2. Cellular level Organelle level Molecular level: Macromolecules Atomic level C, H, O, N, S, P Microscope Cell fractionation -Nucleus -Mitochondria -RER, cell membrane -SER -Cytosol ProteinsCarbohydratesLipidsNucleic acids Studies of cell -Fractionation -Purification/ Identification -Structure/ Function

3. CONTENTS Cell fractionation Electrophoresis Blotting and Hybridization Polymerase Chain Reaction DNA Sequences

4. A lab technique which uses a centrifuge to separate the contents of a cell (organelles) into fractions, after the cell has been gently lysed.  The process to break the cells is “HOMOGENIZATION” and the subsequent isolation of organelles is “FRACTIONATION”.  The centrifugation technique is employed to isolate organelles regarding to their physical characteristics, e.g., size, shape and density. The methods frequently used are “DIFFERENTIAL CENTRIFUGATION” and “DENSITY GRADIENT CENTRIFUGRATION”. Cell fractionation

5. HOMOGENIZATION Cell lysis

6.  Gently disrupt the cells to release cellular components  Physical or non-physical cell lysis methods Physical methods of cell disruption  Disruption of cells results from the shearing forces generated between the cells and either solid abrasive or liquid medium.  Pastel and mortar homogenizer, Abrasive beads, Blender, Pressure homogenization, Osmotic shock, Freezing/ thawing technique, Ultrasonification Non-physical methods of cell disruption  Organic solvents- to destroy membrane  Chaotropic anions- to destabilize membrane  Detergents- to dissolve proteins and lipids membrane  Enzymatic digestion- to digest proteins, carbohydrates and lipids of cell wall HOMOGENIZATION

7. FRACTIONATION Centrifugation

8. Physical methods of cell disruption 1.Pastel and mortar homogenizer 2.Abrasive beads- sands, silica, alumina 3.Blender- special designed blades and chamber 4.Pressure homogenization- cells are imbibed with an inert gas (argon) which will form gas bubbles inside cytoplasm when the cells are suddenly returned to atmospheric pressure, hence rupture the membrane. 5.Osmotic shock- swelling and disrupting of cells in hypotonic solution 6.Freezing/ thawing technique- ice crystals rupture the cells 7.Ultrasonification- ultrasonic wave to break open the plasma membrane and leave the internal organelles intact. Cell Fractionation

9. Non-physical methods of cell disruption 1.Organic solvents- chloroform/methanol mixtures can dissolve membrane lipids (destroy membranes) and release subcellular components. 2.Chaotropic anions- potassium thiocyanate, potassium bromide, lithium diiodosalicylate act to destabilize lipid membranes consequently, the subcellular components are being released. 3.Detergents- solubilize the integral membrane proteins by interacting with the phospholipid bilayer, e.g., SDS (anionic), Deoxycholate (non-denaturing) and Triton X- 100 (non-ionic) 4.Enzymatic digestion- to digest proteins, carbohydrates and lipids of cell wall, Mixture of enzymes: chitinases, pectinases, lipases, proteases, cellulases Cell Fractionation

10. Homogenization medium  Slightly hypo-osmotic or iso-osmotic – to preserve structural integrity of organelles  Osmoticums : sucrose, manitol, sorbitol  Chelating agents : EDTA or EGTA (remove Ca2+ or Mg2+ which are required by membrane proteases)  Protease inhibitor : endopeptidases, exopeptidases  The homogenization should be performed at 4 o C to minimize protease activity Cell Fractionation

11. Fractionation  The most widely used technique for fractionating cellular components is centrifugation technique  Particles of different density, size, and shape sediment at different rate in a centrifugal field. Factors affected the rate of sedimentation:  particle size and shape  the viscosity of suspending medium  centrifugal field * The particle remain stationary when the density of the particle and the density of the centrifugation medium are equal

12. Types of Centrifugation 1. Differential centrifugation 2. Rate-zonal centrifugation 3. Isopycnic centrifugation Cell Fractionation A centrifuge working at speeds in excess of 20,000 RPM is an “ ultracentrifuge ”.

13. Differential centrifugation  Separates particles as a function of size and density  A particular centrifugal field is chosen over a period of time  Larger mass; lower centrifugation force; lesser spin time  Subjected to repeated steps with increasing of centrifugation force Cell Fractionation

14. Differential centrifugation Centrifugation force to pellet the cellular components Cell components Centrifugation force (x g) Nucleus800-1,000 Mitochondria, Lysosome Chloroplast, Peroxisome 20,000-30,000 RER membrane 50,000-80,000 Cell membrane, SER membrane 80,000-100,000 Ribosome150,000-300,000 Cytosol fraction Supernatant

15. Differential Centrifugation Pellet 1Pellet 2Pellet 4Pellet 3

16. Molecules separate according to size and shape centrifugation Rate-zonal centrifugation  Medium  Slightly viscous  Density gradient ; a positive increment in density  e.g., sucrose, GuHCl  Sample is applied on the top of density gradient  Particles separate into a series of bands (zone) in accordance to rate of sedimentation (S), size and shape Rate-zonal Centrifugation

17. CentrifugationFraction collection

18. Isopycnic centrifugation  Based solely on the density of the particles  Separation medium– self-generating density gradient medium (CsCl medium)  Unaffected by the size or the shape of the particles  Mostly used to separate nucleic acids, large glycoproteins Isopycnic Centrifugation

19. What make rate-zonal and isopycnic centrifugations difference?

20. Molecules are separated by electric force F = qE : where q is net charge, E is electric field strength The velocity is encountered by friction qE = fv : where f is frictional force, v is velocity Therefore, mobility per unit field (U) = v/q = q/f = q/6p  r : where  is viscosity of supporting medium, r is radius of sphere molecule + - + - - - -+ E F f v q Electrophoresis

21. Factors affected the mobility of molecules 1. Molecular factors Charge Size Shape 2. Environment factors Electric field strength Supporting media ( pore: sieving effect ) Running buffer - + Electrophoresis

23. Types of supporting media  Paper  Agarose gel ( Agarose gel electrophoresis )  Polyacrylamide gel ( PAGE )  pH gradient ( Isoelectric focusing electrophoresis )  Cellulose acetate Electrophoresis

24. Agarose Gel  purified large MW polysaccharide (from agar)  very open (large pore) gel  used frequently for large DNA molecules

25. Agarose gel staining Ethidium bromide Fluorescence dye Pounseur-S dye Electrophoresis

26. Polyacrylamide Gels  Acrylamide polymer; very stable gel  can be made at a wide variety of concentrations  gradient of concentrations: large variety of pore sizes (powerful sieving effect) Electrophoresis

27.  Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH 3 (CH 2 ) 11 SO 3 - Na +  Amphipathic molecule  Strong detergent to denature proteins  Binding ratio: 1.4 gm SDS/gm protein  Charge and shape normalization SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE ) Electrophoresis

28. 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 Electrophoresis

29. 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 Electrophoresis

30. 2-dimensional Gel Electrophoresis Spot coordination pH MW

31. Hybridization and Blotting

32. Hybridization

33. Hybridization Can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily degraded) Can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily degraded) Dependent on the extent of complementation Dependent on the extent of complementation Dependent on temperature, salt concentration, and solvents Dependent 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) 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, etc. Probes can be labeled with radioactivity, fluorescent dyes, enzymes, etc. Probes can be isolated or synthesized sequences Probes can be isolated or synthesized sequences

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

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

36. RNA Blotting (Northerns) RNA is separated by size on a denaturing agarose gel and then transferred onto a membrane (blot) 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 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 ) Location of probe is determined by detection method (e.g., film, fluorometer )

37. 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.)

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

39. Western Blot Add monoclonal antibodies Rinse again Antibodies will bind to specified protein Stain the bound antibody for colour development It should look like the gel you started with if a positive reaction occurred Block membrane e.g. dried nonfat milk Block membrane e.g. dried nonfat milk Rinse with ddH 2 O Add antibody against yours with a marker (becomes the antigen)

40. Polymerase Chain Reaction (PCR)

41. 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 PCR

42. 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. Background on PCR

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

44. 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 Stabilizes the reaction between: oligonucleotides and template DNAoligonucleotides and template DNA DNA Polymerase and template DNADNA Polymerase and template DNA

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

46. Step 2: Priming Decrease temperature by 15-25 ° 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

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

48. 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 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. The DNA polymerase recognizes the primer and makes a complementary copy of the template which is now single stranded. Approximately 150 nucleotides/sec Approximately 150 nucleotides/sec

49. Potential Problems with Taq Lack of proof-reading of newly synthesized DNA. Lack of proof-reading of newly synthesized DNA. Potentially can include di-Nucleotriphosphates (dNTPs) that are not complementary to the original strand. Potentially can include di-Nucleotriphosphates (dNTPs) that are not complementary to the original strand. Errors in coding result Errors in coding result Recently discovered thermostable DNA polymerases, Tth and Pfu, are less efficient, yet highly accurate. Recently discovered thermostable DNA polymerases, Tth and Pfu, are less efficient, yet highly accurate.

50. 1.Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence. 2.Next, denature the DNA at 94˚C. 3.Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA. 4.Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus ). 5.Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (2 20 ) to 35 trillion copies (2 45 ) of the target DNA. 6.Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely. 7. Cool to 4˚C and store or use amplified PCR product for analysis. How PCR works

51. Step 1 7 min at 94˚CInitial Denature Step 2 45 cycles of: 20 sec at 94˚CDenature 20 sec at 64˚CAnneal 1 min at 72˚CExtension Step 3 7 min at 72˚CFinal Extension Step 4 Infinite hold at 4˚CStorage Thermal cycler protocol Example

52. The Polymerase Chain Reaction

56. PCR amplification Each cycle the oligo-nucleotide primers bind most all templates due to the high primer concentration Each cycle the oligo-nucleotide primers bind most all templates due to the high primer concentration The generation of mg quantities of DNA can be achieved in ~30 cycles (~ 4 hrs) The generation of mg quantities of DNA can be achieved in ~30 cycles (~ 4 hrs)

57. Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg 2+ dNTPs (dATP, dCTP, dGTP, dTTP) OPTIMISING PCR THE REACTION COMPONENTS

58. Organims, Organ, Tissue, cells ( blood, hair root, saliva, semen) Obtain the best starting material you can. Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood Good quality genomic DNA if possible Blood – consider commercially available reagents Qiagen– expense? Empirically determine the amount to add RAW MATERIAL

59. Number of options available Taq polymerase Pfu polymerase Tth polymerase How big is the product? 100bp 40-50kb What is end purpose of PCR? 1. Sequencing - mutation detection -. Need high fidelity polymerase -. integral 3’ 5' proofreading exonuclease activity 2. Cloning POLYMERASE

60. Length ~ 18-30 nucleotides (21 nucleotides) Base composition: 50 - 60% GC rich, pairs should have equivalent Tms Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C] Initial use Tm–5°C Avoid internal hairpin structures no secondary structure Avoid a T at the 3’ end Avoid overlapping 3’ ends – will form primer dimers Can modify 5’ ends to add restriction sites PRIMER DESIGN

61. Use specific programs OLIGO Medprobe PRIMER DESIGNER Sci. Ed software Also available on the internet http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html

62. Mg 2+ CONCENTRATION 1 1.5 2 2.5 3 3.5 4 mM Normally, 1.5mM MgCl 2 is optimal Best supplied as separate tube Always vortex thawed MgCl 2 Mg 2+ concentration will be affected by the amount of DNA, primers and nucleotides

63. USE MASTERMIXES WHERE POSSIBLE

64. How Powerful is PCR? PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified — a boiled bacterial colony. The template DNA need not be highly purified — a boiled bacterial colony. The PCR product can be digested with restriction enzymes, sequenced or cloned. The PCR product can be digested with restriction enzymes, sequenced or cloned. PCR can amplify a single DNA molecule, e.g. from a single sperm. PCR can amplify a single DNA molecule, e.g. from a single sperm.

65. Applications of PCR Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA, etc.) for analysis Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA, etc.) for analysis 1. Gene isolation 2. Fingerprint development Introduce sequence changes at the ends of fragments Introduce sequence changes at the ends of fragments Rapidly detect differences in DNA sequences (e.g., length) for identifying diseases or individuals Rapidly detect differences in DNA sequences (e.g., length) for identifying diseases or individuals Identify and isolate genes using degenerate oligonucleotide primers Identify and isolate genes using degenerate oligonucleotide primers Design mixture of primers to bind DNA encoding conserved protein motifsDesign mixture of primers to bind DNA encoding conserved protein motifs Genetic diagnosis - Mutation detection basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10 -9 bp Genetic diagnosis - Mutation detection basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10 -9 bp

66. Paternity testing Mutagenesis to investigate protein function Quantify differences in gene expression Reverse transcription (RT)-PCR Identify changes in expression of unknown genes Differential display (DD)-PCR Forensic analysis at scene of crime Industrial quality control DNA sequencing Applications of PCR

68. Sequencing of DNA by the Sanger method