Chapt 10
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This document discusses molecular biology techniques. It begins by outlining the learning objectives, which are to list techniques used in molecular biology and describe microscopy, centrifugation, extraction, electrophoresis, and chromatography. It then provides details on each technique, including their purposes and basic procedures. Microscopy is used to produce magnified images of cells and structures. Centrifugation separates substances by density. Extraction isolates molecules like DNA, RNA, and proteins. Electrophoresis separates charged substances using an electric field. Chromatography separates mixtures based on interactions with a solid or liquid medium.
Chapt 10
- 1. GENERAL BIOLOGY HDL 121 MOLECULAR BIOLOGY TECHNIQUE PREPARED BY:MANEGA SCHOOL OF MLT FACULTY OF HEALTH SCIENCE
- 2. MOLECULAR BIOLOGY TECHNIQUE Learning 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. MOLECULAR BIOLOGY TECHNIQUE Topic 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. MOLECULAR BIOLOGY TECHNIQUE 1.1. Molecular biology technique Introduction 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. MOLECULAR BIOLOGY TECHNIQUE 1.2. Technique purpose & basic procedure Technique used in Molecular Biology Microscopic observation Centrifugation Extraction Electrophoresis Chromatography 5 Slide 5 of 10
- 6. MOLECULAR BIOLOGY TECHNIQUE 1.2.1 Microscopic observation 1. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.1 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.1 Microscopic observation Phase 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.1 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.1 Microscopic observation Electron 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.1 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.2 Centrifugation 2. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.2 Centrifugation 13 Slide 13 of 10
- 14. MOLECULAR BIOLOGY TECHNIQUE 1.2.3 Extraction 3. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.3 Extraction DNA Extraction 15 Slide 15 of 10
- 16. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 Electrophoresis 4. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 Electrophoresis Functions 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 Electrophoresis Gel electrophoresis DML 202 General Biology & Human Genetics 11/16/2011 19 Slide 19 of 10 (Chapter 17: Molecular
- 20. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.4 Electrophoresis Limitations 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. Chromatography 5. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. Chromatography Types of Chromatography Paper chromatography Two dimensional paper chromatography Thin-layered chromatography Column chromatography 23 Slide 23 of 10
- 24. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. Chromatography A. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. Chromatography 25 Slide 25 of 10
- 26. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. Chromatography 27 Slide 27 of 10
- 28. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. Chromatography Principle 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. Chromatography Rf 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. Chromatography Functions 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. MOLECULAR BIOLOGY TECHNIQUE 1.2.5. 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. MOLECULAR BIOLOGY TECHNIQUE Topic Slide 34 of 10