DNA Extraction
Yaritza mashes a ripe banana in the school lab and, with a little dish soap, salt, and cold alcohol, she manages to pull out white, mucus-like strands. Her teacher tells her: "What you see is DNA." But a DNA molecule is so thin that nobody can see it with the naked eye. So what are you really looking at? You will find out at the end of the page.
What it means to extract DNA and where it comes from
Eukaryotic cells keep most of their DNA inside the nucleus. A little DNA is also found in the mitochondria and, in plants, in the chloroplasts, but this activity mainly extracts the DNA from the nucleus. To extract DNA is to get it out of there: break the cell's coverings, separate it from the debris, and make it visible. The activity works with almost any tissue (banana, strawberry, onion, lettuce, liver, or chicken breast), because all their eukaryotic cells have a nucleus with DNA.
Before starting, you have to grind the material. This breaks down the tissues and exposes lots of cells to the detergent. The more cells that break open, the more DNA you get.
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…
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?
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…
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?
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…
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.
Lysis: opening the cells
Grinding only breaks the tissue into little pieces. The cells are still whole, with their membrane and nucleus protecting the DNA. To open them you need lysis.
Why does detergent work? Membranes are made of a phospholipid bilayer, which is made of fats. It is the same thing detergent does to the grease on a plate after a sancocho: it breaks it up and lets it mix with water. When the membranes break down, the contents of the cell (DNA, proteins, debris) spill out into the liquid.
The salt provides ions that neutralize the negative charges of the DNA. That way the molecules stop repelling each other and can later clump together.
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…
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.
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…
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.
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…
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.
Materials and safety rules
Even if you use kitchen ingredients, this is a lab activity and you work as in a lab.
| Material | What it is for |
|---|---|
| Beaker | Mixing and grinding the sample with water, salt, and detergent |
| Strainer or filter (with filter paper) | Separating the solid debris from the liquid |
| Test tube | Receiving the filtrate and forming the two layers with the alcohol |
| Glass rod | Collecting the DNA strands |
Safety rules:
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Do not taste or eat anything. Even if the sample is a fruit, lab equipment and reagents are not food.
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Keep the alcohol away from flames and heat sources. Alcohol is flammable and its vapors ignite easily.
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Wear a lab coat and safety goggles, and tell the teacher if anything spills.
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.
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…
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.
Filtering and precipitation with alcohol
After lysis, the mixture is filtered. The solid debris (pulp, pieces of cell wall, and membranes) stays in the filter. A liquid with the DNA dissolved in it passes into the beaker. It is worth getting rid of the solids because they contain no useful DNA and would cloud the result.
The filtered extract looks like cloudy water: you cannot see any DNA. So you add very cold alcohol (for example, isopropyl), little by little down the wall of the tube so that it forms a layer on top of the extract.
Notice the difference. To dissolve means the particles spread out separately among those of the solvent (like the salt in the water of the sancocho). To precipitate means they come out of the liquid and clump into a solid. The change from water to alcohol makes the DNA precipitate, but many proteins and membrane debris stay in the liquid. That is how the DNA is separated from much of the impurities. The DNA does not change chemically: only its physical state changes, like when salt crystallizes again if the water evaporates.
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…
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.
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…
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.
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…
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.
Why the alcohol goes in cold and without stirring
The alcohol must be very cold and must run slowly down the wall of the tube. Each condition has its reason:
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Cold: at low temperature DNA is even less soluble and precipitates more easily. Cold also reduces the movement of the molecules and helps them clump together.
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Slowly, without mixing: alcohol is less dense and floats on top of the extract. That way the two layers stay separate and the DNA precipitates right in the contact zone, where it is collected with the rod. If you stir, everything mixes and the DNA ends up scattered or broken into pieces.
In the contact zone a whitish cloud of tangled strands appears. It is not a single molecule or the DNA of a single cell: it is millions of DNA molecules from millions of cells, along with some proteins, grouped into strands.
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…
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.
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…
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.
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…
During a plant DNA extraction practical, three groups obtain failed results:
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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.
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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.
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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 complete protocol
Each step depends on the one before. Without grinding and lysis there is no free DNA. Without filtering, the solid debris gets in the way. Without the alcohol, the DNA stays dissolved and invisible.
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…
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.
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…
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…
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.
Amount and scale: why DNA can be seen
Now we close the hook. A human cell contains about $6\ \text{pg}$ of DNA, that is, $6 \times 10^{-12}\ \text{g}$. That is an invisible amount. But the strand in the tube is not the DNA of one cell: it is the DNA of millions of cells, and each one contributes its small part.
That is why you have to grind a good amount of material: just a few grams of fruit already contain millions of cells.
And the thickness: a molecule is about $2\ \text{nm} = 2 \times 10^{-9}\ \text{m}$ wide and a strand about $0.5\ \text{mm} = 5 \times 10^{-4}\ \text{m}$. In a line, $\dfrac{5 \times 10^{-4}}{2 \times 10^{-9}} = 2.5 \times 10^{5}$ molecules fit across. Since the strand is round, on the order of tens of billions of molecules fit in its cross section. A huge number of molecules together can be seen.
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…
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…
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.
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…
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.
Improving the extraction: tissue, enzymes, and temperature
School-lab DNA is not pure: it comes out with proteins and lipid debris. There are ways to improve the result.
Depending on the tissue. A tough tissue (onion, leaf) needs more grinding and maybe more detergent than a soft one (strawberry, banana). Plant cells have a cell wall, which grinding helps to break. In addition, plants can have several sets of chromosomes: the cultivated strawberry is octoploid, with eight sets instead of two. More copies of DNA per cell means more DNA in each one, and that is why a larger visible mass is expected than from a diploid cell.
Enzymes. There are proteins stuck to DNA (the histones). A protease enzyme, such as the bromelain in natural pineapple juice or the one in papaya, cuts those proteins. If you add a splash of pineapple juice to the filtrate before the alcohol, the DNA comes out cleaner.
Temperature. Enzymes work well at moderate temperature; if they get too hot they denature and stop working. Boiling the filtrate at $100\ ^\circ\text{C}$ is not a good idea: the heat denatures the proteins, but it also breaks the DNA into pieces and separates its two strands, so sample quality is lost.
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…
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.
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…
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.
Applications and limits of the school method
The DNA you extracted is a crude sample. In a real lab, DNA is purified, measured, and often amplified with the PCR technique (polymerase chain reaction), which makes millions of copies of a fragment. This is used for paternity tests, forensic studies, diagnosis of genetic diseases, and improvement of crops such as cacao, rice, and cassava. It also serves as a bridge to genetic engineering.
With the kitchen method you cannot analyze a forensic sample, for three reasons:
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With only a few dozen cells (a hair with its root), the amount of DNA is tiny and you would not see anything.
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The DNA comes out mixed with proteins and other substances; it is not a pure sample.
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The visible strands do not let you read the genes: for that you have to cut, amplify, and sequence the DNA.
Electrophoresis. DNA samples are cut with enzymes and the fragments are placed in a gel. DNA has a negative charge because of its phosphate groups, so when the current is switched on it migrates toward the positive pole. The gel is a network with pores: short fragments pass through easily and travel farther; long ones get tangled and travel less. The result is bands at different heights. If two samples give the same band pattern, it is very likely they come from the same person.
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…
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: a) If DNA has a net negative charge due to…
To identify a suspect, a laboratory cuts DNA samples with enzymes and subjects the fragments to agarose gel electrophoresis:
- 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.
- 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.
- 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.