Molecular Genetics

M.Mubashar ali

Roll# 01

BS zoology

6th Semester

+923026243208

Preparation and isolation of genomic

DNA

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CONTENTS

Introduction

Apparatus

Chemical

Procedure (Steps)

1. Growing and harvesting a bacterial culture

2. Preparation of a cell extract

3. Purification of DNA from a cell extract

4. Concentration of DNA samples

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INTRODUCTION

Bacteria are prokaryotic organisms having DNAwithout any membrane bounded nucleus ,their DNAis circular and present in cytoplasm .This DNA iscalled Genomic DNA

Genomic DNA will often be required as a source ofmaterial from which to obtain genes to be cloned.

Genomic DNA may be obtain from a culture ofbacteria , from a plant, from animal cells.

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ApparatusTest tubes

Petri dish

Wire loop

Autoclave

Beaker

Conical flask

Physical balance

Stirring rod

Centrifuge

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CHEMICALSFor culturing of bacteria:

Nutrient agar ,Nutrient broth( M9 medium and Luria-Bertani (LB) medium.

For isolation:By lysozyme, 50ml ethylenediamine tetra acetate(EDTA), or a combination of both.Sodium dodecyl sulphate (SDS)1 : 1 phenol( pH 8.0) and chloroformProteinase K enzymeRibonuclease enzymeChromatographic matrix or resinMonovalent cations such as sodium ionsEthanol

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PROCEDURE

The procedure for genomic DNA preparation from aculture of bacterial cells can be divided into fourstages(Figure 1):

1. Growing and harvesting a bacterial culture

2. Preparation of a cell extract

3. Purification of DNA from a cell extract

4. Concentration of DNA samples

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Fig 1: The basic steps in preparation of total cell DNA from a culture of bacteria.

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STEPS1.Growing and harvesting a bacterial culture

Culture medium must provide a balanced mixture of theessential nutrients at concentrations that will allow thebacteria to grow and divide efficiently

Two type of liquid mediums used for growing

M9 mediumdefined medium in which all the components are known.It provide essential elements such as nitrogen, magnesium,and calcium, as well as glucose to supply carbon andenergy.Have growth factors such as trace elements and vitamins.The bacterial culture has to be grown under preciselycontrolled conditions.

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Luria-Bertani (LB) mediumUndefined medium, meaning that the preciseidentity and quantity of its components are notknown.

Contain tryptone and yeast extract, are complicatedmixtures of unknown chemical compounds.

Tryptone supplies amino acids and small peptides.

Yeast extract (a dried preparation of partially digestedyeast cells) provides the nitrogen requirements, alongwith sugars and inorganic and organic nutrients.

LB need no further supplementation and support thegrowth of a wide range of bacterial species.

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The composition of two typical media for the growth of bacterial cultures.

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Harvesting a bacterial culturePerformed by spinning the culture in a centrifuge

Fairly low centrifugation speeds will pellet the bacteriaat the bottom

Bacteria from a 1000 ml re suspended into a volume of

10 ml or less.

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2.Preparation of a cell extractThe bacterial cell is enclosed in a cytoplasmicmembrane and surrounded by a rigid cell wall

cell lysis is brought about by exposure to chemicalagents

involves one agent attacking the cell wall and anotherdisrupting the cell membrane

brought about by lysozyme, ethylenediaminetetraacetate (EDTA), or a combination of both.

Lysozyme is present in egg white and in secretionssuch as tears and saliva, and which digests thepolymeric compounds that give the cell wall itsrigidity.

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EDTA (Ethylene Diamine tetra accetic acid)a chelatingagent removes magnesium ions that are essential forpreserving the overall structure of the cell envelope,and also inhibits cellular enzymes that could degradeDNA.

Detergent such as sodium dodecyl sulphate (SDS) isalso added it aid the process of lysis by removing lipidmolecules and thereby cause disruption of the cellmembranes

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the final step in preparation of a cell extract is removalof insoluble cell debris

cell extract can be pelleted by centrifugation leavingthe cell extract as a reasonably clear supernatant.

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3.Purification of DNA from a cell extract

In addition to DNA, a bacterial cell extract containssignificant quantities of protein and RNA and shouldbe removed

Two methods are used for removing protein and RNA

Removing contaminants by organicextraction and enzyme digestion

to deproteinize a cell extract is to add phenol or a 1 : 1mixture of phenol and chloroform

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The layers then separated by centrifugation,precipitated protein molecules are left as a whitecoagulated mass at the interface between theaqueous and organic layers

The aqueous solution of nucleic acids can then beremoved with a pipette.

Several phenol extractions one after the otherbecause protein content is so great that a single phenolextraction is not sufficient to completely purify thenucleic acids

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It results in a certain amount of breakage of the DNAmolecules

the cell extract treated with a protease such aspronase or proteinase K before phenol extraction.

This enzymes break polypeptides down into smallerunits, which are more easily removed by phenol

Effective way to remove the RNA is with the enzymeribonuclease, which rapidly degrades thesemolecules into ribonucleotide subunits.

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Removal of protein contaminants by phenol extraction.

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Using ion-exchange chromatography to purify DNA from a cell extractSeparates molecules according to how tightly theybind to electrically charged particles present in achromatographic matrix or resin

DNA and RNA are both negatively charged, as aresome proteins, and so bind to a positively chargedresin

The electrical attachment is disrupted by salt, removalof the more tightly bound molecules requiring higherconcentrations of salt

The resin in a glass or plastic column and then add thecell extract to the top

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The extract passes through the column, and becausethis extract contains very little salt all the negativelycharged molecules bind to the resin

If a salt solution of gradually increasing concentrationis now passed through the column, the different typesof molecule will become unbound in the sequenceprotein, RNA, and finally DNA.

Different salt solutions are used to unbound theprotein and RNA, leaving just the DNA bound,followed by a second of a higher concentration whichunbound the DNA, now free from protein and RNAcontaminants.

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4.Concentration of DNA samplesIt is important to increasing the DNA concentrationfrequently used method of concentration is ethanolprecipitationIn the presence of salt of monovalent cations such assodium ions and at a temperature of −20°C or less,absolute ethanol efficiently precipitates polymeric nucleicacidsEthanol precipitation has the added advantage of leavingshort-chain and monomeric nucleic acid components insolutionA glass rod pushed through the ethanol into the DNAsolution. When the rod is removed,DNA molecules adhereand can be pulled out of the solution in the form of a longfiberThe precipitate can be collected by centrifugation

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Collecting DNA by ethanol precipitation. (a) Absolute ethanol is layered on top of a concentrated solution of DNA. Fibers of DNA can be withdrawn with a glass rod. (b) For less concentrated solutions ethanol is added (at a ratio of 2.5 volumes of absolute ethanol to 1 volume of DNA solution) and precipitated DNA collected by centrifugation.

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THE END

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