. CONTENTS • Enzymes • Electrophoresis • Blotting and Hybridization • Polymerase Chain Reaction • DNA Sequences

5. Enzymes • Large molecules made of various amino acids • Act as catalysts to speed up reactions w/out being destroyed • Increase the rate of reaction • Highly specific • Lowers energy of activation level • Activity lost if denatured • May contain cofactors such as metal ions or organic (vitamins)

6. Name of Enzymes • End in –ase • Identifies a reacting substance 1. sucrase– reacts sucrose 2. lipase - reacts lipid • Describes function of enzyme 1. oxidase– catalyzes oxidation 2. hydrolase– catalyzes hydrolysis

7. Classification of Enzymes Class Reactions catalyzed • Oxidoreductoasesoxidation-reduction • Transferasestransfer group of atoms • Hydrolaseshydrolysis • Lyasesadd/remove atoms to/from a double bond • Isomerasesrearrange atoms • Ligasescombine molecules

8. Enzyme Action: Lock and Key Model • An enzyme binds a substrate in a region called the active site • Only certain substrates can fit the active site • Amino acid R groups in the active site help substrate bind • Enzyme-substrate complex forms • Substrate reacts to form product • Product is released

9. Lock and Key Model + + E + S ES complex E + P P S S P

10. Enzymes use in Molecular Genetics 1. Restriction endonucleases/enzymes 2. Ligase 3. DNA polymerase

11. Restriction Enzymes Molecular scissors which isolated from bacteria where they are used as Bacterial defense against viruses Molecular scalpels to cut DNA in a precise and predictable manner Enzyme produced by bacteria that typically recognize specific 4-8 base pair sequences called restriction sites, and then cleave both DNA strands at this site A class of endo-nucleases that cleavage DNA after recognizing a specific sequence Members of the class of nucleases

12. Nuclease Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and RNA molecules • Endonuclease • Cleave nucleic acids at internal position • Exonuclease • Progressively digest from the ends of the nucleic acid molecules

13. Endonuclease

14. Restriction Enzymes • There are already more than 1200 type II enzymes isolated from prokaryotic organism • They recognize more than 130 different nucleotide sequence • They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence • Palindromic sequence: • The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand • To Avoid confusion, restriction endo-nucleases are named according to the following nomenclature

15. Nomenclature • The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated • The second and third letters are usually the initial letters of the organisms species name. It is written in italic • A fourth letter, if any, indicates a particular strain organism • Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery

16. Nomenclature

17. Specificity

18. Restriction Product

19. Restriction enzymes Restriction enzymes can be grouped by: • number of nucleotides recognized (4, 6,8 base-cutters most common) • kind of ends produced (5’ or 3’ overhang (cohesive=sticky), blunt=flush) • degenerate or specific sequences • whether cleavage occurs within the recognition sequence

20. A restriction enzyme (EcoRI) 1. 6-base cutter 2. Specific palindromic sequence (5’GAATTC) 3. Cuts within the recognition sequence (type II enzyme) 4. produces a 5’ overhang (sticky end)

21. Ligase Any of a class of enzymes that act as catalysts in chemical reactions in which molecules are linked together, as in the synthesis and repair of DNA or in the formation of recombinant DNA Any of a class of enzymes that catalyze the linkage of two molecules, generally utilizing ATP as the energy donor (synthetase).

22. Function of DNA ligase The enzyme, DNA ligase, repairs the millions of DNA breaks generated during the normal course of a cell's life, for example, linking together the abundant DNA fragments formed during replication of the genetic material in dividing cells.

23. Ligase

24. DNALigase Mechanism

25. DNALigase Mechanism

26. Human DNA Ligase • Human DNA ligase III gene encodes both nuclearand mitochondrial enzymes. • DNA ligase plays a central role inDNA replication, recombination, and DNA repair.

27. DNA Polymerase • an enzyme that is template based and has both 5’->3' DNA polymerase activity and 3’->5' exonuclease activity. • highly processive, meaning it synthesizes long stretches of DNA without dissociating from the DNA template. • an open right hand, composed of a thumb domain that binds to thioredoxin, a finger domain in which catalytic activity resides, a palm domain that cradles the DNA, and a terminal exonuclease domain 

28. Three main features of the DNA synthesis reaction • DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and he 5’-phosphate of the dNTP. • Energy for this reaction is derived from the release of two of the three phosphates of the dNTP. • DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening process. • rate ≤ 800 dNTPs/second • low error rate 3. Direction of synthesis is 5’ to 3’

29. DNA elongation

30. DNA elongation

31. Types of DNA polymerase • Polymerase Polymerization Exonuclease Exonuclease #Copies • (5’-3’) (3’-5’) (5’-3’) • I Yes Yes Yes 400 • II Yes Yes No ? • III Yes Yes No 10-20 • 3’ to 5’ exonuclease activity : ability to remove nucleotides from the • 3’ end of the chain • Important proofreading ability • Without proofreading error rate (mutation rate) is 1 x 10-6 • With proofreading error rate is 1 x 10-9 (1000-fold decrease) • 5’ to 3’ exonuclease activity : the ability in DNA replication & repair.

32. Eukaryotic enzymes • Five common DNA polymerases from mammals. • Polymerase  (alpha): nuclear, DNA replication, no proofreading • Polymerase  (beta): nuclear, DNA repair, no proofreading • Polymerase  (gamma): mitochondria, DNA repl., proofreading • Polymerase  (delta): nuclear, DNA replication, proofreading • Polymerase  (epsilon): nuclear, DNA repair (?), proofreading • Different polymerases for the nucleus and mtDNA • Some polymerases proofread; others do not. • Some polymerases used for replication; others for repair. • Polymerases vary by species.

33. In this illustration, DNA ligase (in color) encircles the DNA double helix. Researchers investigating an important DNA-repair enzyme now have a better picture of the final steps of a process that glues together, or ligates, the ends of DNA strands to restore the double helix.The enzyme, DNA ligase, repairs the millions of DNA breaks generated during the normal course of a cell's life, for example, linking together the abundant DNA fragments formed during replication of the genetic material in dividing cells.

34. GEL ELECTROPHORESIS The motion of disperse charged particle relatives to a fluid under the influence of a spatially uniform electric field First observed by Reuss, 1807 • For separating disperse charged biological molecule of any size/length • Uses electricity • Uses a matrix • Uses buffer solution

35. Electrophoresis - • Factors affecting the mobility of molecules: • 1. Molecular factors • Charge • Size • Shape • 2. Environment factors • Electric field strength • Matrix (pore: sieving effect) • Running buffer +

36. Electrophoresis

37. Types of matrix (supporting media) • Paper • Agarose • 1. purified large MW polysaccharide (from agar) • 2. very open (large pore) gel • 3. used frequently for large DNA molecules • Acrylamide • 1. a white odorless crystalline solid chemical compound • 2. soluble in water, ethanol, ether, chloroform • 3. used to synthesize poly-acrylamide which find many uses as • water soluble thickeners • Starch • Cellulose acetate

38. DNA Agarose Gel An analytical technique used to separate DNA by size • Electric field induces DNA to migrate toward the anode due to the net negative charge of the sugar phosphate backbone of the DNA • Longer molecules migrate more slowly • Visualized using a fluorescence dye special for DNA such as ethidium bromide

39. Polyacrylamide Gels • acrylamide polymer • very stable gel • can be made at a wide variety of concentrations • large variety of pore sizes (powerful sieving effect)

40. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) • Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH3(CH2)11SO3- Na+ • Amphipathic molecule • Strong detergent to denature proteins • Binding ratio: 1.4 g SDS/g protein • Charge and shape normalization

41. Isoelectric Focusing Electrophoresis (IFE) • Separate molecules according to their isoelectric point (pI) • At isoelectric point (pI) molecule has no charge (q=0), hence molecule ceases • pH gradient medium

42. 2-dimensional Gel Electrophoresis • First dimension is IFE (separated by charge) • Second dimension is SDS-PAGE (separated by size) • So called 2D-PAGE • High throughput electrophoresis, high resolution

43. 2-dimensional Gel Electrophoresis • Spot coordination • pH • MW

44. Blotting and Hybridization

45. Blotting • Transfer the DNA from the gel to a solid support • Transferring of DNA, RNA, Protein to an immobilizing binding matrix such as nitrocellulose paper or nylon Northern blot (RNA) Eastern blot (???) Western blot (Protein) Southern blot (DNA)

46. Blotting • Two methods : • Capillary transfer • Electrophoretic transfer

47. SOUTHERN BLOTTING • The technique was developed by E.M. Southern in 1975. • The Southern blot is used to detect the presence of a particular piece of DNA in a sample. • The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome • The key to this method is hybridization. • Hybridization-process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

48. SOUTHERN BLOTTING There are 2 important features of hybridization: • The reactions are specific The probes will only bind to targets with a complementary sequence. • The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

49. Southerns Blotting (DNA Blotting) • DNA fragments created by restriction digestion are separated on an agarose gel • Separated fragments are denatured and transferred to a membrane (blot) by blotting • Probe is hybridized to complementary sequences on the blot and excess probe is washed away • Location of probe is determined by detection method (e.g., film, fluorometer)

50. Southern blotting