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CSI

Crop Science Investigation
Grades 4-8

TEACHER GUIDE

Have you ever wondered what a forensic scientist has to do to analyze DNA evidence? DNA (the building blocks of life) is present in the cells of all living organisms. In forensic science DNA is usually extracted from human cells to identify unknown parentage, crime scene suspects, missing victims and to investigate genetic diseases. Since 1985, "DNA fingerprinting" has been used in legal cases around the world to link suspects to the scene of a crime.

 

Today, your class will be able to extract DNA from fruit to see what it looks and feels like. This is similar to what a scientist has to do before they can then use the information contained in this DNA to solve crimes, although they don't use the same protocol.

OBJECTIVES

This experiment is designed to:

  • Demonstrate how DNA is extracted from cells
  • Develop an interest in the forensic science behind DNA evidence

Preparation for experiment

Time Required: ~20 minutes

Student #: 10

Kiwifruit Extraction Solution

(Teacher Pre-Lab):

MATERIALS

  • 10 mL of clear shampoo (suave daily clarifying shampoo)
  • 1.5 g of table salt
  • Distilled H2O
  • Test tube for each student pair for allocation of extraction solution.

PROCEDURE (This procedure can be modified for amount depending on the size of a class.)

  1. Mix 90 mL of distilled water and 1.5 g of salt.
  2. Add shampoo until solution volume is 100 mL. Stir slowly to avoid foaming of the shampoo.
  3. Measure 20 mL of solution into test tubes (1 per student pair)

Student Procedure (per student pair)

Time required: ~ 45 minutes

MATERIALS

Each student station should have the following:

  • 1 test tube of extraction solution per student station (prepared by teacher)
  • 1 liter Zipper bag (one per student station)
  • Freshly cut Kiwifruit and skinned (each fruit cut into 12 pieces, allocate ~ 30 g per student station into zipper bag)
  • Beaker (one per 5 student pairs)
  • Hot water plate with beaker of water set at a constant 60 degrees Celsius (one per 5 student pairs)
  • Cheese cloth or coffee filter (cut to fit over beaker)
  • Tape
  • Ice water bath (one per 5 student pairs)
  • Ice cold 70% or greater ethanol (2 mLs per student)
  • 1 small test tube (1 per student)
  • 1 paper clip bent into an "L" shape to fit into the test tubes and retrieve the DNA (1 per student)

PROCEDURE

  1. Add extraction solution to the zipper bag with kiwifruit. Close bag, let as little air in as possible.
  2. Mush the kiwi thoroughly for 5 minutes, without breaking the bag.
  3. Place the zipper bags with fruit and extraction solution into the hot water bath for about 10-15 minutes. Occasionally shake the bag to distribute heat
  4. Put the "mashed" bags of kiwifruit and solution into the ice bath for 1 minute. Remove and mush the kiwifruit more. Repeat this procedure 5 times.
  5. Tape the cheese cloth/coffee filter over the beakers. Filter this mixture through the filter. All student groups can combine their solutions at this point. Let the solution drain for 5 minutes.
  6. Aliquot approximately 2 mL of the kiwifruit solution into each test tube, one for each student.
  7. Carefully, without disruption of the test tube contents add approximately 2 mL of ice-cold ethanol to each tube. Do this by letting the drops run slowly down the side of the test tube and rest on top of the kiwifruit mixture.
  8. Let the solution sit for 2 minutes without disturbing it. The DNA will appear as transparent, slimy, white mucus.

ANALYSIS

Recap with your students the pre-laboratory preparation.

What was the purpose of the shampoo? The soap/detergent destroys the cell and nuclear membranes to allow the DNA to get out. It does this by dissolving lipids and proteins that hold the membranes together The detergent causes lipids and proteins to precipitate out of the solution leaving the DNA in the solution.

What was the purpose of the salt? The salt enables the DNA strands to stick together. It also causes proteins and carbohydrates to precipitate.

Why was it necessary to "mush" the kiwifruit? Pressing the kiwi fruit along with the soap helps destroy the cell membranes while physically breaking apart the cell walls.

Why do we cool the mixture? Cooling protects the DNA from its' self. DNases or enzymes that destroy DNA are present in the cell's cytoplasm. The DNA's nuclear membrane is destroyed by the soap and the DNA is now susceptible to the DNases. These enzymes are temperature sensitive and cooling the solution slows down the process of degradation. Dnases are in cells to protect the organisms from viruses.

What happened when the cold ethanol was added? The alcohol will dissolve everything but the DNA. This occurs because DNA is not soluble in alcohol, the other components of the mixture stay in solution. The DNA will become apparent as white mucous strands that can be spooled with a glass pipette or rod.

Would it make a difference if it were warm ethanol added? The colder something is, the more likely it will precipitate or solidify. Cooling the ethanol increases the amount of DNA that is precipitated. The temperature has nothing to do with the DNases at this point; it has to do with precipitating or solidifying the DNA out of the solution.

QUESTIONS & ANSWERS

  1. To purify a molecule, such as DNA, what must you isolate it from in the case of kiwi fruit? All parts of the cell except for DNA. The cell wall, cell membrane, nuclear membrane, mitochondria, vacuoles, endoplasmic reticulum, Golgi apparatus, lysosomes, etc.
  2. What are the steps required to isolate the DNA? The first step is to break apart the cells by physical (pressing) and chemical (soap) means. This process breaks down the cell walls, membranes and nuclear membranes. Cooling the DNA stops the cell from destroying its own DNA with the cell's DNases. Next, filter to separate out the cell parts we don't need. Finally, precipitate out the DNA through chemical means (the ethanol).
  3. What can we do with DNA once we have extracted it? DNA can be used for DNA fingerprinting to solve a crime, identify parentage, and check for genetic defects. We could look at the kiwifruit DNA to see if it has been genetically modified, by comparing it to a known modified and unmodified sample. DNA can also be used for transformation, to insert it into a foreign organism to give that organism a specific trait.

REFERENCES

  1. www.accessexcellence.org
  2. www.biotech.iastate.edu
  3. www.biology.arizona.edu