In order to isolate DNA, the solution or substance used as extraction liquid has to do a lysis process within the cells, meaning that it needs to break down the cells. Because plant cells like strawberries and tomatoes contain a cell wall, it is harder for the solution to penetrate the cell membrane and obtain the genetic material. In this experiment we will extract DNA from two plants and see if the solutions were able to breakdown the cell wall.
What is the real appearance of DNA?
To perform DNA isolation from both tomato and strawberry pulp with the same extraction method.
Comparing tomato and strawberry DNA samples, and determine if there is any observable difference among them.
Despite the various characteristics that make species so different from each other, there is one specific molecule that is present in every single living thing without exception, which is DNA. Deoxyribonucleic acid, also known as DNA, is a molecule that every organism, from amphibians, to reptiles, to mammals, contain within their cells, and it is of significant importance because it’s the blueprint for everything that happens within the organism. Not only does it code for the different features that make us different from one another, but it is also the one responsible for indicating other structures and molecules how to behave and which functions to perform in order to maintain life. Because of DNA, organisms can fully developed and grow, as well as carry out their function in the environment.
Having stated the importance of DNA, scientists have long made experiments regarding this molecule. Evaluating it makes it useful to obtain all the codes and genetic material that makes up an organism, meaning that all of its composition information is available. At the same time, its analysis involves a detailed process because it is a very small molecule, only observable through a microscope. Its capacity of chromosome arrangement makes it possible for cells to contain the complete set. Finally, the scope of this report will be to evaluate the experimentation on DNA extraction in two species of plants; tomatoes and strawberries.
This experiment is of great importance because it shows us the components that make up our bodies and the structures that are crucial for all of the functions of our body to perform accurately. It gave us the opportunity to explore one of the fundamental molecules, the one that makes up everything we know, DNA. At the same time, research on this type of molecule has had a lot of relevance, because there are still many things that we don’t know and need to understand about its complexity. Throughout the extraction process, scientists can analyze and manipulate it, and it allows them to detect genetic disorders like different types of trisomies, fingerprints, create GMOs, and even produce products for the medical industry such as insulin, antibiotics, and when focused in the areas of genetics, it allows us to understand how heritable traits are passed down from our parents on to us, and how DNA is what makes the 7 billion people that live on earth so different. As biology students, getting to learn about such a significant component in our lives makes us understand more about life itself.
As in any other laboratory session, it is required that students wear lab coats at all times, and it is recommended that people with long hair tie it back to avoid any inconvenience. For this experiment in particular, students must be careful and keep cautious when using sharp objects like scalpels, and glass materials like test tubes, measuring cylinders and beakers. Since we are going to be using ethanol in this experiment, we need to make sure that it is measured carefully in its respective container, because it may cause irritation if it is exposed to the skin. As for the saline solution, students must be careful with the exposure they hold to this material, especially eye contact.
When exposed to freezing ethanol, saline solution and dishwasher soap, there will be a lysis process in the strawberry and tomato cells, and their genetic material (DNA) will be released.
Independent variables: Detergent, saline solution and freezing ethanol.
Dependent: Observable differences within the tomato and strawberry DNA samples that we obtain during the experiment (quantity extracted, purity of the sample as well as DNA concentration)
DNA (deoxyribonucleic acid) is an organic chemical with elaborate structure found in prokaryotes, eukaryotes, and many viruses. DNA is responsible for storing information about our bodies and coding genetic information for transmitting inherited traits from parents to offsprings. The actual chemical was discovered in 1869, but the fact that it aids in the process of genetic inheritance was not proven until 1943. Rosalind Franklin, James Watson, and Francis Crick were the scientists who determined the structure of DNA.
Fig. 1, Francis Crick and James Watson.
Since DNA is present in almost all of the living thing on the planet, we can say that it can be found in both eukaryotic and prokaryotic cells. In prokaryotic cells, it is located in an area called the nucleoid. In eukaryotic cells, the majority of the genetic material is found inside the cell’s nucleus, but there is also an independent section of the molecule that can be found in other organelles called mitochondria and chloroplasts, the organelles responsible for our respiration process. responsible for respiration, being these mitochondria and chloroplasts. Within the nucleus, this complex molecule is ridiculously tightly packed for it to be able to fit, because the DNA itself is incredibly large. (Khan Academy, 2018)
Fig. 2, DNA structure
.For the genetic material to be “packed” accurately, it must be wrapped around a protein called a histone, and the loops that come up as a result are called nucleosomes. Furthermore, these two stack together to form a DNA complex called chromatin. When all of these loops are looped around together, we get a molecule called chromosomes. Even though chromosomes are the biggest form in which genetic material is arranged, it is so small that it is few times that people are able to see it under a microscope.
DNA is a double helix polymer, in other words, a spiral with two DNA strands tangled up around one another. Each strand of the molecule is made up of monomer nucleotides. These consist of a sugar molecule, a phosphate group and one of the four nitrogenous bases. these bases are divided into two groups. The purines, adenine and guanine, and pyrimidines, cytosine and thymine. These groups of molecules are joined by a covalent bond in the phosphate of one nucleotide and the sugar of the next, which is why it is often known as a phosphate-sugar backbone. This is what carries the four bases and the sequence of these, are the ones than encode all the information about or bodies. The sequence of amino-acids that is encoded in DNA residues in proteins, using the genetic code. The fact that DNA is what codes proteins, in other words, tells them what to do, is what makes DNA so important. This molecule controls how the organism behaves, reacts and develops , and also the traits that it passes down generations of species. (US National Library of Medicine, 2019)
Fig. 3 Amino acid sequencing.
Fig. 4. Chromosomal structure.
The genetic code are the set of rules that information in DNA sequences follows in order to be translated into proteins. It defines a mapping between tri-nucleotides sequences called codons and amino acids, because every group of three nucleotides are what specify one amino acid (Science Daily, n.d.) The vast majority of genes are coded with the same code, the “standard genetic code” but there are many variants, so this one is not a universal code. An example of genetic coding that varies from the standard genetic code is the process of protein synthesis that happens in the mitochondria of the cell. The portion of the genome that codes for protein is referred as gene. Thesare short sections of DNA that carry information for particular characteristics like eye colour.Different sets of genes carry information for different physical characteristics. There are many genes in a chromosome. In a cell nucleus, DNA is organised into coiled strands called chromosomes. Human beings have 46 chromosomes in each cell. Half of our chromosomes are inherited from one parent and half from the other. As humans, therefore, we have 23 chromosomes from each parent.
This explains why organisms can share characteristics from both parents. (British Broadcasting Company, 2019)
Chromosomal structure is made up of duplicate held together by centromeres, which are a point of connection of a protein fibre. This connection is generally located in the middle that separate the long and the short arms, and aid with the process of cell division, making it possible for sister chromatids to attach to each other. This way, alleles will be able to separate in the division process, and can be further on combined with other ones to form a combination of traits. The similarities that are shown between members of the same species are due to common genes that code for the same characteristics or traits. However, every organism is unique and different from each other because alleles get combined and create an exclusive set of genes that will vary the gene expression.
Fig. 5 Chromosomes in cell division.
As we have previously stated, the genome is not the same in all the biological classifications. The chromosome number often varies in number, shape and content. We are able to study and observe these trends through their karyotype. A karyotype is an image of the chromosomes of a living thing. They are key to knowing the differences in chromosomes in different species, for example, the fact that there are 48 chromosomes in chimpanzees but 24 in tomatoes. In addition to this, It also gives an idea of chromosome classification. It includes how are chromosomes divided; how many of them can be found within the nucleus of every cell or how many of them there are for every type. The most common is diploid nuclei, in which chromosomes are arranged in pairs. Illustrated by the case of humans, which are diploid, we have a total of 46 chromosomes, arranged in 23 pairs. Consequently, a polyploid nuclei is the one that has more than two homologous sets chromosomes in each cell, and it is more common in some plants and crops. There is a wide range of polyploidy, including triploids like bananas, and like potatoes. (New World Encyclopedia, 2008). In the case of humans, the National Human Genome Institute stated that the complete DNA sequence consists of 3 billion base pairs that form 46 chromosomes of each cell. (23 pairs). 22 of these 23 pairs are autosomes, and the 23rd pair is known as the sec chormosome. This is the chromosome that influences the sex that a fetus will develop to be.
Fig. 6 Human Karyotype
Fig 7. DNA structure.
As mentioned before, DNA belongs to the biomolecule classification of nucleic acids. Its structure consists of three basic parts: a pentose sugar (a five carbon atom), a phosphate group and a nitrogenous base. Nucleotides are linked together by covalent bonds, and at the same time, the nitrogenous bases are joined by hydrogen bonds. There are four nucleotides; Adenine, Cytosine, Thymine and Guanine(A, C, T, G) and they can be linked together in any order because their phosphate and sugar structure are the same. DNA molecules in prokaryotes can be found in the cell’s cytoplasm, and in the nucleus for eukaryotes. This last organelle is characterized for being surrounded by an extra membrane that provides it with protection, aside from the plasma membrane that encloses the cell. That being said, eukaryotes have two membranes; one for nucleus or genetic material protection and another one for general cell delimitation.
2 small Ziploc bags
2 fresh strawberries
1 fresh tomato
2 big gauzes (cheesecloths)
Fig. 8, Materials
Completely line the funnel with cheesecloth. Insert the funnel into the empty tall drinking glass.
Fig. 9 step 1
Remove and discard the green tops from the strawberries.
Put the strawberries into a clean resealable plastic sandwich bag and push out all of the extra air. Seal the bag tightly.
With your fingers, squeeze, and smash the strawberries for two minutes
Fig 10 & Fig 11, step 4
Mix one half teaspoon of salt, one third of water, and one tablespoon of dishwashing liquid in an empty beaker. This is your extraction liquid.
Fig 12 & 13 step 5
Add three tablespoons of the extraction liquid you prepared to the strawberries in the bag. Push out all the extra air and reseal the bag.
Fig 14, step 6
Squeeze the strawberry mixture with your fingers for one minute.
Fig 15, step 7
Pour the strawberry mixture from the bag into the funnel. Let it drip through the cheesecloth and into the tall glass until there is very little liquid left in the funnel (only wet pulp remains).
Fig 16, step 8
Pour the filtered strawberry liquid from the tall glass into the small glass jar so that the jar is one quarter full.
Fig 17, step 9
Measure out one-half cup of cold rubbing alcohol
Tilt the jar and very slowly pour the alcohol down its side. Pour until the alcohol has formed approximately a one-inch-deep layer on top of the strawberry liquid. You may not need all of the one half cup of alcohol to form the one-inch layer. Do not let the strawberry liquid and alcohol mix.
Fig 18, step 11
Study the mixture inside of the jar. The strawberry DNA will appear as gooey clear/white stringy stuff.
Fig 19, step 12
Repeat the procedure with the fresh tomato. If the tomato isn’t mature yet, or if it doesn’t expel much liquid, add a few tablespoons of water.
Take pictures of the results.
Fig 21 & 21, step 14
Result #1 Strawberry DNA. Fig 22 strawberry sample.
This sample shows the Strawberry DNA that we extracted. The DNA can be very clearly seen in this sample. We did however observe that our sample was a little bit dirty with bubbles that originated when mixing the dishwasher soap with the fruit puree that could’ve been avoided by mixing the substances with more moderation. This can denote the presence of the ethanol substance, but the limit between its concentration and the rest of the sample is still clear. It is noticeable that the middle quartile range of the test tube is the one that contains the most genetic material. Now referring to the genetic material extracted, DNA molecules can be recognized as the cloudy, white, transparent bulbs that are seen in the majority of the model. Regarding the illustrated color, this one has a dark pink or red colour, mainly because it is the same tone as the strawberry tissue. In this case, the dishwasher soap didn’t have any effect on influence on the sample´s color.
Result #2 Tomato DNA. Fig 23 Tomato sample.
This picture shows a sample of DNA that we extracted from tomato cells. the DNA can be clearly identified. In the middle we can see clearly that the sample lightens in colour, which shows us mildly the genetic material. Where the tomato breaks and makes a horizon with the ethanol, is where we can very clearly see the DNA. we however, had an inconvenience where the cheese cloths were really thin, and the solution did not purify as well as we would’ve liked it to. The top and foggy-like part of the sample shows the ethanol layer, and the bubbles under it denote the limit or separation. What is found is the middle are the DNA molecules. They can be identified because of their cloud-like shape, and the mixture concentration. The sample showed an orange almost salmon colour to it, because the fruit is originally red but the detergent was green the final appearance must have derived from such combination. Compared to other samples seen in the laboratory, this sample one got a brighter tone. This could have happened because the amount of dishwasher soap could have varied moderately.
As we had expected in the established hypothesis, the dishwasher soap, the saline solution and the ethanol together performed the lysis process on the cells, and that led to the release of DNA molecules from the cells of the strawberry and tomato tissue, as well as its precipitation on the sample. As discussed previously in this report, cell membranes are made of a phospholipid bilayer that gives the cell’s nucleus a layer of protection and moreover, needs to be broken down in order to perform DNA extraction. In addition to this, It was also previously discussed that the genetic material in vegetable cells is preserved more rigorously, as they possess a cell membrane made up of various polysaccharides like is cellulose. This brings us to conclude that these two things need a very specific set of components in order to be able to break this layer down.
Firstly, i will assess the chemical components that , helped break down the cell and release the DNA. Dishwasher soap has a substance called “Laurel Sulfate” which aids in removing proteins and fats, just as when used to wash the dishes. This substance helps pull the fats and proteins apart from the membrane that covers the nucleus. In this specific case, the properties of lipids can clearly be identified. Another factor that aids in us being able to see the genetic material, is that DNA molecules have a negative charge, which means that the molecules naturally repel each other due to their charge. Because of this, the saline solution neutralizes the negative charges making them come together, which is what makes it a big piece of DNA that we can observe without the help of a microscope or advanced laboratory equipment. Once the detergent and the salt have an effect on the cell membrane and break it down in order to release DNA, it is transparent, meaning that it cannot be seen. Ethanol is what makes it possible for us to be able to see the genetic material. This process is called precipitation. However, it is important for the ethanol to be really cold, because if it isn’t, the effect might not happen with the noticeable expected results. To conclude, these substances are the ones to breakdown the membranes containing DNA, and are what make us observe it and analyze it.
When comparing both samples together, a significant remark is how despite the fact that both have a cellular wall structure aside from the main membrane, the observations that we made allow us to conclude that the DNA extraction from strawberry cells is more abundant than the one extracted from tomato cells. In this laboratory we don´t count with the necessary equipment to quantify a determined amount of extraction, but indeed, the molecules that have a foggy and cloudy appearance are bigger and dominant in the first sample. I believe that this happened because of the chromosome and gene arrangement that was previously described. Since strawberries are generally octoploids, while tomatoes are diploids, tomatoes have 12 sets, all of which have two chromosomes. In total, they have 24 chromosomes. On the other hand, strawberries have 7 sets. Despite this number being smaller, being octoploid means that they have 8 chromosomes for each pair. Therefore, they end up having 56 chromosomes in total. The fact that this amount is greater means that there will be more genetic information in such cells that are going to be useful for analysis. Additionally, ripe strawberry cells have the advantage of containing enzymes known as pectinases and cellulases that aid in breaking down the cell walls.
Throughout the DNA extraction experiment, there were several different outcomes and results. The hypotheses established were confirmed: based merely on observation and qualitative practices, the DNA molecules were better identified in the strawberry cells, as compared to the tomato cells. We were able to confirm our hypothesis; we could prove that agents including detergent or dishwasher soap, saline solution and ethanol are essential to carry out a lysing process. The examination evaluated the process by which the cell wall and the cell membrane were broken down, the lipids and the proteins were separated, and the role of precipitation was observed, all with the purpose of releasing a section of the genetic material in an organism’s cells. The experimentation also confirmed the roles and the importance of the alcohol, the dishwasher and the salt to perform membrane dissolution and other processes, such as the membrane lysis and the precipitation. If it weren’t for these materials, the extraction could not have been successfully carried out.
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