PREFACE

The project entitled Extraction of DNA from fruits was undertaken to gain information and knowledge regarding isolation of DNA. The report presented here is focused on properties of DNA and its structure. The text is supplemented with photographs.

ACKNOWLEDGEMENT
I wish to express our sense of appreciation and indebtedness to our guiding team who offered encouragement and cooperation. First of all I would like to thank our principal Prof. Sandhya Singh Kaushik for giving us such a good platform. I express my sincere gratitude to our honourable Prof. Uma Jaiswal for her keen interest in our project guidance and constant encouragement. Her profound experience and supportive nature was of great help. I am also thankful to incharge of botany section Prof. Madhubala Tyagi for providing necessary facilities to conduct the project.

CONTENTS

Objective Introduction Requirements Procedure Observation and conclusion Reference

Aim:To extract DNA from fruits.

Introduction: A DNA molecule is composed of two polynucleotide chains that are coiled around each other to form a rigid double helix. The double helical structure of DNA is very stable at room temperature because the hydrogen and hydrophobic bonds between the stacked bases hold the two polynucleotide chains together. However, if a solution of DNA is heated to a critical temperature, these bonds are broken and the two polynucleotide strands separate by a process called denaturation. DNA denaturation is accompanied by a decrease in the viscosity of the solution because single stranded DNA molecules form flexible coiled structures that no longer retain the rigid native structure of the DNA double helix. If the DNA is cooled rapidly, the molecules will remain as single stranded polynucleotides. However, if the solution is cooled very slowly, restoration of DNA helix will occur. The reassembly of the two separated polynucleotide strands is called renaturation. Macromolecules such as DNA, RNA and protein are not soluble in alcohol solution and precipitate upon addition of alcohol. In general globular proteins and RNA form fine, nonfibrous precipitate in alcohol. In contrast the rod like DNA molecules precipitate in alcohol as long fibers that can be spooled on to a glass rod. The ability of DNA to form fibers in alcohol depends on the physical properties of the DNA molecules. For example, DNA that has been broken into small pieces by DNAse I digestion will not form fibers, nor will single stranded DNA that has been prepared by heat denaturation of the DNA double helix. Isolated DNA in a test tube is also a long, stiff molecule. When alcohol is added to a DNA solution, the fibers precipitate and can be spooled on to a glass rod. DNA is an extremely long molecule that is very thin yet quite rigid. The isolation of intact DNA molecules from a cell is difficult because of the relative ease with which these long rod-like molecules can be broken. Even the injection of a solution of DNA through the needle of a hypodermic syringe can cause extensive break down of DNA molecules. Requirements: Plant material: Banana and apple. Glassware: Test tubes, beakers ( 250 cc ,100 cc, 50 cc), measuring cylinders (10 ml), glass rod, slides and cover slips Chemicals: 95% denatured ethyl alcohol, table salt, water Others: Absorbent paper tissues, liquid detergent, sieve, ice cubes, microscope

Procedure: The procedure described below exploits the fact that the external membrane of cells and that of their nuclei are composed of fatty substances that can be broken down using a simple detergent. The first operation in this procedure is to breakup the fruit into a pulp or mush so that the cells are separated each from other as much as possible thereby exposing them to the action of the detergent. Secondly, the detergent is added to the pulp of the fruit so as to release the DNA from the cell membranes, which encapsulate it. Thirdly, it is necessary to filter the mixture to separate the nucleic acids from the remains of the cellular membrane. Finally, the DNA is precipitated in the alcohol where it becomes visible. The DNA you obtain using this procedure can be observed with a microscope. Preliminary Operations The day before the experiment, prepared some ice cubes. Atleast two hours before, placed in a freezer a sealed vapor-tight plastic bottle with 100cc of 95% ethyl alcohol. This container was closed tightly to prevent alcohol vapors from being released since they are flammable. 15 minutes before starting the procedure warmed a pot of tap-water to Preparation of the Extraction Solution As mentioned previously, the DNA is held inside the nucleus of the cells of the fruit we are using. To free the DNA, it will be necessary to breakdown the membranes of the cells as well as those of the nuclei. As these membranes are made up of phospholipids, which are molecules rich in fats, we dissolved them by using a simple household detergent. We also used a little table salt, which helps to eliminate the protein, called histones, on which the DNA is wrapped. Poured 3 g of salt and 80cc of hot tap water in a 100cc beaker. Mixed until the salt was completely dissolved. With the pipette, took 10 cc of liquid detergent and added it to the solution. Added tap water to obtain a total volume of 100cc. While avoiding to produce bubbles, mixed to homogenize the solution. The extracting solution was ready. c

Preparation of the Pulp This operation serves to separate the cells from each other and to expose them to the action of the extraction solution. Placed 100g of banana/apple on a chopping board and crushed it to obtain a pulp. Poured the pulp in a 250cc beaker.

Extracting the DNA The aim of this operation is to breakdown the membranes of the cells and their nuclei to free the DNA Poured the extracting solution on the pulp. Mixed the pulp so as to distribute the extracting solution and to make the temperature uniform. After 15 minutes, placed the beaker in water with ice-cubes. Mix the pulp to make the temperature uniform. After 5 minutes, removed the beaker from the cold water and prepared to filter. Filtration The filtration process is used to collect the liquid rich in DNA and to separate it from the cellular remnants and the other tissues of the fruit, which will be discarded. Placed the sieve on the bowl. Took a filter paper, soaked it and placed it in the sieve. Poured a little pulp on the filter, taking care that it goes through the filter paper. Mixed with care to help the filtration and avoided ripping the filter paper.

Precipitating the DNA DNA is quite soluble in water and invisible, while it is insoluble in alcohol wherein it precipitates and become visible. By adding alcohol to the DNA filtrate solution in the tube, the DNA is rendered visible. Slowly, poured in the tube of the previous step some icy alcohol by avoiding it mix with the filtrate. The volume of the alcohol has to be about the same of that of the solution. Let the tube rest for 5 minutes to allow to the DNA to precipitate and accumulate in the tube.

Observation and conclusion At the interface between alcohol and the filtrate a milky substance, which increased in volume as time progressed was observed. This milky substance was the DNA of the banana.

REFERENCES
BOOKS REFERRED:

Lehninger,E.2008
Principles of biochemistry(fifth edition). Strayer, Text book of biochemistry.

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