Experiment 10

Objectives

DNA (deoxyribonucleic acid) is the central genetic material in living organisms, including plants, animals, bacteria and viruses. In the present experiment you will isolate DNA from a plant source, spinach. The DNA isolated using these techniques can be used for many different biochemical processes, including cloning, sequencing, transforming into other cells, and characterized according to content of the different nucleotides that make up this molecule. Based on standard base-pairing (A pairs with T; G pairs with C) you will be able to look at one strand of DNA and determine its complimentary strand as used for DNA replication.  In addition, you will be able to determine the sequence of RNA made from a particular region of DNA (this is called transcription in the cell). From the sequence of RNA (especially mRNA), you can determine what amino acids are inserted into a protein. Thus, the overall objective of this experiment is to look at DNA and see how it can be used in the cell.

Background

The standard genetic material in the cell is DNA. There are actually two different forms of nucleic acid that can exist in a cell or organism (including plants, animals, viruses and bacteria). One of these is DNA, which stands for deoxyribonucleic acid.  The other is RNA which is ribonucleic acid. For most organisms RNA is produced using a specific process called transcription from a piece of DNA. There are, however, a few viruses which only use RNA, both for its genetic makeup and for making new RNA molecules used for making protein. 

Historically, DNA has been prepared using simple techniques when isolated from a cell. The cells, or tissues, are usually disrupted using mechanical techniques to break things apart. In addition, addition of other chemicals aid in this process.  In the experiment we will be doing today, the spinach will be broken down into cells, or smaller clumps of cells, from the whole leaf by use of a blender. After the spinach "juice" is produced, it will be treated with a detergent referred to as SDS (sodium dodecyl sulfate; also named sodium lauryl sulfate and you may recognize the name on the ingredients of shampoo) which will help dissolve organic material. SDS dissolves the lipids which are part of the membrane surround the cell much like normal soaps or detergents dissolve grease. In addition, SDS also dissolves proteins essentially destroying the structural integrity of the cell. The DNA, since it is soluble in aqueous mixtures is released from the cell as a very long polymer (each strand of DNA contains a single polymer of greater than 1 billion nucleotides). In addition, a protease will be added to digest cellular protein that has been made soluble by the SDS described above.  The protease will progressive digest the released proteins (much like the proteases in your stomach and intestine digest protein from your diet) of the cell and make the isolation of pure DNA possible.

Besides doing the actual experiment to isolate DNA, you will learn to put a double-stranded piece of DNA together from the nucleotide sequence for a strand of DNA.  This is the procedure followed in the cell during replication using common base pairing.  In addition, you will be given the chance to make a piece of RNA from a DNA strand following the process used to make mRNA used in protein synthesis.  From the mRNA that you produce, you will be able to put together a small protein using the Universal Genetic Code table.

Procedure

For the isolation of DNA, follow the procedure outlined here.

1. Obtain from your instructor about 10.0 mL (up to as much as 15.0 mL) of the extract (juice) of spinach prepared by blending spinach leaves with an aqueous buffer (5 mM EDTA).
2. Add 0.5 mL of a 10% SDS detergent solution to your spinach extract to disrupt the plant cells and to solubilize lipid and protein from the spinach extract.
3. Add one, and only one, drop (using a plastic Pasteur Pipette) Proteinase K (a potent protease which breaks down most proteins) that will digest the protein of the extract into smaller peptides that can be easily isolated away from the DNA
4. Incubate the spinach/SDS/protease mixture at 37 ^oC for at least 60 minutes.

While the above mixture is incubating, you can do the nucleic acid exercises described below. These exercises teach you about normal base pairing in DNA and RNA and how mRNA sequences are converted in protein. Don't be afraid to ask your instructor for some assistance if needed.

After your DNA has incubated for the 60 minutes, you will need to isolate the DNA from your reaction mixture. This is done using either isopropyl alcohol or ethanol. Because DNA is usually soluble in aqueous mixtures, due to the phosphate-sugar-phosphate bonds in the backbone of the DNA, in order to isolate the DNA you must reduce its solubility in water.  This is accomplished by adding alcohol (either isopropyl alcohol or ethanol). The addition of the alcohol makes the DNA essentially insoluble in an alcohol/water mixture.

• Pour the contents of your extraction mixture into a 100-mL beaker.
• Add twice the volume of extraction mixture of alcohol. You will be using ice-cold ethanol that has a salt (sodium acetate) added to you. The sodium ions complex with the negatively charged phosphate part of the DNA chain, helping to neutralize the phosphate ion. Along with the alcohol, the salt helps to reduce the solubility of the DNA.

After adding the alcohol, you should start to observe, near the interface of the two liquid phases, a stringy white substance.  This is the DNA that you wish to isolate.

• Take a glass stirring rod (this rod may have a rubber policeman on one end; be sure to use the glass end of this stirring rod), and gently start to stir at the interface where you see the white stringy material so that the DNA starts to accumulate as a fibrous white mass. Continue gentle stirring to accumulate all of the DNA wad into a single mass.
•  Using a pair of forceps, reach in and grab the "snot-wad" of DNA and transfer it to another 50-mL beaker that has about 30 mL of isopropyl alcohol in it. Using your forceps, you should use an up-and-down motion to wash your DNA wad. At this point you should have a good amount of DNA. The contaminating material that washes free from the fibrous DNA will most likely be smaller pieces of DNA or of protein.

If you wish to keep some of the DNA as a souvenir, take a pair of scissors and physically cut the wad of DNA into two pieces. transfer the DNA into a small 1.5-mL micro centrifuge tube with about 1 mL of isopropyl alcohol in it. The DNA can be kept for years in this condition.

DNA, RNA and Protein Exercises

For this part of the exercise, you will need to use your knowledge of nucleic acid base pairing to get the correct responses. For DNA base pairs, "A" pairs with "T" and "G" pairs with "C." Therefore, using the DNA sequence given your answer for the DNA Pair Strand would start as "AAA" because the original DNA sequence starts with "TTT" and "A" pairs with "T" and so forth.

DNA Sequence:    3’- TTT TAC TCA GGC ACG GCA CCT AAT GTA ACT AA -5'

DNA Pair Strand:  5'- ________________________________________________ -3'

The RNA sequence uses "U" in place of "T" found in DNA, so, you can show the correct mRNA sequence either by copying the "DNA Pair Strand" above and simply replace any "T" bases with "U" bases. Alternatively (and this is not bad since it gives you a chance to determine base pairs again), you can use the original DNA sequence and base pair "U" with any "A" sequence in the DNA.

mRNA Sequence: 5'- ________________________________________________ -3'

For the protein sequence you need to use the Universal Genetic Code table that is in your textbook. Look at the mRNA sequence above, and then starting at the left side, scan the sequence of the mRNA until you encounter a triplet codon "AUG" which is required to start protein synthesis. The "AUG" codon encodes the amino acid methionine (MET), so that every protein starts with a MET amino acid. Then, for each succeeding codon (consisting of three nucleotides - a triplet), find the appropriate amino acid being encoded. Write that sequence of amino acids into the Protein Sequence below. When you encounter, instead of an amino acid, a terminator codon (UGA, UAA or UAG), that stops the synthesis of protein.

Protein Sequence:     ________________________________________________

Good luck and have fun.