Chapt 10


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  • Delicate: require careful handling
  • Percolates: passess
  • Affinity: connection
  • Chapt 10

    2. 2. MOLECULAR BIOLOGY TECHNIQUELearning Outcomes After completing this lecture, students will be able to: (a) List few techniques used in molecular biology field (b) Know & able to describe - Microscopic observation - Centrifugation - Extraction - Electrophoresis - Chromatography Topic Slide 2 of 10 © 2010 Cosmopoint
    3. 3. MOLECULAR BIOLOGY TECHNIQUETopic Outlines 1.1. Molecular biology technique 1.2. Technique purpose & basic procedure 1.2.1 Microscopic observation 1.2.2 Centrifugation 1.2.3 Extraction 1.2.4 Electrophoresis 1.2.5 Chromatography Slide 3 of 10 © 2010 Cosmopoint
    4. 4. MOLECULAR BIOLOGY TECHNIQUE1.1. Molecular biology techniqueIntroduction Molecular biology is the study of biology at a molecular level (eg. Replication, transcription & translation of the genetic material) Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interactions between DNA, RNA & protein biosynthesis & learning how these interactions are regulated 4 Slide 4 of 10
    5. 5. MOLECULAR BIOLOGY TECHNIQUE1.2. Technique purpose & basic procedureTechnique used in Molecular Biology Microscopic observation Centrifugation Extraction Electrophoresis Chromatography 5 Slide 5 of 10
    6. 6. MOLECULAR BIOLOGY TECHNIQUE1.2.1 Microscopic observation1. Microscopic Observation Microscope: instrument designed to produce magnified visual or photographic images of objects too small to be seen with the naked eye The microscope must accomplish three tasks: (a) produce a magnified image of the specimen (b) separate the details in the image (c) render the details visible to the human eye or camera Multiple-lens (compound microscopes) designs with objectives & condensers 6 Slide 6 of 10
    7. 7. MOLECULAR BIOLOGY TECHNIQUE1.2.1 Microscopic observation Some common types of microscopes which can be used in the study of cells are (a) Light (optical) microscopes (b) Phase contrast microscopes (c) Transmission electron microscope (d) Scanning electron microscope 7 Slide 7 of 10
    8. 8. MOLECULAR BIOLOGY TECHNIQUE1.2.1 Microscopic observationPhase contrast microscope Many cell details cannot be seen using an ordinary optical microscope. This is because there is very little contrast between structures. They have similar transparency & are not coloured Special phase contrast condensers & objective lenses are added to the light microscope. Light rays travelling through material of different densities are bent & altered giving a better contrast. 8 Slide 8 of 10
    9. 9. MOLECULAR BIOLOGY TECHNIQUE1.2.1 Microscopic observation Phase contrast microscopes enable living, non- pigmented specimen to be studied without fixing & staining This type of microscope give better contrast but do not improve resolution 9 Slide 9 of 10
    10. 10. MOLECULAR BIOLOGY TECHNIQUE1.2.1 Microscopic observationElectron microscope (EM) Uses an electron beam instead of light rays Electrons have short wavelengths ( ~ 0.0005 nm). This give a high resolving power to the EM which can resolve two objects that are only ~ 1 nm apart Electrons are negatively charged & can be focussed by the use of electromagnets in the EM. Magnification range from 15x to 200,000x There are two main types of EM: Transmission EM & Scanning EM 10 Slide 10 of 10
    11. 11. MOLECULAR BIOLOGY TECHNIQUE1.2.1 Microscopic observation Transmission electron microscope (TEM) - study the ultra-structure of a cell Scanning electron microscope (SEM) - produce 3-dimentional view of objects - eg. cells, tissue & small organism 11 Slide 11 of 10
    12. 12. MOLECULAR BIOLOGY TECHNIQUE 1.2.2 Centrifugation2. Centrifugation A piece of equipment, generally driven by a motor, that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle (separate substances or greater & lesser density) There are many different kinds of centrifuges, including those for very specialised purposes. 12 Slide 12 of 10
    13. 13. MOLECULAR BIOLOGY TECHNIQUE1.2.2 Centrifugation 13 Slide 13 of 10
    14. 14. MOLECULAR BIOLOGY TECHNIQUE 1.2.3 Extraction3. Extraction Molecules that can be extracted are: (a) DNA (b) RNA (c) protein DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis 14 Slide 14 of 10
    15. 15. MOLECULAR BIOLOGY TECHNIQUE 1.2.3 ExtractionDNA Extraction 15 Slide 15 of 10
    16. 16. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 Electrophoresis4. Electrophoresis Is a technique used to separate substances with different charges Eg. Proteins in an electric field Other mixture include amino acids & nucleic acid fragments especially DNA fragments for fingerprinting The medium used can be paper, gel layer or in a column 16 Slide 16 of 10
    17. 17. MOLECULAR BIOLOGY TECHNIQUE1.2.4 Electrophoresis Electrodes are placed on both end of wet paper or gel on a piece of glass In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel Proteins can be separated on the basis of size by running the DNA through an agarose gel Proteins can be separated on the basis of size by using an SDS-PAGE gel, or on the basis of size and their electric change by using what is known as a 2D gel electrophoresis 17 Slide 17 of 10
    18. 18. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 ElectrophoresisFunctions Very useful to separate proteins, as they are delicate. Enzymes separated by this technique are still active. To diagnose diseases as when blood plasma proteins are separated, extra proteins found could be antibodies (Ab) formed to combat certain pathogens. The Ab are compared with standard ones & extracted to determine the actual type For DNA-fingertyping, which is used to identify individual in forensic science 18 Slide 18 of 10
    19. 19. MOLECULAR BIOLOGY TECHNIQUE1.2.4 Electrophoresis Gel electrophoresis DML 202 General Biology & Human Genetics 11/16/2011 19 Slide 19 of 10 (Chapter 17: Molecular
    20. 20. MOLECULAR BIOLOGY TECHNIQUE1.2.4 Electrophoresis Principle of gel electrophoresis. Influence of charge and particle size on the electrophoretic mobility of proteins or other macromolecules like nucleic acids. A. Separation by charge, B. Separation by particle size, C: Addition of A and B, D: Compensation of A and B. 20 Slide 20 of 10
    21. 21. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 ElectrophoresisLimitations Only small amounts of substance can be separated Substances which are of no charge or too similar in charges cannot be separated 21 Slide 21 of 10
    22. 22. MOLECULAR BIOLOGY TECHNIQUE1.2.5. Chromatography5. Chromatography Is a technique used to separate mixtures of chemicals of similar nature (eg. photosynthesis pigments) by allowing their common solvent flowing over them in a solid medium such as paper Can separate other mixtures, which include proteins, amino acids, nucleic acids, nucleotides, fatty acid, monosaccharides & disaccharides The solid media are paper, gel layer or column of cellulose & achrimide polymer 22 Slide 22 of 10
    23. 23. MOLECULAR BIOLOGY TECHNIQUE1.2.5. ChromatographyTypes of Chromatography Paper chromatography Two dimensional paper chromatography Thin-layered chromatography Column chromatography 23 Slide 23 of 10
    24. 24. MOLECULAR BIOLOGY TECHNIQUE1.2.5. ChromatographyA. Paper chromatography  Initially, a small but concentrated amount of mixture e.g leaf extract is applied on one end of the paper. The paper is hung on a common solvent such as petroleum ether.  When the solvent goes up, the solute will separate as indicated by the different coloured spots 24 Slide 24 of 10
    25. 25. MOLECULAR BIOLOGY TECHNIQUE1.2.5. Chromatography 25 Slide 25 of 10
    26. 26. MOLECULAR BIOLOGY TECHNIQUE1.2.5. Chromatography B. Two dimensional paper chromatography Can be done in 2 dimensions with a square piece of paper when there are too many solutes in the mixture. Is done1st with one solvent then the paper is turned 90° to be separated with another solvent giving a better separation such as with a mixture of amino acids 26 Slide 26 of 10
    27. 27. MOLECULAR BIOLOGY TECHNIQUE1.2.5. Chromatography 27 Slide 27 of 10
    28. 28. MOLECULAR BIOLOGY TECHNIQUE1.2.5. ChromatographyPrinciple It involves passing a mixture dissolved in a “mobile phase” through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated. Methods used to separate and/or to analyze complex mixtures based on differences in their structure and/or composition The components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed 28 Slide 28 of 10
    29. 29. MOLECULAR BIOLOGY TECHNIQUE1.2.5. Chromatography Test molecules which display tighter interactions with the support will tend to move more slowly through the support than those molecules with weaker interactions Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography Repeated sorption/desorption acts that take place during the movement of the sample over the stationary bed determine the rates. The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column 29 Slide 29 of 10
    30. 30. MOLECULAR BIOLOGY TECHNIQUE1.2.5. ChromatographyRf Important character of the solute in a certain solvent The ratio of the distance moved by the solute to that moved by the solvent Distance moved by the solute Rf = Distance moved by the solvent 30 Slide 30 of 10
    31. 31. MOLECULAR BIOLOGY TECHNIQUE1.2.5. Chromatography Rf is a constant used to determine the position of an unknown solute if the Rf under the same condition is known Used to identify an unknown spot in the chromatogram 31 Slide 31 of 10
    32. 32. MOLECULAR BIOLOGY TECHNIQUE1.2.5. ChromatographyFunctions The technique is simple & can be easily carried out to separate chemicals of similar nature. It takes a short time to carry out. A simple separation of leaf pigments only takes less than 30 minutes It requires only simple apparatus such as paper and dropper to apply the mixture on the paper 32 Slide 32 of 10
    33. 33. MOLECULAR BIOLOGY TECHNIQUE1.2.5. Chromatography Limitations Only small amounts of substances can be separated at one time When the solutes are too similar like certain amino acids, they are not separable by this technique 33 Slide 33 of 10
    34. 34. MOLECULAR BIOLOGY TECHNIQUE Topic Slide 34 of 10