Dna Extraction

Objective : To extract the DNA from onion samples.
Introduction :
Nuclues has DNA molecule in it. It is packaged into thread like structure known as chromosomes. Each and every chromosome is made up of DNA tightly coiled many times around proteins which is known as histones. Histones supports the structure. Chromosome cannot be visible even under the microscope if the cells are not dividing.

DNA consist of 2 long polynucleotide which is composed of 4 types of nucleotide units. Nucleotides are composed of 5 carbon sugar attached to the phosphate group and a nitrogen containing base. The base might be Adenine,Cytosine,Guanine,or Thymine. Nucleotides are linked together by covalent bond through the sugar and phosphate. The 3d structure of DNA is the double helix. It is formed from the 2 polynucleotide chains. Both chains are held together by hydrogen bonding.
Genes carry biological information that must be copied accurately for transmission to the next generation each time a cell divides to form two daughter cells. Two central biological questions arise from these requirements: how can the information for specifying an organism be carried in chemical form, and how is it accurately copied? The discovery of the structure of the DNA double helix was a landmark in twentieth-century biology because it immediately suggested answers to both questions, thereby resolving at the molecular level the problem of heredity. We discuss briefly the answers to these questions in this section, and we shall examine them in more detail in subsequent chapters.

DNA encodes information through the order, or sequence, of the nucleotides along each strand. Each base—A, C, T, or G—can be considered as a letter in a four-letter alphabet that spells out biological messages in the chemical structure of the DNA. Organisms differ from one another because their respective DNA molecules have different nucleotide sequences and, consequently, carry different biological messages.

As discussed above, it was known well before the structure of DNA was determined that genes contain the instructions for producing proteins. The DNA messages must therefore somehow encode proteins . This relationship immediately makes the problem easier to understand, because of the chemical character of proteins. The properties of a protein, which are responsible for its biological function, are determined by its three-dimensional structure, and its structure is determined in turn by the linear sequence of the amino acids of which it is composed.

The linear sequence of nucleotides in a gene must therefore somehow spell out the linear sequence of amino acids in a protein. The exact correspondence between the four-letter nucleotide alphabet of DNA and the twenty-letter amino acid alphabet of proteins—the genetic code—is not obvious from the DNA structure, and it took over a decade after the discovery of the double helix before it was worked out. This code in detail in the course of elaborating the process, known as gene expression, through which a cell translates the nucleotide sequence of a gene into the amino acid sequence of a protein.

Transcription is the process by which DNA is copied (transcribed) to mRNA, which carries the information needed for protein synthesis. Transcription takes place in two broad steps. First, pre-messenger RNA is formed, with the involvement of RNA polymerase enzymes. The process relies on Watson-Crick base pairing, and the resultant single strand of RNA is the reverse-complement of the original DNA sequence. The pre-messenger RNA is then "edited" to produce the desired mRNA molecule in a process called RNA splicing. The mechanism of transcription has parallels in that of DNA replication. As with DNA replication, partial unwinding of the double helix must occur before transcription can take place, and it is the RNA polymerase enzymes that catalyze this process.

Unlike DNA replication, in which both strands are copied, only one strand is transcribed. The strand that contains the gene is called the sense strand, while the complementary strand is the antisense strand. The mRNA produced in transcription is a copy of the sense strand, but it is the antisense strand that is transcribed.
Ribonucleotide triphosphates (NTPs) align along the antisense DNA strand, with Watson-Crick base pairing (A pairs with U). RNA polymerase joins the ribonucleotides together to form a pre-messenger RNA molecule that is complementary to a region of the antisense DNA strand. Transcription ends when the RNA polymerase enzyme reaches a triplet of bases that is read as a "stop" signal. The DNA molecule re-winds to re-form the double helix.
The mRNA formed in transcription is transported out of the nucleus, into the cytoplasm, to the ribosome (the cell's protein synthesis factory). Here, it directs protein synthesis. Messenger RNA is not directly involved in protein synthesis − transfer RNA (tRNA) is required for this. The process by which mRNA directs protein synthesis with the assistance of tRNA is called translation.

The ribosome is a very large complex of RNA and protein molecules. Each three-base stretch of mRNA (triplet) is known as a codon, and one codon contains the information for a specific amino acid. As the mRNA passes through the ribosome, each codon interacts with the anticodon of a specific transfer RNA (tRNA) molecule by Watson-Crick base pairing. This tRNA molecule carries an amino acid at its 3′-terminus, which is incorporated into the growing protein chain. The tRNA is then expelled from the ribosome. Figure 7shows the steps involved in protein synthesis.

Apparatus and Materials :
Onion,Detergent,Salt solution,Alcohol,Test tubes ,Glass rods,Filter paper

Procedure :

1. A piece of onion is grind up in small amount of water. 2. some detergent and salt solution added while Stiring gently for 5 minutes. 3. The liquid from the mixture into a clean test tube is transferred. 4. cold alcohol is added by tilting the test tube at a 45-degree angle and very slowly pour the alcohol down the side of the tube. The alcohol trickle down the side and come to rest on the top of the water so that it forms a separate layer (don’t let the layers mix). 5. Test tube is placed in its rack and did not move for at least 15 minutes. The DNA will began precipitating out immediately between the two layers of liquid. 6. After 15 minutes, the DNA was found to float on the top of the test tube. Wooden stick is used to spool the DNA out.

Results :

Two layers of solution were formed whereby the layer below is a clear solution and the layer on top is where the DNA resides. DNA is a very long molecule but compared to the holes in the filter paper, the molecule is still small enough to pass through

Discussion :

In an onion, the DNA is tightly packed in the nucleus of each cell. In order to get at this we must first blend the onion, which increases the onions surface area, Why we need to mash the onion? What we want to do by mashing the onion is to either break the cell walls (releasing the DNA into the juice) or at the very least expose the cell walls so the detergent can break them down..This buffer solution is used in this lab for several reasons.
First of all, the saltiness and acidity (pH) of the solution is very close to that in living things; as a result, the DNA will like to dissolve into this solution. The phosphate groups on the outside of DNA carry a negative charge. These negative charges are attracted to and are neutralized by cations such as sodium. When sodium is added to DNA it forms a protective "shell" around it. On the other hand, protein molecules precipitate from solution in the presence of salt.
Secondly, the detergent is added to help break down cell walls in the onion cells. Cell walls in living things are made of long polar molecules with a “greasy” end and a charged end. Because detergent is used to break apart greasy particles in your clothes, it will also work to tear apart the “greasy” molecules in cell walls. It will be important that these cell walls break down in this lab, because inside the cell is where the DNA is.
Before adding the alcohol, The clear solution you have in this test tube consists of dissolved DNA fragments, as well as some other biochemical compounds such as RNA and some proteins. DNA is a very long molecule, but compared to the holes in a piece of filtering paper, the molecule is still small enough to pass through.
The rubbing alcohol is used to extract the DNA from the onion juice. The reason you want the rubbing alcohol to stay on top of the onion juice is because by doing that the liquid will form two distinct layers. Generally, molecules are attracted to the boundaries of two liquids - sometimes the concentration of large molecules can be much higher at the boundary between two liquids.
DNA is insoluble in ethanol (ethyl alcohol). As ethanol is added to a solution containing DNA, the DNA will come out of solution and stick to whatever is around. If the DNA is attracted to the surface, we can pull most of it out. However, if the alcohol and onion juice mixes too much, there will be too much alcohol throughout the whole liquid, and the DNA won’t be attracted to the surface, making it much harder to pull any out of the tube.

DNA spools onto the stick or glass rod because the exposed ends have polar chemical groups on them. Glass and wood are also polar, so the ends of the DNA are attracted to the stirrer. By winding the stirrer, you are basically just reeling in the DNA molecules.
When you pull the DNA through the nonpolar alcohol layer, it clumps together because it would rather be attached to polar materials such as the stick or even itself. Remember, “like dissolves like”, meaning that polar compounds will tend to want to stay in polar environments while nonpolar compounds will want to stay in nonpolar environments. In this case, DNA, a polar compound, sticks to itself simply because it prefers a polar environment (itself) to a nonpolar environment (the rubbing alcohol).

Conclusion :
DNA is like a microfilm onto which thousands of coded pieces of information were recorded even before birth, along with the genes which serve to programme cellular activity such as the production of proteins, lipids and enzymes, or pigments like melanin, found at the heart of the skin. The acid is used to dissolve the DNA in it. Detergent is used to break the cell walls and alcohol to separate the 2 layers. Single DNA strand is hard to see compared to a pile of DNA strand.

Reference :

1. http://www.accessexcellence.org/AE/AEC/CC/DNA_extractions.php 2. http://misterguch.brinkster.net/mlx031.pdf 3. http://www.sciencebuddies.org/science-fair-projects/project_ideas/BioChem_p001.shtml

QUESTIONS

1. What does the DNA look like?

DNA is like a microfilm onto which thousands of coded pieces of information were recorded even before birth, along with the genes which serve to programme cellular activity such as the production of proteins, lipids and enzymes, or pigments like melanin, found at the heart of the skin. The DNA forms white precipitate in the mixture. When the DNA is spooled out by the wooden stick, it seems to be like a small thread-like substance.

2. What do you think was the specific purpose of adding each of the following: (i) detergent,salt and alcohol . Detergent was added to break down the cell walls in the onion cells. Cell walls in living things are made up of long polar molecules with a ‘greasy’ end and a charged end. Because detergent is used to break the ‘greasy’ particles in your clothes, it will also work to tear apart the ‘greasy’ molecules in cell walls. The salt shields the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution. The alcohol was used to extract the DNA from the onion juice. The reason why the alcohol is required to stay on top of the onion juice is because by doing that the liquid will form two distinct layers. As important as DNA molecules to life, they are still extremely fragile and break apart easily when removed from cells. To slow down the rate at which the DNA breaks up, the alcohol was cooled down. Chemical reactions always take place slower in cold solutions than in warm ones because there is less energy around to make the reaction take place.

3. Why onion is suitable for DNA extraction? An onion is used because it has a low starch content, which allows the DNA to be seen clearly.

4. What is the component in the detergent that helps in the DNA extraction?

Sodium Dodecyl Sulfate, or SDS. is a sodium-containing detergent. It has the chemical formula of C H NaO S. Detergents are used to break down cell walls and membranes. They work by chemically poking holes in the cell membranes or walls. Once holes are poked in the membranes, the membranes can be further disrupted mechanically, as with a blender. After that, it is easier to get the contents of the cell out, including the DNA.