ANDC DU/Biology Protocols/Genomic DNA

Introduction to DNA
Deoxyribose Nucleic Acid (DNA). is very long and contains the genetic code needed to direct a cell’s activities. DNA was first isolated by Friedrich Meischer in 1869. He extracted a substance from the cells’ nuclei that he called 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
 * 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 1953 Rosalind Franklin carried out x rays diffraction of DNA molecule which suggested that DNA molecule either helical or corkscrew in shape

Finally Watson and 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 nucleotides spiralling tightly around an imaginary axis to form a double helix. Deoxyribose sugar-phosphate backbones are on the outside of the helix, and the four different nitrogen bases are paired in the interior of the helix. Double strands are held together by hydrogen bonds between the complementary bases. There is a crucial relationship between the two strands: where there is adenine (A) on one strand, it can only pair with thymine (T) on the other strand, and guanine (G) always pairs with cytosine (C). There is a complementarity between the two strands.

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

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

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.

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.

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.

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.

= Isolation of Genomic DNA from E. coli. =

Requirements
Biological

Overnight grown culture of E.coli.

Chemicals

A. Luria Broth medium:

Tryptone1.0g

Yeast extract0.5g

Nacl0.5g

Glucose0.1g

Dissolve the components in 75ml of distilled water, adjust the pH to 7.6 and finally make the volume to 100ml. Sterilize by autoclaving

B. GTE mix:

50mM Glucose

50mM Tris

10mM EDTA

Sterilize by autoclaving

C. 10mM NaCl

Sterilize by autoclaving

D. 10% SDS pH 7.2:

Dissolve 10g of SDS in 80 ml of distilled water adjust pH to 7.2. Raise the volume to 100ml.

Do not Autoclave.

Glassware/Plastic ware

All glassware and plastic ware used should be sterilized


 * Conical flasks 100 ml
 * Petri plates
 * Eppendroffs
 * Micro tips
 * Pipettes

Instruments/ equipment


 * Orbital shaker
 * pH meter
 * Weighing balance
 * Centrifuge
 * Micropipettes

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 centrifuging process. If time is used efficiently, the remainder of the lab can be completed within a 70-minute period.

Procedure

 * Take 1.5 ml of overnight grown culture of E.coli in microfuge tube.
 * Centrifuge at 10,000 rpm for 5 minutes to obtain pellet
 * Discard the supernatant and add 200 µl of GTE mix. Break the pellet.
 * Incubate at room temperature for 5 minutes.
 * Add 400 µl of 1% SDS and keep on ice for 5 minutes.
 * Add 60 µl of 10mM NaCl and 700 ul of isopropanol
 * Invert mix.
 * Centrifuge at 10,000 rpm for 20 minutes.
 * Decant the alcohol and air dry.
 * Dissolve in 50 µl of TE buffer.
 * Centrifuge at 10,000 rpm for 5 minutes
 * Take the supernatant in a fresh microfuge tube and subject it to gel electrophoresis.

Discussion
Nucleic acids are the most polar of the biopolymers and are therefore soluble in polar solvents and precipitated by nonpolar 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 indicate 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.
 * 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.