ANDC DU/Biology Protocols/DNA Spooling

Isolation of Genomic DNA from Plant Tissue (Cauliflower) by Spooling Method

Introduction to DNA

Deoxyribonucleic Acid (DNA) is a genetic material which contains all the information or genetic instructions required for metabolism, reproduction and hereditary characters of an organism. DNA is often compared to a set of blueprints or a recipe, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.

Chemically, DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.

Within cells, DNA is organized into structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms (animals, plants, and fungi) store their double-stranded DNA inside the cell nucleus, in mitochondria or chloroplasts. The DNA in the nucleus is linear, whereas the DNA in mitochondria and chloroplasts is circular. In prokaryotes (bacteria and archaea), however, DNA is found in the cell's cytoplasm and is called nucleoid. It is circular and is relatively free of associated protein, but the DNA in the nucleus of eukaryotes is associated with basic proteins, called histones. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.

DNA was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it nuclein and later on nucleic acid since the substance isolated was acidic in nature. This discovery of DNA paved the way for many scientists whose work contributed to the understanding of DNA.

In 1920s P.A. Levene found that DNA molecule contain three main components:

• Five carbon sugar - Deoxy ribose in DNA - Ribose in RNA • Phosphate group • Nitrogenous base - Purines: Adenine and Guanine - Pyrimidines: Cytosine, Thiamine and Uracil

He concluded that DNA molecule is a polymer and each unit is made up of above mentioned components which he called nucleotides. Later on Chargaff showed that four nucleotides are not present in equal proportions in DNA but there is always an equal proportion of purines and pyrimidines.

In 1937 William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure. In 1928, Frederick Griffith discovered that DNA carries genetic information, when Oswald Avery, along with co-workers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle in 1943. DNA's role in heredity was confirmed in 1952, when Alfred Hershey and Martha Chase in the Hershey-Chase experiment showed that DNA is the genetic material of the T2 phage.

In 1953 Rosalind Franklin carried out X-ray diffraction of DNA molecule which suggested that DNA molecule either helical or corkscrew in shape. Finally in 1953, James Watson and Francis Crick gave the most acceptable model of DNA structure.

According to this model:

• DNA does not exist as a single strand but as two chains of polynucleotide spiralling tightly around an imaginary axis to form a double helix.

• The double strands run anti-parallel to each other and are held together by hydrogen bonds.

• Each spiral of DNA molecule has 10 nucleotides and is 34 Å long. The distance between two nucleotides is 3.4 Å.

• The diameter of a DNA molecule is 20 Å and is constant.

• Each nucleotide consists of a nucleoside and a phosphate group. Each nucleoside, in turn, consists of a sugar molecule and a nitrogenous base.

• Sugar present in the DNA molecule is deoxyribose and the bases are of two kinds-pyrimidines and purines.

• Deoxyribose sugar-phosphate form the external backbone of the helix, and the four different nitrogenous bases are paired in the interior of the helix by hydrogen bonding.

• Pyrimidine bases are Thymine (T) and Cytosine (C), while purines are Adenine (A) and Guanine (G).

• Pyrimidines always pair with purines, viz. A pairs with T by double hydrogen bonds and G pairs with C by triple hydrogen bonds.

• The total molar amount of pyrimidines and purines is always equal, i.e., A+G=T+C.

Simultaneously, Maurice Wilkins and his colleagues also worked on DNA structure and reported the same. In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine. Later, Chargaff showed that four nucleotides are not present in equal proportions in DNA but there are always an equal proportion of purines and pyrimidines.

DNA is found both in prokaryotes and eukaryotes. In prokaryotes, DNA is double-stranded and circular and is found throughout the cytoplasm and is called nucleoid. In eukaryotes, DNA is located in the nucleus and in mitochondria or chloroplasts. The DNA in the nucleus is double-stranded and linear, whereas the DNA in mitochondria and chloroplasts is like prokaryotic DNA, double-stranded and circular. The DNA in prokaryotes is relatively free of associated protein, but the DNA in the nucleus of eukaryotes is associated with basic proteins, called histones. Now that the structure of DNA has been studied for over 100 years and has basically been accepted, procedures have been devised to isolate almost pure DNA from its other components.

Principle of DNA Isolation

The isolation of DNA is one of the most commonly used procedures in many areas of bacterial genetics, molecular biology and biochemistry. Purified DNA is required for many applications such as studying DNA structure and chemistry, examining DNA-protein interactions, carrying out DNA hybridizations, sequencing or PCR, performing various genetic studies or gene cloning. The isolation of DNA from bacteria is a relatively simple process. The organism to be used should be grown in a favorable medium at an optimal temperature, and should be harvested in late log to early stationary phase for maximum yield

There are several basic steps in DNA extraction. The five steps are as follows:

1. Homogenization or disruption of cells: The cell must first be lysed (broken open) to release the nucleus in eukaryotes or nuleoid in prokaryotes. Cells are broken by grinding, tissue homogenization, or treatment with Iysozyme.

2. Inhibition of DNAase: At this point the DNA must be protected from enzymes that will degrade it, causing shearing. Many of the nucleases present in cells can digest nucleic acids. When the cell is disrupted, the nucleases can cause extensive hydrolysis. Nucleases apparently present on human fingertips are notorious for causing spurious degradation of nucleic acids during purification. Chelating agents are added to remove metal ions required for nuclease activity.

3. Dissociation of nucleoprotein complexes: DNA-protein interactions are disrupted with SDS, phenol, or broad spectrum proteolytic enzymes as pronase or proteinase K. Alkaline pH and high concentration of salts improve the efficiency of the process.

4. Removal of contaminating materials: Contaminating molecules especially proteins are removed by treatment with phenol or chloroform-isoamyl alcohol or phenol chloroform. Proteins can also be removed by salting out proteins by sodium acetate.

5. Precipitation of DNA: Once the DNA is released, it must be precipitated in alcohol. The DNA in the aqueous phase is precipitated with cold (0oC) ethanol. The precipitate is usually redissolved in buffer and treated with phenol or organic solvent to remove the last traces of protein, followed by reprecipitation with cold ethanol. RNA is removed by limited treatment with deoxyribonuclease-free ribonuclease.

Aim

Isolation of Genomic DNA from plant tissue (Cauliflower) by spooling method.

Objectives

The goal of this exercise is to demonstrate the isolation of DNA from living tissues. The students will be able to: • isolate DNA from cauliflower tissue. • make observations regarding the results of the isolation. • develop a hypothesis from their observations regarding the nature of the molecule. • design an investigation to test their hypothesis.

Introduction

This laboratory protocol will demonstrate several basic steps required for isolation of chromosomal DNA from cells. To extract the chromosomal DNA, both the cell membrane and the nuclear membrane must be lysed, or broken open. This is accomplished by disrupting the membranes with a solution of detergent and salt, creating a cell homogenate. Once the DNA is released from the nucleus, it must be protected from nucleases, enzymes which will degrade the DNA. Keeping the cell homogenate cold and various chemical components of the homogenization medium help restrict the action of these nucleases. The final step of this protocol involves precipitation of DNA from the homogenate. When the homogenization medium is added in the first step, the positive ions of the salts (sodium chloride and sodium citrate) bind to the negatively charged DNA backbone, creating a DNA molecule with a neutral charge. When a cold polar solvent, like ethanol, is added to the DNA solution, the DNA is precipitated out of solution, leaving other cell components behind (proteins, lipids, polysaccharides, general cell debris).

DNA can be isolated and precipitated using tissue from cauliflower. The cells are disrupted by manually homgenizingin homogenization medium. Ice cold ethanol/isopropanol is added to this homogenate to precipitate the DNA. The DNA can then be isolated by slowly stirring a glass stirring rod in the homogenate, literally spooling the DNA onto the glass rod.

Requirements

Biological

Cauliflower, kept at -70 ^oC for at least 3-4 hrs

Chemicals

• SDS extraction Buffer (100ml):

-1M Tris, pH 7.210.0 ml

-0.5 M EDTA04.0 ml

-3 M NaCl16.7 ml

-10% SDS17.0 ml

-Raise the volume to 100 ml with autoclaved water.

• 5M potassium acetate
• Chloroform
• Isoamyl alcohol
• Isopropanol or ethanol

Glassware/Plasticware

All glassware and plastic ware used should be sterilized

• Conical flasks (100 ml)
• Petri plates
• Eppendorffs
• Microtips
• Pipettes

Instruments/ Equipment

• pH meter
• Weighing balance
• Centrifuge
• Micropipettes
• Mortar and pestle

Time Required

This lab generally requires more than one lab period. It is suggested that the solutions should be prepared in advance and stored in refrigerator, and have students begin the procedure with the homogenization process. If time is used efficiently, the remainder of the lab can be completed within a 70- 90 minute period.

Procedure

• Take the 5 g of frozen sample.
• Grind the tissue completely using mortar and pestle.
• Put the homogenate in10 ml of preheated (65^oC) extraction buffer for 30 minutes.
• Add 5 ml of potassium acetate and invert mix for 5 minutes.
• Add equal volume of (25 ml) chloroform : isoamyl alcohol (24:1)and invert mix for 5 minutes.
• Centrifuge at 3000 rpm for 20 minutes at room temperature.
• Collect the supernatant and repeat the extraction step once more.
• To the final supernatant add equal volume of chilled isopropanol and mix gently by inversion.
• Spool out the DNA by glass rod and dissolve the DNA in 5-10 ml of TE buffer.

Result

Discussion

Nucleic acids are the most polar of the biopolymers and are therefore soluble in polar solvents and precipitated by non-polar solvents. The extracted DNA was dissolved in TE buffer. The quality of DNA was judged by the electrophoresis. On electrophoresis one band was observed near the well which clearly indicates that the band is of high molecular weight and thus it’s a genomic DNA. Since there only one band is seen on the gel, so no shearing of DNA has taken place.

Precautions

• Use caution when operating the centrifuge.
• Dispose of used reagents according to local ordinances.
• Wear latex gloves and goggles during the procedure.
• The laboratory surfaces should be very clean during all procedures used in this activity.
• Use thoroughly clean instruments and glassware. Rinse all equipment with isopropyl alcohol or acetone.
• Ethanol is highly flammable; use caution.

Question

1. Why is it important to keep the cell homogenate and the solutions cold? 2. The homogenizing solution contains high concentrations of liquid detergent and salt. What does the detergent do to the cells? Why is it important to have a lot of salt in the solution? 3. What do you know about the DNA molecule that makes it possible to spool it out of the homogenate? Why don’t other molecules in the homogenate do this? 4. Can you think of a way to prove that the molecule you have isolated is DNA? Design an experiment to show this.

Work in progress, expect frequent changes. Help and feedback is welcome. See discussion page.