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Extraction and Analysis of Onion DNA

I. Introduction

“DNA is a very long, threadlike macromolecule made up of a large number of deoxyribonucleotides, each composed of a base, a sugar, and a phosphate group. The bases of DNA molecules carry genetic information, whereas their sugar and phosphate groups perform a structural role.“

-Lubert Stryer, Biochemistry 4th Edition

The sugar in the DNA molecule is deoxyribose, shown in Figure 1. The bases are either purines (adenine & guanine) or pyrimidines (thymine & cytosine), shown in Figure 2. The DNA polymer is formed is a chain of alternating phosphate and deoxyribose units with bases attached as side units on deoxyribose. In nature, DNA forms a double helix with strands of complementary bases as shown in Figure 3. In the double-helix form, adenine & thymine stick together by hydrogen bonding as shown in Figure 4. Chemical agents or higher temperatures can unwind (denature) the helix by overwhelming the hydrogen bonds. Physical observation of this effect is possible using UV/Vis absorbance because the hydrogen bonding affects the ring structure in the purines and pyrimidines and changes their absorbance (Figure 5).

In this experiment we will isolate DNA from bananas, redissolve it in aqueous solution, and then attempt to measure detect chemical denaturing with urea. Due to the relatively poor solubility of DNA, we will try several aqueous matrices including deionized water, 50 mM NaCl, and tris/EDTA (TE) buffer. As we do this work, care must be taken to avoid harsh physical and chemical treatment of the DNA which can render it insoluble.

II. Characteristics of DNA

Absorbance Measurements

  • Double-Stranded DNA: 1 absorbance unit at 260 nm = 50 µg/ml
  • Single-Stranded (denatured) 1 absorbance unit at 260 nm = 33 µg/ml
  • Linear range 5 – 100 µg/ml

Solubility

  • DNA is slightly soluble in distilled water
  • DNA should be nicely soluble in 50 mM NaCl
  • DNA is soluble in TE buffer which controls pH with a Tris buffer and uses EDTA to reduce effects of Ca2+ and Mg2+.

IV. Measurements

Prepare a protocol (work with other students if possible) to accomplish the following measurements:

  1. Determine the mass of extracted DNA. To do this, you will need to allow the extracted DNA to dry for 10-20 minutes. Do not heat it or wash with excessive amounts of Ethanol or it may not redissolve.
  2. Measure blank spectra of the following (use H2O as the reference solution):
  3. DI Water;
  4. Water + 50 mM NaCl;
  5. Water + 50 mM Urea;
  6. Buffer solution.
  7. Blank spectra are necessary because we don’t know exactly what we are looking for and need to know what the sample matrix looks like.
  8. Prepare a solution of DNA in each of the solutions. You will probably need to prepare a more concentrated solution and dilute it. Weigh the amount of DNA removed from your bulk sample instead of trying to weigh a very tiny amount.
  9. Measure sample spectra of DNA extract in each of the solutions. The area of interest is primarily 200 – 300 nm but note any peaks in the rest of the spectrum.
  10. Determine the effect of Urea on the DNA absorbance. If no effect, try again with double the Urea concentration.
  11. If time allows, you might investigate methods to precipitate DNA. Addition of 0.1 M NaCl and pH manipulation are possibilities.

Stock Solutions

  • 0.1M Urea
  • TEA Buffer
  • 0.1 M NaCl


Figure 3: Polymeric form of DNA

Prelab Exercises

Photocopy your prelab and turn in the ORIGINAL before lecture on Tuesday, August 6.

1. Expected physical properties of DNA: Describe what you think the extracted DNA sample will be like. Will it be a thin liquid, crystalline solid, gel, color, dense, conductive electrolyte?

2. Solution Dilution: Stock solutions of NaCl and Urea will be available. If a 0.1M Urea solution is available and you have a 200 g/mL DNA solution, how would you make 10 mL of 50mM Urea and 50 g/mL DNA?

3. Measuring DNA: If you want to know the mass of DNA extract, how will you weigh it? Use your answers to #1 to help you decide. You may wish to investigate the term “tare.”

  1. Solution Preparation: Design protocols for preparation of:
  2. A DNA stock solution (be mindful of it’s limited solubility). How much will you need? What is a practical amount of DNA to weigh?
  3. 50 mM Urea in water (make about 50 mL so you can rinse the cuvette with it)
  4. 50 mM NaCl in water (make about 50 mL so you can rinse the cuvette with it)
  5. 50 mM Urea solutions with your DNA (10 mL should be plenty)
  6. 50 mM NaCl with your DNA (10 mL should be plenty)

Assume that you will have 0.25M stock solutions of NaCl and Urea available.