Grade by Grade
D. Molecular Genetics · Molecular Genetics

Investigation: Extracting DNA from Plant Tissue

D2.3 A1.1 A1.4 A1.5 A1.6 A1.8 A1.10 A1.11 A1.13About 75 minutes
Learning goals
  • I can extract DNA from plant tissue and explain the purpose of every step in terms of cell structure and chemistry.
  • I can plan a fair test of a variable that affects DNA yield, with a hypothesis, controlled variables and repeated trials.
  • I can record and analyse data, including means, ranges and sources of error.
  • I can follow safety procedures for flammable alcohol and describe how a school or university lab does the same extraction.

Words to know

lysis
Breaking open a cell, here by mashing and by dissolving membranes with detergent.
extraction buffer
The solution (water, detergent, salt) used to break open cells and keep DNA dissolved.
protease
An enzyme that breaks down proteins by hydrolysing peptide bonds; papain and bromelain in meat tenderizer are examples.
precipitate
To come out of solution as a solid. DNA precipitates in cold alcohol.
filtrate
The liquid that passes through a filter.
yield
The amount of product obtained, here the amount of DNA.
controlled variable
A factor kept the same in all trials so that it cannot explain a difference in results.

DNA you can hold

A single DNA molecule is about 2 nm wide, far too thin to see with any light microscope. Yet with kitchen materials you can pull out a clump of DNA big enough to wind around a stick. In this lesson you will do it, and then go further than a demonstration: you will explain why every step works and design a fair test of something that might increase the yield. That is curriculum expectation D2.3, plus the investigation skills of strand A.

Remember from Grade 11

In the Grade 11 lesson DNA, Genes and Chromosomes you may have extracted DNA from strawberries with soap, salt and cold alcohol. This time you will use a different specimen, add a protein-digesting enzyme, explain each step with Grade 12 chemistry, and run a controlled investigation.

The chemistry behind each step

1. Break the cells apart (mechanical lysis). Plant cells have a tough cellulose cell wall. Mashing or blending tears the walls and frees the cell contents.

2. Dissolve the membranes (chemical lysis). The plasma membrane and the nuclear envelope are phospholipid bilayers (you studied the fluid mosaic model in the Biochemistry unit). Detergent molecules have a nonpolar tail and a polar head, like phospholipids. They pull phospholipids out of the bilayer into small clusters, so the membranes fall apart and the DNA is released.

3. Add salt. DNA's backbone is full of negatively charged phosphate groups, which repel each other and attract water. Positive sodium ions (Na⁺) cluster around the phosphates and shield those charges. This lets DNA strands come together and later come out of solution. Salt also helps separate DNA from proteins.

4. Remove proteins (optional). In the nucleus, DNA is wound around histone proteins. A protease breaks peptide bonds and frees the DNA. Unseasoned meat tenderizer contains plant proteases (papain from papaya or bromelain from pineapple). Proteases in the extract also break down DNases, enzymes that would cut DNA.

5. Precipitate with cold alcohol. DNA dissolves well in water but not in alcohol. When cold alcohol is layered on top of the extract, DNA comes out of solution at the boundary. The cold temperature lowers DNA's solubility further and slows enzymes. Clumps of millions of DNA molecules form white strands, often carrying tiny gas bubbles that make them float up.

1. Mashbreaks cellwalls2. Detergentdissolvesmembranes3. Saltshields DNA's− charges4. Proteasedigestshistones5. Cold alcoholDNA comes outof solutionStep 4 is optional at home (unseasoned meat tenderizer); labs use a purified enzyme.
The logic of DNA extraction: break open the cells, dissolve the membranes, free the DNA from proteins, then make it precipitate where it can be seen.
ice-cold alcohol layerwhite, stringy DNAinterface between layersfiltered extract (water-based)less densemore dense
Alcohol is less dense than the water-based extract, so it floats on top. DNA is insoluble in alcohol, so it comes out of solution at the boundary and rises in white strands.
Worked example

Example 1: Planning a fair test

Plan an investigation to answer: Does adding a protease (meat tenderizer) increase the amount of DNA extracted from banana? Write a hypothesis and identify the independent, dependent and controlled variables.

  1. Hypothesis: If a protease is added to the banana extract, then more DNA will precipitate, because the protease digests the histone proteins bound to the DNA, freeing more DNA to clump together.
  2. Independent variable: protease added (yes or no).
  3. Dependent variable: amount of DNA, measured as the height in mm of the white DNA layer in identical narrow glasses after 5 minutes.
  4. Controlled variables: the same banana (same ripeness, mass), same buffer recipe and volume, same mashing time, same filtrate volume (10 mL), same volume and temperature of alcohol, same waiting time, same glass shape.
  5. Control group: filtrate with no tenderizer. Repeated trials: at least 3 for each group.

Hypothesis with a reason; IV = protease or not; DV = DNA layer height (mm); many controlled variables; a no-protease control; 3+ trials each.

Hands-on activity

Investigation: Banana DNA, with and without a protease

Grown-up note: an adult should handle the rubbing alcohol (isopropyl alcohol, 91% or 99%). It is flammable and poisonous to drink: keep it in a closed container in the freezer for at least an hour, away from any flame, stove or heater, and use it in a ventilated room. Wear safety glasses or glasses if you have them. Nobody tastes anything in this activity.

You need: half a ripe banana, a zip-top bag, a measuring cup, 100 mL of warm tap water, 1 tablespoon (15 mL) of clear dish soap, ½ teaspoon of table salt, a coffee filter and a funnel (or a filter folded inside a glass), 6 identical narrow clear glasses or test tubes, a ¼-teaspoon measure, unseasoned meat tenderizer powder, about 80 mL of ice-cold rubbing alcohol (adult), a wooden skewer, a ruler with millimetres, a timer, and a notebook.

  1. Buffer: stir the water, soap and salt gently (avoid foam).
  2. Lyse: put the banana and all of the buffer in the bag. Seal and mash for 3 minutes. Let it stand 10 minutes.
  3. Filter into a clean glass. This takes several minutes; don't squeeze the filter.
  4. Label 3 glasses P (protease) and 3 glasses C (control). Pour 10 mL of filtrate into each.
  5. Add ¼ teaspoon of meat tenderizer to each P glass and stir gently for 10 seconds. Stir each C glass the same way with nothing added. Wait 10 minutes.
  6. The adult slowly pours 10 mL of cold alcohol down the inside of each glass so it forms a layer on top.
  7. Wait 5 minutes without moving the glasses. Measure the height of the white DNA layer in each, in mm. Record in a table.
  8. Wind some DNA onto the skewer and look at it closely.
  9. Pour the alcohol mixtures into the sink with plenty of running water (or follow your local disposal advice), and wash everything.

Record: a data table (group, trial, height in mm), the mean for each group and the range. Note anything unusual (foam, a trial where alcohol mixed in).

Worked example

Example 2: Analysing the results

A student got these DNA layer heights (mm). Control: 6, 7, 5. Protease: 9, 11, 10. Calculate the mean and range of each group, state a conclusion, and give two sources of error.

  1. Control mean = (6 + 7 + 5) ÷ 3 = 6.0 mm; range 5 to 7 mm, so 6.0 ± 1 mm.
  2. Protease mean = (9 + 11 + 10) ÷ 3 = 10.0 mm; range 9 to 11 mm, so 10.0 ± 1 mm.
  3. The ranges do not overlap, and the protease mean is 4.0 mm higher (about 67% more). The data support the hypothesis that a protease increases the DNA yield.
  4. Sources of error: (1) the DNA layer has a fuzzy edge, so readings are only to about ±1 mm; (2) some meat tenderizers contain salt, which could increase precipitation by itself (a confounding variable; check the ingredients or add the same amount of salt to the controls); (3) alcohol poured too fast mixes with the extract and lowers the yield; (4) only 3 trials each.
  5. Precision: heights measured with a ruler marked in mm justify whole millimetres; means can be given to one decimal place (6.0, 10.0) as averages.

Control 6.0 ± 1 mm; protease 10.0 ± 1 mm. The protease increased the yield in this sample. Main errors: fuzzy measurement, possible salt in tenderizer, pouring technique, few trials.

024681012mmno tenderizermean 6.0with tenderizermean 10.0height of DNA layer (sample data, 3 trials each; bars show the range)
Sample data for the meat-tenderizer investigation. The ranges (error bars) do not overlap, which supports a real difference. Three trials is still a small sample.

How a school or university lab does it

The steps are the same, but the materials are more controlled:

  • The extraction buffer contains a detergent such as SDS, NaCl, a pH buffer, and EDTA. EDTA binds magnesium ions, which DNases need, so DNA is protected from being cut.
  • A purified protease (for example, proteinase K) removes proteins.
  • Mixtures are spun in a centrifuge to remove cell debris quickly.
  • DNA is precipitated with ice-cold 95% ethanol, spooled on a glass rod, then dried and dissolved in buffer.
  • Purity is checked with a spectrophotometer. DNA absorbs ultraviolet light most strongly at 260 nm, and proteins at 280 nm. A ratio A260/A280 of about 1.8 indicates fairly pure DNA.

Your kitchen DNA is not pure: it contains RNA, proteins and carbohydrates (bananas have plenty of starch and pectin). But the principle is exactly the one used to prepare DNA for PCR and sequencing in the lesson Copying, Sorting and Reading DNA.

Just for fun

The bananas in Canadian grocery stores are almost all one variety, the Cavendish, and it is triploid: three sets of chromosomes. Odd numbers of chromosome sets cannot pair evenly in meiosis, which is a big part of why it makes no hard seeds. So the DNA in your glass came from a plant that farmers grow from shoots of other banana plants, not from seeds.

Your turn

1

Why does detergent release DNA from a cell?

2

Explain the role of salt in terms of electric charge.

3

Why is the alcohol ice-cold?

4

Write a testable hypothesis for an investigation on the effect of mashing time on DNA yield.

5

In the protease investigation, why must the control glasses also be stirred for 10 seconds?

6

Give two safety rules for using rubbing alcohol.

More practice: warm-up

1

What does a protease digest?

2

Is DNA soluble in water or in alcohol?

3

What structure must be broken open in plant cells but not animal cells?

4

What is the dependent variable in the protease investigation?

5

Why is the extract filtered?

More practice: on your own

1

Why does EDTA protect DNA in a lab extraction buffer?

2

A student's DNA spectrophotometer reading gives A260/A280 = 1.4. What does this suggest, and what step could be improved?

3

Three trials give 8, 12 and 4 mm. Calculate the mean and comment on the reliability.

4

Why can you see extracted DNA but not a single DNA molecule?

5

A student uses seasoned meat tenderizer that contains salt and spices. Explain why this could make the conclusion invalid, and how to fix it.

Challenge

1

Strawberries are octoploid (8 sets of chromosomes) and bananas are triploid (3 sets). Predict which gives more DNA per gram, and explain why ploidy alone may not decide it.

2

Design an investigation to find the best alcohol temperature for DNA precipitation. Include the levels of the independent variable, how you would control the temperature, and how you would present the data.

3

A forensic lab must extract DNA from a tiny blood stain. Red blood cells in mammals have no nucleus. Where does the DNA come from, and why does the lab's method use PCR afterward?

Check your understanding

Quiz

1.

What is the main role of detergent in DNA extraction?

2.

Why does DNA appear at the boundary between the extract and the alcohol?

3.

Salt helps DNA precipitate because sodium ions:

4.

In an investigation of the effect of a protease on DNA yield, which is the control group?

5.

A student's results are 7, 8 and 7 mm (no protease) and 7, 9 and 8 mm (protease). Which conclusion is best supported?

6.

A spectrophotometer ratio A260/A280 of about 1.8 indicates:

For parents and teachers

Big idea (D2.3 and strand A): extracting DNA is a chain of steps, each with a chemical reason (break walls, dissolve membranes, shield charges, remove proteins, precipitate). The investigation part asks your teen to plan and evaluate a fair test.

Safety: please handle the rubbing alcohol yourself. It is flammable and poisonous. Keep it away from the stove and any flame, keep the room ventilated, and store it out of reach. Nobody tastes anything. Wash up afterwards.

How to help: Before the experiment, ask your teen to state the hypothesis and list three controlled variables. Afterwards, ask "How sure are you? What could have gone wrong?"

Common difficulties:

  • Writing a hypothesis with no reason ("because...").
  • Saying the detergent "kills" the cells instead of dissolving membranes.
  • Concluding that something is "proven" from three trials.
  • Forgetting that seasoned tenderizer contains salt (a hidden variable).