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 • Restriction Enzymes • Electrophoresis • Blotting and Hybridization • Polymerase Chain Reaction • DNA Sequences

5. 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

6. 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

7. Endonuclease

8. 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

9. 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

10. Nomenclature

11. Specificity

12. Restriction Product

13. 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

14. 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)

15. 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

16. 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 +

17. Electrophoresis

18. 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

19. 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

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

21. 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

22. 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

23. 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

24. 2-dimensional Gel Electrophoresis • Spot coordination • pH • MW

25. Blotting and Hybridization

26. 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 Southern blot DNA Northern blot RNA Western blot Protein

27. 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.

28. 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.

29. 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)

30. Southern blotting

31. Some Applications of DNA Blots • Map restrictions sites near a particular locus for gene isolation or allele analysis (e.g., RFLP restriction fragment length polymorphism) • Identity of closely related genes • Confirmation of gene transfer or gene disruption • Detection of foreign DNA

32. RNA Blotting (Northern) • RNA is separated by size on a denaturing agarose gel and then transferred onto a membrane (blot) • 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)

33. Applications of RNA Blots Detect the expression level and transcript size of a specific gene in a specific tissue or at a specific time. Sometimes mutations do not affect coding regions but transcriptional regulatory sequences (e.g., UAS/URS, promoter, splice sites, copy number, transcript stability, etc.)

34. Western Blot • Protein blotting • Highly specific qualitative test • Can determine if above or below threshold • Typically used for research • Use denaturing SDS-PAGE • Solubilizes, removes aggregates & adventitious proteins are eliminated Components of the gel are then transferred to a solid support or transfer membrane weight Paper towel Wet filter paper Transfer membrane Paper towel

35. Add antibody against yours with a marker (becomes the antigen) Stain the bound antibody for colour development It should look like the gel you started with if a positive reaction occurred Western Blot • Block membrane e.g. dried nonfat milk Rinse with ddH2O Add monoclonal antibodies Rinse again Antibodies will bind to specified protein

36. Hybridization Pairing of complementary DNA and/or RNA and/or protein

37. Hybridization • It can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily degraded) • It depended on the extent of complementation • It depended on temperature, salt concentration, and solvents • Small changes in the above factors can be used to discriminate between different sequences (e.g. small mutations can be detected) • Probes can be labeled with radioactivity, fluorescent dyes, enzymes. • Probes can be isolated or synthesized sequences

38. In-situ hybridisation Hybridization which is performed by denaturing the DNA of cell squash on a microscope slide so that reaction is possible with an added of probe • Chromosome in-situ hybridisation • DNA probe detects sequences in chromosomes • Map gene sequences • Tissue in-situ hybridisation • RNA probe detects sequences in cells and tissues • Identify sites of gene expression • Analyse tissue distribution of expression

39. Oligonucleotide probes • Single stranded DNA (usually 15-40 bp) • Degenerate oligonucleotide probes can be used to identify genes encoding characterized proteins • Use amino acid sequence to predict possible DNA sequences • Hybridize with a combination of probes • TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) - could be used for FWMDC amino acid sequence • Can specifically detect single nucleotide changes

40. Detection of Probes • Probes can be labeled with radioactivity, fluorescent dyes, enzymes. • Radioactivity is often detected by X-ray film (autoradiography) • Fluorescent dyes can be detected by fluorometers, scanners • Enzymatic activities are often detected by the production of dyes or light (x-ray film)

41. Polymerase Chain Reaction(PCR)

42. Polymerase Chain Reaction Powerful technique for amplifying DNA Amplified DNA are then separated by gel electrophoresis

43. PCR • A simple rapid, sensitive and versatile in vitro method for selectively amplifying defined sequences/regions of DNA/RNA from an initial complex source of nucleic acid - generates sufficient for subsequent analysis and/or manipulation • Amplification of a small amount of DNA using specific DNA primers (a common method of creating copies of specific fragments of DNA) • DNA fragments are synthesized in vitro by repeated reactions of DNA synthesis (It rapidly amplifies a single DNA molecule into many billions of molecules) • In one application of the technology, small samples of DNA, such as those found in a strand of hair at a crime scene, can produce sufficient copies to carry out forensic tests. • Each cycle the amount of DNA doubles

44. Background on PCR • The Ability to generate identical high copy number DNAs made possible in the 1970s by recombinant DNA technology (i.e., cloning). • Cloning DNA is time consuming and expensive • Probing libraries can be like hunting for a needle in a haystack. • Requires only simple, inexpensive ingredients and a couple hours • PCR, “discovered” in 1983 by Kary Mullis, • Nobel Prize for Chemistry (1993). • It can be performed by hand or in a machine called a thermal cycler.

45. Three Steps • Separation Double Stranded DNA is denatured by heat into single strands. Short Primers for DNA replication are added to the mixture. • Priming DNA polymerase catalyzes the production of complementary new strands. • Copying The process is repeated for each new strand created All three steps are carried out in the same vial but at different temperatures

46. Step 1: Separation • Combine Target Sequence, DNA primers template, dNTPs, Taq Polymerase • Target Sequence 1. Usually fewer than 3000 bp 2. Identified by a specific pair of DNA primers- usually oligonucleotides that are about 20 nucleotides • Heat to 95°C to separate strands (for 0.5-2 minutes) • Longer times increase denaturation but decrease enzyme and template Magnesium as a Cofactor Mg stabilizes the reaction between: • oligonucleotides and template DNA • DNA Polymerase and template DNA

47. Heat Denatures DNA by uncoiling the Double Helix strands.

48. Step 2: Priming • Decrease temperature by 15-25 °C • Primers anneal to the end of the strand • 0.5-2 minutes • Shorter time increases specificity but decreases yield • Requires knowledge of the base sequences of the 3’ - end

49. Selecting a Primer • Primer length • Melting Temperature (Tm) • Specificity • Complementary Primer Sequences • G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches • 3’-end Sequence • Single-stranded DNA

50. Step 3: Polymerization • Since the Taq polymerase works best at around 75 ° C (the temperature of the hot springs where the bacterium was discovered), the temperature of the vial is raised to 72-75 °C • The DNA polymerase recognizes the primer and makes a complementary copy of the template which is now single stranded. • Approximately 150 nucleotides/sec