Bio 240 lab report #3 - Grade: A PDF

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Amplification of Genes in Human cheek cells using the haeiii restriction enzyme and gel electrophoresis...

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AMPLIFICATION OF GENES IN HUMAN CHEEK CELLS USING THE HAEIII RESTRICTION ENZYME AND GEL ELECTROPHORESIS

GENETICS LAB GROUP 8 wcu

Abstract Every person is unique in their own way, meaning individuals inherit different genomes that affect their phenotypes. An example would be a person homozygous dominant for the TAS2R38 gene versus an individual who is recessive for the gene. This is the real cause or each individual’s uniqueness; there is a 0.1% difference that makes us, us. By amplifying a certain DNA fragment, scientists are able to discover what genotype an individual may obtain. Once that DNA sequence has been amplified, HaeIII is added to the DNA. This restriction enzyme is able to recognize a specific DNA sequence, in most cases the DNA is a palindrome, and can cut both of the DNAs strands. This DNA is loaded into a well and is ready to be run through gel electrophoresis. Electrophoresis will run for 45 minutes, after which the gel is then put under a UV light. Individuals are now able to read their genotype to the TAS2R38 gene to see if they are tasters of non-tasters. Out of 20 students, 14 had the TAS2R38 gene and were able to taste bitterness; six individuals did not have the gene and were not able to taste bitterness. A majority agreed that their results from the PTC paper were conclusive to the results from the gel electrophoresis.

Introduction Amplifying a specific DNA fragment of an individual has been essential in the past couple of years. One small sequence about four to eight nucleotides long, can determine if the individual obtains a certain trait or not. Humans share about 99.9% of DNA with one another; a small amount of differences can cause each person to be unique.1 For example, humans have a 1% difference in DNA with dolphins; just a small difference can cause several different outcomes in phenotypes. The TAS2R38 gene is inherited in a Mendelian fashion, causing

variation and codes for one of the bitterness taste receptor proteins.1 Amplifying the TAS2R38 gene, an individual can find out more information about themselves than if they are a taster or not. The bitter taste of 6-n-propylthiouracil has been known to be associated with a person’s body mass index and can be linked to certain food preferences.2 Using an amplified gene can also give indications on family relations, similar to using blood types to identify if an individual is related to other family members. Areas that exhibit variability in humans are known as single nucleotide polymorphisms (SNPs) and are a change in the genomic DNA sequence. If SNPs occur in a non-protein coding region, then there is no phenotypic expression. When SNPs occur in a protein-coding region, there is an amino acid change which may alter the protein function. The TAS2R38 gene produces a G-protein rich receptor that responds to phenylthiocarbamide (PTC). This gene, in particular, is a great indication why some individuals like or hate different kinds of vegetables; since some of the same molecules are found in vegetables as in the TAS2R38 gene/PTC. Once the gene has been amplified, restriction enzymes are then added to help recognize the specific DNA sequence that needs to be cut. When the restriction enzyme reads the specific sequence, it then is able to cut both strands of DNA. Since these areas are a palindrome, the enzyme is able to cut both strands. A palindrome is when the sequence on a DNA strand reads the exact same on both strands when read from the 5’ to 3’ ends.1 HaeIII is the restriction enzyme used in this experiment and is able to recognize the sequence 5’-GGCC-3’. When the enzyme recognizes this sequence it then cuts in between the G and C nucleotides. This specific enzyme is used to determine an individual’s genotype because of the recognition site the SNPs contain, that allow the enzyme to work.

If an individual is homozygous dominant for the TAS2R38 gene, then the DNA has a HaeIII restriction site causing the DNA to be cut. If an individual’s heterozygous for the gene, then about half of their DNA is cut, instead of the entire sequence. If an individual is homozygous recessive, then the DNA does not have an HaeIII restriction site and is not able to bind and cut the sequence. To test this, the amplified DNA sequence will be treated with Haelll restriction enzyme, loaded into a well and set up for the agarose gel electrophoresis. Since DNA is negatively charged, the DNA fragments will migrate towards the positively charged terminal. Small fragments are able to move faster through the gel matrix than the larger fragments.3 This process is applied to the DNA for about 45 minutes, enabling the small fragments to move to a specific frame on the gel. When the run is complete, a UV light is put to the gel, and the genotype for the individual can be read. The objective for this experiment is to allow an individual to learn his or her genotype for the TAS2R38 gene. I hypothesize that I will be either homozygous dominant or heterozygous for the TAS2R38 gene; since I had tasted a mild taste on the PTC paper, I believe I cannot be recessive in any way.

Methods Each individual in the laboratory was given a number at the start of the lab; this number was to be used to label every test tube to help maintain or track each person’s specific DNA. Every person was given a 1.5mL Eppendorf tube to start off and on the tube each individual had to write their number and their initials. Each person was provided with a plastic cup containing 10mL of saline solution. This solution was to be poured into the mouth and swished around vigorously for approximately 10-15 seconds. As each individual finished this task, the solution was to be spit back into the plastic cup. Until a pipet was obtained, the cup had to be swirled in

order to mix the cells up. A p1000 pipet set to 750 ml was then needed to transfer 1.5mL of the solution to the 1.5mL Eppendorf tube each student was originally given. The Eppendorf tube was then to be placed, along with other students, in a balanced configuration centrifuge. The centrifuge was then spin for about two minutes on full speed to create a visible pellet at the bottom of the test tube. When the centrifuge had ceased, each person then had to pour out the supernatant liquid on top of the pellet in the sink. Using a different tip, the same pipet was used to put more solution from the plastic cup into the 1.5mL Eppendorf tube. The test tube was spun one more additional time in the centrifuge, pouring out the supernatant in the sink again. This time leaving 0.1mL of the supernatant left in the test tube. Using a micropipette, mix the pellet with the supernatant making sure there is no pellet visible in the end. After this was completed, the instructor then added 200uL of well mixed chelex. The resulting sample was boiled at 99 oC for ten minutes in order for the denaturation process to occur. The test tube was then put in a micro centrifuge and spun for about 60 seconds at full speed. Using a p20 pipette, 20ml of solution was then transferred to a 0.5mL Eppendorf tube. Labeling the new test tube with the individual's initials and their number. These test tubes are stored on ice until they are needed again. Each person in the laboratory was then handed a PCR tube, already containing a master mix of buffer, Taq Polymerase, Mg++ ions and dNTPs. Using a fresh pipet tip on a p20 pipette, 2mL of the individual’s DNA were added to the reaction tube. Making sure there was DNA added, the tube was tapped on the desk to make sure the two solutions mix properly. After this step was finished, each student was given two pieces of paper: a control and a PTC paper. Each student had to put these papers in their mouth and record what they had tasted. After each student recorded their answer, the instructor then handed out a fresh 0.2mL Eppendorf tube and this tube

was to be labeled “U” along with the students assigned number; the original PCR product was then to be labeled “D” for digested. Taking the “D” test tube, the instructor would then add 1uL of enzyme HaeIII directly to the PCR liquid. Using a p20 pipette, mixing of the two solutions with an up down motion had to be done to make sure the solutions were in fact mixed. The “D” test tube was then added to a thermal cycler that was programmed for 30 minutes. At the start of the lab, the instructor had already prepared the agarose gel; this gel contains Ethidium Bromide, allowing the genes to be seen under a UV light at the end of the lab. In an orderly fashion, each student was to receive their “D” and “U” test tubes and then load 10uL of each into adjoining wells in the gel. The gel would then run for approximately 45 minutes; this game the small fragments time to move through the gel’s matrix. After the 45 minutes concluded, each person was then able to look at the gel under a UV light to determine their genotype.

Results Once the gel electrophoresis was completed and everyone saw their genotype, the instructor photographed the gels; these are the two pictures below. Individuals who have two bands on the 221bp fragment are considered homozygous recessive. Individuals who have one band on the 221bp fragment and one band on the 177bp fragment are known as homozygous dominant. Those who poses two bands on the 221bp fragment and one additional band on the 177bp fragment are considered heterozygous. In the table below, is the sum of how many individuals are in each genotypic category. Out of a class of 20 individuals, ten are heterozygous, four are homozygous dominant, and six are recessive for the TAS2R38 gene. The majority of the class has the taster gene, but more individuals being heterozygous than homozygous dominant; I personally was heterozygous for the TAS2R38 gene.

Table 1. The three different genotypes and how many individuals obtained each genotype Genotypes

Number of individuals who

Total number of individuals

Heterozygous Homozygous dominant Homozygous recessive

obtain this phenotype 10 4 6

in the laboratory 20 20 20

Picture 1. The large gel that a majority of the class loaded their DNA

Picture 2. The small gel in which only three individuals loaded their DNA

Discussion For an individual who is a non-taster or recessive, then when their chromosome seven DNA is amplified by PCR, there is no restriction site for the HaeIII restriction enzyme to attach.3 Thus, resulting in the DNA to not be cut by the HaeIII restriction enzyme; This is exactly what is occurring in the individuals that are recessive for the gene. For an individual who is a taster, then when their chromosome seven DNA is amplified by PCR, the HaeIII restriction enzyme will be able to recognize the DNA sequence 5’-GGCC-3’.3 HaeIII will cut this sequence on both strands (DNA is a palindrome) in between the G and C nucleotides; this is what’s occurring in the individuals who are homozygous dominant for the taste gene. Individuals who are heterozygous, their DNA is only cut halfway causing three bands to show up on the gel instead of just two like the homozygous dominant. These results should correlate with the PTC paper that everybody had

tasted in the beginning of the lab. For my hypothesis, I hypothesized that I would end up being heterozygous or homozygous dominant for the TAS2R38 gene; since I had tasted mild on the PTC paper. Looking at my genotype on the gel, I am indeed heterozygous for the taste gene; concluding in an accurate hypothesis. In every experiment, there is always room for error. Such as tasting the PTC paper; if someone thinks they tasted or didn’t taste something then that could affect how that individual thinks for the rest of the lab, until they get their results. Another error could possibly be the pipetting; if each person did not pipet the right amount into the well or into the solutions, then the DNA could possibly not show up in the gel after the 45 minutes was up. Also, another error could occur if the individual did not carefully keep track of which test tube was which, accidently inserting the wrong test tube solutions; this would also cause the DNA to probably not show up in the end. Knowing exactly what to pour out and when, and when to mix things up is a huge part of this lab; if that were to be messed up in any way then your DNA would then be compromised. Knowing the process on how to amplify a gene is very crucial; this allows geneticists to be able to take any unique gene not only for a human, but an animal as well to find the genotype. Geneticists have now come to find that a unique gene could be tied to more phenotypes than originally thought. In a study conducted by the Society for the Study of Amphibians and Reptiles, geneticists were able to amplify DNA from the skin of a Forest Dwelling Tree Frog. With a couple of skin swabs, they were able to obtain Brain-derived neurotrophic factor, tyrosinase precursor, and three types of mitochondrial DNA.4 With this information, the Society of the Study of Amphibians and Reptiles can conclude different environmental and physical effects can indeed effect unique genes as a whole. This is true with humans as well; the results of

a unique gene can also help determine if a child really does belong to the parents, since these unique sets of genes are inherited in a Mendelian fashion. This is the same idea as using blood to help identify an individual into their family. This can be beneficial in the long run for health reasons because if both parents carry a specific gene you could determine if the child would have or carry this gene as well. Some of these unique genes can be linked to different phenotypes. Such as the TAS2R38 gene, it can be linked to body mass index as well as certain food preferences.2 Every individual on this planet is different because of that 0.01% people do not share with one another; that small difference is what really makes the personality of an individual.

References

1. Lab 5A: Isolation and Amplification of a Fragment of Mitochondrial DNA, BIOL240-77, Western Carolina University, November 6th, 2017

2. Barbarossa, Iole Tomassini, et al. "A Rapid Screening Method for the Identification of a Single-Nucleotide Polymorphism in the Carbonic Anhydrase VI Gene in Studies of Sensitivity to the Bitter Taste of 6-N-Propylthiouracil." Genetic Testing and Molecular Biomarkers, vol. 15, no. 10, Oct. 2011, pp. 721-724. EBSCOhost, doi:10.1089/gtmb.2011.0040.

3. Lab 5B:Restriction Enzyme Digest and Gel Electrophoresis, BIOL240-77, Western Carolina University, November 13th, 2017

4. STREICHER, JEFFREY W., et al. "Extracting and Amplifying DNA from Skin Swabs of a Forest Dwelling Tree Frog Species (Nyctixalus Pictus)." Herpetological Review, vol. 45, no. 3, Sept. 2014, pp. 421-424. EBSCOhost, proxy195.nclive.org/login? url=http://search.ebscohost.com/login.aspx? direct=true&db=a9h&AN=112002442&site=ehost-live&scope=site....