DNA Extraction

Explain in your own words what extracting DNA from a eukaryotic cell means, and name two simple materials from which cells can be obtained for the practical (for example, cheek cells or a fruit). Why should the material be thoroughly mashed before starting?

In the DNA extraction practical, washing-up liquid, salt and very cold alcohol are used. Explain the role of each reagent: which cellular structures the detergent dissolves, what the salt is used for, and what happens to the DNA when it comes into contact with the alcohol.

Reason why almost any tissue works for extracting DNA, for example a lettuce leaf or a piece of chicken, as long as its cells have a nucleus. What do all those cells have in common?

Washing-up liquid dissolves cell membranes because these are made of a phospholipid bilayer. Explain the analogy with what the detergent does when washing a greasy plate and what then happens to the contents of the cell.

Look at the diagram of the test tube at the end of the DNA extraction practical. Identify what the labels ?1, ?2 and ?3 indicate (the alcohol layer, the aqueous extract with the dissolved DNA, and the DNA strands appearing at the interface), and explain why DNA concentrates precisely in that area.

After mashing the material with the extraction mixture, the practical continues by filtering everything. Explain what is retained by the filter, what passes into the beaker and why it is useful to get rid of the solid part.

In the tube, the DNA goes from being dissolved in the extract to forming a precipitate of strands when the alcohol is added. Explain whether the DNA molecule has changed chemically in this process or only its physical state has changed, and compare it with what happens to salt when it precipitates in water.

The alcohol in the practical must be very cold and should be poured carefully so that it forms a layer on top of the extract without mixing with it. Explain the advantage of the alcohol being cold for the solubility of DNA, and why the two layers should not mix.

DNA is soluble in the salty aqueous extract but insoluble in cold alcohol, while many proteins and membrane remains stay in the liquid. Explain the difference between dissolving and precipitating, and why this change in solubility allows the DNA to be separated from most impurities.

List the laboratory equipment needed for the DNA extraction practical (beaker, strainer or filter, test tube, glass rod) and explain what each item is used for.

Although the practical uses kitchen ingredients, it is carried out in a laboratory. Name two safety rules you must follow (for example, do not taste anything and keep the alcohol away from any flame) and explain the reason for each one.

These steps of the DNA extraction practical are out of order: add the very cold alcohol down the side of the tube without shaking; mash the material; prepare the mixture of water, salt and detergent; filter the mixture; collect the DNA strands with a rod. Put them in order and justify why each step should be in that position.

Design an experiment to find out from which fruit, among banana, kiwi and strawberry, the most DNA is obtained using the same protocol. State which variables you would keep constant so that the comparison is fair and which one you would measure to compare the results.

At the end of the practical, a white cloudy layer formed by strands appears at the interface between the extract and the alcohol. Describe what is observed and explain what this material actually is — it is not a single molecule or the DNA of a single cell — and why it is visible to the naked eye when a single DNA molecule is not.

A human cell contains about $6\ \text{pg}$ of DNA ($6 \times 10^{-12}\ \text{g}$). Estimate how many cells would be needed to obtain $1\ \text{mg}$ of DNA and reason why the practical requires mashing a good amount of material.

A DNA molecule is about $2\ \text{nm}$ thick, while the strands seen in the practical measure a few tenths of a millimeter, for example $0.5\ \text{mm}$. Estimate how many DNA molecules packed together fit across the thickness of one strand and relate the result to the fact that the precipitated DNA is visible to the naked eye.

A classmate suggests extracting human DNA from a drop of blood. Reason why red blood cells are not suitable for this practical and which blood cells should be used instead.

You want to extract DNA from a strawberry and an onion using the same protocol. Reason what adjustments you might need in mashing or in the amount of detergent depending on the hardness of each tissue, and predict from which one you expect to obtain more visible DNA, knowing that the strawberry is octoploid: its cells have eight sets of chromosomes instead of two.

A group of students wants to adapt the plant DNA extraction protocol to animal tissue (chicken breast). They propose eliminating two steps: the initial mechanical grinding (because meat is softer than a plant cell wall) and the addition of detergent (because animal cells lack a cell wall).

Critically evaluate their two proposals: justify whether it is correct to dispense with each of these steps based on animal cell organization, and predict what they would obtain if they omitted the detergent.

At the end of the practical with strawberry and kiwi, a student compares both tubes and states: "The DNA of both fruits looks exactly the same (a whitish, filamentous mass). This proves that at the molecular level, DNA of all species is identical and that extraction cannot be used to differentiate between them". Refute their conclusion by distinguishing which part of the DNA molecule is universal to all living things and which part actually contains the information that allows a laboratory to sequence and identify the species.

During a plant DNA extraction practical, three groups obtain failed results:

• Group A: Blended the plant tissue in a blender at maximum power for ten minutes and does not observe strands, only a cloudy liquid without defined filaments.

• Group B: Added 96% alcohol at room temperature ($22\ ^\circ\text{C}$) instead of precooling it to $-20\ ^\circ\text{C}$, and observes hardly any precipitate at the interface.

• Group C: Vigorously stirred the test tube with a glass rod right after pouring the alcohol, and the fibers that were beginning to clump broke apart.

Justify what occurred at a physical or molecular level in each case to explain the failure of the extraction.

The DNA obtained with the school protocol is not pure: it precipitates along with proteins (histones) and lipids. One team proposes adding fresh pineapple juice (rich in bromelain, a protease enzyme) to the filtrate before adding the alcohol. Another team proposes heating the filtrate to $100\ ^\circ\text{C}$ for $15$ minutes to eliminate protein impurities.

Evaluate both proposals: justify which of the two is appropriate by explaining the action of the protease on the histones, and predict the counterproductive effect that boiling the sample would have on the DNA molecule.

In a forensic investigation, a tiny biological sample is collected (a hair with a root containing barely a few dozen cells). A student claims: "We can use the homemade strawberry protocol: we crush it, add dish soap, salt, and alcohol, and then read the genes with a magnifying glass." Refute their claim by identifying three insurmountable limitations of this school method and justify which advanced techniques (such as PCR amplification or enzymatic purification) are essential in a real laboratory to analyze the sample.

To identify a suspect, a laboratory cuts DNA samples with enzymes and subjects the fragments to agarose gel electrophoresis:

1. a) If DNA has a net negative charge due to its phosphate groups, deduce with reasoning towards which electric pole (positive or negative) the fragments will migrate when the current is turned on.
2. b) If a sample contains DNA fragments of $300$ base pairs and others of $2500$ base pairs, predict which ones will end up furthest from the loading well in the gel matrix and justify the physical reason.
3. c) Interpret what it would mean if the suspect's banding pattern matches the sample found at the crime scene exactly in position and number.