Chapt 10

GENERAL BIOLOGY

SCHOOL OF MLTFACULTY OF HEALTH SCIENCE

PREPARED BY:MANEGA

HDL 121MOLECULAR BIOLOGY

TECHNIQUE

MOLECULAR BIOLOGY TECHNIQUE

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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

Topics

© 2010 Cosmopoint


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Topic Outlines

1.1. Molecular biology technique

1.2. Technique purpose & basic procedure1.2.1 Microscopic observation1.2.2 Centrifugation1.2.3 Extraction1.2.4 Electrophoresis1.2.5 Chromatography

© 2010 Cosmopoint


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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

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1.1. Molecular biology technique


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Technique used in Molecular BiologyMicroscopic observationCentrifugationExtractionElectrophoresisChromatography

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1.2. Technique purpose & basic procedure


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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 cameraMultiple-lens (compound microscopes) designs with

objectives & condensers

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1.2.1 Microscopic observation


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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

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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.

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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

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Electron microscope (EM)

Uses an electron beam instead of light raysElectrons 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 200,000xThere are two main types of EM: Transmission EM &

Scanning EM

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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

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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.

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1.2.2 Centrifugation


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1.2.2 Centrifugation


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3. Extraction

Molecules that can be extracted are:

(a) DNA

(b) RNA

(c) proteinDNA extraction is a routine procedure to collect DNA for

subsequent molecular or forensic analysis

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1.2.3 Extraction


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DNA Extraction

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1.2.3 Extraction


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4. Electrophoresis

Is a technique used to separate substances with different charges

Eg. Proteins in an electric fieldOther mixture include amino acids & nucleic acid

fragments especially DNA fragments for fingerprintingThe medium used can be paper, gel layer or in a

column

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1.2.4 Electrophoresis


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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

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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

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Gel electrophoresis

04/10/2023

DML 202 General Biology & Human Genetics

(Chapter 17: Molecular Biology Technique)

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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.

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Limitations

Only small amounts of substance can be separatedSubstances which are of no charge or too similar in

charges cannot be separated

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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

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1.2.5. Chromatography


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Types of Chromatography

Paper chromatographyTwo dimensional paper chromatographyThin-layered chromatographyColumn chromatography

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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

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B. Two dimensional paper chromatographyCan 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

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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

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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

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Rf Important character of the solute in a certain solventThe ratio of the distance moved by the solute to that

moved by the solvent

Distance moved by the soluteRf =

Distance moved by the solvent

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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

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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

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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

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THANK YOU

Chapt 10

Technology

Transcript of Chapt 10