Extracting DNA from Fruit in Various Stages of Ripeness

INTRODUCTION This life science based experiment will test strawberries in their various stages of ripeness, in order to see which stage will yield the most extractable DNA. An extraction kit will be designed from common household items, such as salt and detergent, in order to purify the DNA so that it is visible to the naked eye and can be weighed. Three degrees of strawberry will be tested: strawberries that have not fully ripened yet, identified by their firm bodies that are still a mixture of green and red; strawberries that have ripened fully, identified by their firm-but-not-hard bodies and bright red color; and strawberries that are overly ripe, which can be identified by their mushy and easily bruised bodies, as well as their dark red color.

PROBLEM STATEMENT Which degree of strawberry ripeness will yield the most extractable DNA: under ripe, ripe, or over ripe?

SUMMARY OF PROJECT PLAN First, the 1/2 teaspoon of salt, 1/3 cup of water, and 1 tablespoon of detergent needed for the DNA extraction liquid will be mixed and set aside. Three strawberries of the first stage of ripeness will be placed into a plastic bag and mashed into a pulp. Three tablespoons of the extraction liquid will be added to the bag and blended via the same mashing process. The strawberry mixture will then be poured into a nylon-covered funnel set over a small glass, until the liquid and pulp have been separated. One teaspoon of the strawberry mixture’s liquid will then be poured into a test tube. 5 ml of chilled rubbing alcohol will be poured into the test tube after, so that it forms a layer atop the strawberry liquid. A droplet of blue dye will be added to the mix, so that it settles on the DNA between the layers and dyes it blue, making it easier to identify the DNA. The blue DNA will then be measured using milliliter markings on the test tube, and recorded.

RELEVENCE Deoxyribonucleic Acid – better known as DNA – is a set of instructions that can be found in the cells of every living thing. The study of all DNA is very important. Without it, key medical discoveries that save countless lives every day would not be made. Using DNA, we are able to discover diseases a baby could inherit from its parents before birth, to detect whether a suspect is guilty or innocent, and to find chromosomal defects in patients with Downs Syndrome.

The study of strawberry DNA specifically is also important, and can be applied to several real world scenarios. For instance, scientists are able to isolate particular proteins and chemicals that have been rumored to slow the spread of cancer. They are also able to clone proteins known for turning strawberries red and creating the strawberries’ flavor.

The study of extractable strawberry DNA at various stages in maturation can also be applied to real world scenarios. Scientists are able to compare the growing process and maturation of strawberries to that of other fruits. It can also be used to advise consumers of when strawberries are at their peak, so that they are able to get the optimal amount of nutrients out of the fruit.

A1. Literature Review

Two studies were found that related specifically to this one. The first is an experiment conducted in 2009 by William S. Boyd. The second is another experiment conducted in 2005 by Kaeleigh Thorp.

William S. Boyd – Extracting DNA from Fruit in Stages of Ripeness

SUMMARY The objective behind Boyd’s experiment was to find out whether ripe fruit would yield more extractable DNA than unripe or overripe. His experiment involved bananas, kiwis, and strawberries. The result was that, in the case of the kiwis and strawberries, ripe fruit did in fact yield more extractable DNA. However, he found that unripe bananas yield more extractable DNA than ripe and overripe. He concluded that, as fruit ripens, the nutrients break down and it begins to decompose, which destroys cells containing extractable DNA.

CONNECTION As is the case with this experiment, Boyd wanted to know which stage of ripeness would yield the most DNA.

COMPARING AND CONTRASTING Procedures – Many of the procedures in Boyd’s experiment were similar yet different. Instead of putting the fruit in a bag and mashing it with his fingers, the fruit was blended in a food processor. The extraction liquid was chilled instead of the alcohol. The strawberry mixture was drained through nylon, but it was filtered and before being poured into the test tube instead of being filtered directly into the test tube. A graduated eyedropper was used to distribute the alcohol instead of pouring the alcohol down the side of the tube (Boyd, 2009).

Materials – Many of the materials in Boyd’s experiment were also similar. He used salt, water, and detergent to make his extraction liquid, which are the same materials as the extraction liquid in this study. He used alcohol to bring the DNA fibers together, blue dye to enhance the visibility and measurability of the extracted DNA, and a graduated test tube for measurements. However, there were some notable differences. He added pineapple juice to his extraction liquid, and his experiment used bananas and kiwis as well as strawberries, instead of strawberries alone (Boyd, 2009).

Kaeleigh A. Thorp – Extracting DNA from strawberries

SUMMARY The objective behind Thorp’s experiment was to determine whether unripe, ripe, or overripe fruit would yield more extractable DNA. Her experiment used primarily strawberries. She hypothesized that ripe strawberries would yield the most extractable DNA, as under-ripe strawberries were not yet fully developed and overripe strawberries were too far into the decomposition process. Her findings supported her hypothesis, as the ripe strawberries did yield more extractable DNA (Thorp, 2007).

CONNECTION Thorp’s experiment had the same objective as this study – to find out what stage of ripeness would produce the most extractable DNA in Strawberries (Thorp, 2007).

COMPARING AND CONTRASTING Procedures – The procedures of Thorp’s experiment differed very little from this study. She chilled her extraction liquid by sitting it in a bowl of water and ice cubes, where this study did not require the extraction liquid be chilled. She used a blender to mash the fruit, instead of mashing it in a bag using fingers, and added water to it – also something this study did not require. Lastly, again instead of using a plastic bag and fingers, she used a glass extraction rod to mix the extraction liquid with the blended strawberries (Thorp, 2007).

Materials – Thorp used nylon to filer the strawberry mixture, added blue dye to increase visibility and measurability, and used a graduated test tube for measurements, which are all in congruence with this study. However, instead of using salt, water, and detergent to make her own extraction liquid, Thorp used a premade ‘Powdered Buffer’ made up of sodium chloride, sodium bicarbonate, and papain enzyme. She also used a premade ‘Cell Blaster’, containing sodium dodecyl sulfate (Thorp, 2007).

A2a. Experimental Design Steps

Preparation:

Put the rubbing alcohol in a freezer or refrigerator, so that it will be cold enough to use later.

Step 1: Extraction Liquid

Combine a 1/2 teaspoon of salt, 1/3 cup of water, and 1 tablespoon of detergent in a jar to use as an extraction liquid. Mix it well and set it aside.

Step 2: Prepare DNA for Extraction

• Take 3 strawberries and place it in a plastic bag.
• Push out all excess air and seal tightly.
• Mash the strawberry into a pulp by squeezing the bag with fingers. Do this for 2 minutes.
• Pour 3 tablespoons of the extraction liquid into the plastic bag.
• Push out all excess air and seal tightly.
• Mix the strawberry and extraction liquid by squeezing the bag with fingers. Do this for 1 minute.

Step 3: Separate Liquid from Solid

• Stretch the nylon over the funnel.
• Place the tube of the funnel into a glass.
• Pour the strawberry pulp and extraction liquid over the nylon-lined funnel.
• Let the liquid drip into the glass for 30 seconds, or until the nylon stops dripping.
• Throw away the nylon and pulp.

Step 4: Extract the DNA

• Pour the liquid into the test tube, filling it 1/4th of the way.
• Retrieve the rubbing alcohol from the freezer.
• Carefully tilting the test tube, pour the rubbing alcohol so that it runs slowly down the side – instead of directly into the strawberry liquid – and forms a layer on top of the strawberry liquid.
• Make sure the alcohol and the strawberry liquid do not mix, as the DNA collects between the layers.
• Add one drop of blue dye to the mixture.
• Take a moment to marvel at the blue gel-like substance (DNA made visible) that forms between the layers.

Step 5: Measure Extracted DNA

• Using the graduated milliliter lines on the test tube, measure and record the amount of blue gel-like substance.

Step 6: Repeat Process

• Thoroughly clean the cups, jar, test tube and funnel using water and paper towels.
• Repeat all of the steps with other strawberries, making sure to record the amount of DNA so a comparison can be made.

A2b. Reasoning

This method of experimental design was chosen because it called for fewer and more readily accessible supplies, and also because it had fewer and uncomplicated steps.

The reasoning behind the method of testing this question was that overly complicated steps allow a higher margin for error. A simpler method provides fewer chances for mistakes to be made.

There were several other studies consulted that had methods of testing similar to what is used in this experiment, but there were no other studies that had methods of testing that were the same. The method of testing in this experiment was developed using bits and pieces of other studies.

The way this question is being tested is a better way than others because it was developed from bits and pieces of other more complicated studies, making it simpler.

A2c. Sequence of Events

The first step in collecting the data is adding one drop of blue dye to the layers of strawberry mixture and alcohol. The dye will collect between the layers and highlight the extracted DNA sitting in the middle, forming a blue gel-like substance. This gel-like substance will be measured and recorded using the millimeter markings on the graduated test tube.

A2d. Tools

• Measuring cups
• Measuring spoons
• Small jar
• Graduated test tube
• Funnel
• Nylon
• Drinking Glass
• 9 Unripe strawberries
• 9 Ripe strawberries
• 9 Overripe strawberries
• 1/2 teaspoon of salt
• 1/3 cup of water
• 1 tablespoon of detergent
• 9 Resealing plastic bags
• 1 drop of blue dye

A3. Variables

A dependant variable is what the scientist measures, and is the part of the experiment that relies on changes made by the independent variable.

An independent variable is what the scientist varies, and is the part of the experiment that decides the outcome of the dependant variable.

A controlled variable is what the scientist keeps the same, and the part of the experiment that must not change in order to ensure that the results are measurable.

DEPENDENT VARIABLE: Extracted DNA

INDEPENDENT VARIABLE: Strawberries in three stages of development: under-ripe, ripe, and overripe

CONTROLLED VARIABLE: The amount of strawberries, the amount of extraction liquid, the amount of alcohol, the amount of blue dye, the test tube and all other equipment.

A4. Threat Reduction to Internal Validity

Threats to the internal validity of this study have been reduced by the simple testable question, the properly identified variables, the control for outside influences, and the solid experimental procedure.

MATURATION The experiment will be started and completed in a single day, and will take a maximum of two hours. That will allow sufficient time for each trial to be conducted carefully and for the utensils to be cleansed – while assuring that there will be no time for the subjects to change before measurements.

REPEATED MEASUREMENTS The experiment will be repeated three times for each type of strawberry, with a new set of materials each time, equating in exactly nine trial runs. Each sample will be disposed of after results are recorded, before the next trial was done – none of the samples will be reused, nor will they come into contact with each other.

INCONSISTENCE IN INSTRUMENTATION  In every trial run, the measurements will be taken using the same graduated test tube, glass, jar, and measuring spoons. Every measurement made will be taken in a way identical to the one before it, so that the outcome of the experiment is not compromised.

EXPERIMENTAL MORTALITY The experiment is designed so that it cannot be completed without all of the subjects, meaning none of the subjects can drop out or be eliminated without completely derailing the study. This way, the trials will remain the same, and the results will not be compromised.

EXPERIMENTER BIAS The experiment did not involve and could not come to any result that the experimenter would benefit directly from. The experimenter remained objective throughout the study.

CONTROLLED VARIABLES There are several controlled variables that limit the factors that could skew the results. The tools for measurement remain the same throughout the trials so that there is no chance of new tools not providing the same results. The amount of strawberries stays the same – three per trial – so that the amount of extractable DNA is not distorted by one trial having more strawberries than the others. The amount of blue dye remains the same throughout the trials so that a larger amount of dye won’t make the results seem bigger than they are.

A5. Hypothesis:

I predict that the ripe strawberries will produce more extractable DNA than both the under-ripe strawberries and the overripe strawberries.

This prediction is based on observation. The under-ripe strawberry is still underdeveloped and very firm, meaning that it will likely produce less juice when mashed up – less juice, less DNA. On the flipside, the overripe strawberry is overdeveloped and in a state of degradation, meaning that the DNA will likely be broken down and harder to extract. The ripe strawberry will produce more juice than the overripe, and will not be as susceptible to bruising and damage as the under-ripe, meaning it will likely produce more extractible DNA.

B. Process of Data Collection

The data was collected by first adding one drop of blue dye to the layers of strawberry mixture and alcohol in the graduated test tube. The dye gathered between the layers and around the extracted DNA that sat in the middle, so that it appeared to be a blue gel-like substance. This made the extracted DNA easier to see, which in turn made it easier to measure. The DNA was then measured and recorded using the millimeter markings on the graduated test tube.

PROCESS OF RECORDING DATA:

TOOLS USED FOR COLLECTION:

1 drop of blue dye

Graduated test tube

UNIT OF MEASUREMENT USED:

Millimeter

METHOD OF RECORDING:

Unripe

Ripe

Over-Ripe

Trial #1

3/4 ml

3 1/4 ml

1/2 ml

Trial #2

1/2 ml

2 3/4 ml

1/4 ml

Trial #3

1 ml

3 1/2 ml

1/2 ml

B1. Appropriate Methods

The methods described above were the best to conduct the experiment on this testable question because they relied less on scales. In many other studies, the ulterior way of measuring was to take a wooden rod, spool the DNA, and weigh it on a milligram scale. The wooden rod would be previously weighed and subtracted from the weight of the DNA spooled rod (science buddies). With the method used here, the rod is cut out of the picture – only the DNA itself is measured. By doing this, we ensure that differently-weighted rods cannot skew the weight of the DNA.

The drop of blue dye made it easier to see the extracted DNA. It was important that the DNA be clear so that the measurements were at their utmost accuracy. The graduated test tube made it so the DNA did not have to be spooled or moved before measuring, which kept the specimens together and limited the chances of losing or damaging the specimens. Millimeters were the practical unit of measurement, as the amount of extracted DNA is very small.

C. Results

The unripe strawberries were very firm and still mostly green. They were harder to mash up. The first trial including the unripe strawberries yielded 3/4 ml of extractable DNA. The second trial yielded less with 1/2 ml of extractable DNA. The third trial was the most successful, yielding 1 ml of extractable DNA.

The ripe strawberries were softer and bright red all over. They were easier to mash. The first trial including the ripe strawberries yielded 3 1/4 ml of extractable DNA. The second trial yielded less with 2 3/4 ml of extractable DNA. The third trial once again was the most fruitful, yielding 3 1/2 ml.

The over-ripe strawberries were very soft, a darker red, and covered in bruises. They were the easiest to mash up. The first trial including the over-ripe strawberries yielded 1/2 ml of extractable DNA. The second trial yielded a mere 1/4 ml of extractable DNA. The third trial produced the same results as the first, with 1/2 ml of extractable DNA.

As the graph above shows, the ripe strawberries yielded a much larger amount than unripe and over-ripe strawberries. A single parallel is drawn between the unripe and over-ripe strawberries as they both yielded 1/2 ml of extractable DNA in separate trials – unripe reaching 1/2 ml in Trial 2, over-ripe reaching 1/2 ml in trial 1.

D. Conclusion

The graph above displays how great the leap in extracted DNA was between the strawberry types. Although the unripe yielded higher results than the over-ripe strawberries in two of the trials (Trials #1 and #3), they both produced a minimal amount of extractable DNA when compared to the ripe strawberries.

The unripe strawberries did not do as well because they are not yet mature. They provided less juice when mashed up for the extraction process, which provided fewer strands of DNA.

The over-ripe strawberries did the worst because they are on the downgrade of maturation. While they provided plentiful juice for extraction, the DNA strands were destroyed in the process of decay.

The ripe strawberries yielded the highest amounts of extractable DNA because they are at the hit the highest point of maturation. They provided the right amount of juice for the extraction process, and because they were at their peak, the DNA strands were intact.

D1. Confirmation of Hypothesis

I predicted that the ripe strawberries would produce more extractable DNA than both the under-ripe strawberries and the over-ripe strawberries. Based on my findings, with the ripe strawberries producing high amounts of extractable DNA where the unripe and over-ripe strawberries produced low amounts, it is evident that the ripe strawberries did yield the most extractable DNA. Therefore, I accept my initial hypothesis.

D2. Experimental Design as Key Factor

Experimental design is a key factor in science inquiry because it is the part in which groups are given their set treatments. In other words, experimental design is what decides if Group A will get Treatment B and Group C will get Treatment D, or if Group A will get Treatment D and Group C will get Treatment B. Without experimental design, the groups won’t be assigned their proper treatments, and a statistical analysis cannot be made.

If an experimental design is poorly constructed, it might miss some key components that affect the outcome altogether. For instance, if an experimental design lacks a control, nothing remains constant and some variables may not be counted for. Results of the experiment can be inconclusive, and when that happens, the study is rendered invalid.

D3. Replication

Replication is the process of repeating the steps of a procedure, so that an experiment can be duplicated again and again with the same results.

Replication is important because there is always the possibility that results in a study have been skewed, or an experiment has been conducted wrong. Repeating the process and including several trials provides a way to prove that results are correct and to procure an average when averages are called for.

This study is replicable because the instructions are clear and precise so that replication of the experiment as a whole is made easy, and the supplies needed are easy to find and easy to use.

D3a. Evaluation of Validity

Validity is important in science experiments because it proves the experiment was done correctly and the results were recorded accurately. Having a strong sense of validity means that the variables were measured reliably and strong causal links between the variables were found.

REPLICATION This study is replicable in that there were three trials to each study. To confirm which one yielded more DNA than the rest, each type of strawberry was tested in three separate trials – that way there were nine collective results each to consider instead of three.

This study uses that replication to prove its analysis of the data.

RELIABILITY This study is reliable thanks to that use of replication. Each type of strawberry was tested in three separate trials – three for unripe, three for ripe, three for over-ripe – to make sure the results were constant instead of a onetime occurrence. The results remained the in the same vicinity throughout the trials, proving that they are reliable.

EXPERIMENTAL DESIGN The experimental design remains valid thanks to its simplicity. There was very little margin for error, and so repeating each trial using the same methods and measurements was quite simple.

FUTURE QUESTIONS AND STUDIES Future studies might be expanded to use more than just strawberries. For example, one such study could compare ripe bananas to ripe strawberries, or ripe strawberries to ripe kiwis. Other studies might not involve strawberries at all, but replicate this study with a different fruit. For instance, would the results be the same with other fruits? Would ripe bananas yield more extracted DNA than unripe or overripe bananas?