Lactase Synthesis Lab Report
Introduction On average Americans eat 627 lbs. of dairy products each year (“USDA ERS - Dairy Data,” n.d.). When consumed the principle carbohydrate in dairy, lactose a disaccharide sugar, is either digested in the small intestine by lactase or is passed to the large intestine where it is broken down by bacteria. The lactose disaccharide is composed of the monosaccharides glucose and galactose. Lactase in an enzyme that catalyzes the breakdown of lactose into its two monosaccharides in a hydrolysis reaction so they can be absorbed through the intestinal mucosa membrane (Olds & Sibley, 2003). For the majority of mammals, lactase activity is significantly reduced after being weaned from their mother’s milk. Since most mammals do not continue
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to consume lactose after weaning, the reduction in lactase production has no significant effect. Humans continue to ingest lactose even after weaning, yet the majority of human’s experience this reduction in lactase post weaning resulting in lactose being needing to be broken down by bacteria in the large intestine. When the bacteria in the large intestine metabolize lactose a mixture of hydrogen, methane, and carbon dioxide gas is produced from lactose fermentation. When this fermentation occurs after dairy is consumed symptoms of abdominal cramps, bloating, flatulence, and diarrhea would be experienced (Biology 225 lab manual, S2017). This person would demonstrate the phenotype of lactose intolerance due to the fact that they are lactase non-persistent, meaning they no longer produce the enzyme lactase. Someone who is lactose tolerant and does not exhibit these symptoms after consuming dairy are lactase persistent, meaning they still have active lactase in their gut to break down lactose.
The gene that codes the enzyme lactase is called the LCT gene.
The mutation investigated in this experiment that is associated with lactase persistence is the located upstream of the LCT gene. In this mutation a single nucleotide polymorphism changes a cytosine into a thymine that then can be detected using the polymerase chain reaction technique (Biology 225 lab manual, S2017). In this experiment amplification Refractory Mutation System PCR was used to detect this nucleotide change. Two primers were used in two different PCR reactions, one to detect the wild type allele and the other to detect the mutant allele. These primers are allele specific so the mutant type primer has the reverse complement of the single nucleotide polymorphism responsible for lactase persistence and the wild type primer has the reverse compliment of the gene responsible for lactase non-persistence (Biology 225 lab manual, S2017). Lactase persistence is a dominant mutation, but only about 30% of the world’s population is lactase persistent, these people are typically from Northern Europe, and parts of Africa and Asia where people have been consuming dairy for millennia. The other 70% of the world’s population is lactase non-persistent which is the recessive wild-type allele (Biology 225 lab manual, S2017). The objective of this experiment is to determine the predicted phenotype of lactase persistence or lactase non-persistence of all biology 225 lab students and compare their predicted phenotype to …show more content…
their self-reported phenotype. My self-reported phenotype is lactase tolerance, therefore my genotype would correspond to the phenotype of lactase persistence. Since lactase persistence is the mutant allele and lactase non-persistence is the wild type allele the majority of the lab students should be lactase non-persistent.
Materials and Methods The genomic DNA used for this experiment to test for lactase persistence and lactase non-persistence was crudely isolated from cheek cells using buccal wash protocol. Then following the procedure outlined in the lab manual, the sample was centrifuged at a low speed, the supernatant was removed, the cells were vortexed to be resuspended, and transferred into a microfuge tube to be microfuged at a high speed. Next most of the remaining supernatant was removed and the cells were resuspended. Chelex beads were then added to the sample and the sample was heated on a heat block. Once removed and let cool the sample was spun on the microfuge to pellet the cell debris and chelex (Biology 225 lab manual, S2017). The supernatant which now contained the DNA was transferred to a new tube to be used for PCR. Two PCR reactions were done one with the wild type primer and the other with the mutant primer. In two separate tubes with their respective primers, the primers were combined with the genomic DNA sample, and mix containing water, buffer, deoxy ribonucleotides, and Taq polymerase, following the procedure outlined in the lab manual (Biology 225 lab manual, S2017). The samples were then placed on the cool rack and in the PCR for a “hot-start” PCR experiment. After the genomic DNA was amplified by PCR the amplified samples were electrophoresed to identify their products based on molecular weight. The wild type and mutant primers were combined with loading die and loaded onto 1.5% agarose gels along with a molecular weight marker. Once the power supply was turned on and the gels ran they were illuminate and photographed for analysis.
Results
The predicted genotypes all of the biology 225 lab students were determined by analyzing the results of the argose electrophoesis of the PCR products. The resulting gel with my DNA sample can be seen in figure 1. Lane 1 on both rows contained the molecular weight marker. My PCR product containing the wild type primer was in lane 2, and my PCR product with mutant type primer was in lane 3 (figure 1). In the lane with my PCR product and wild type primer there was no band and in the lane with my PCR product and mutant type primer there was a band present (Figure 1). The results of the electrophrese on agarose gels of all the students PCR products were used in determining their predicted genotype and whether they were heterzyogous, homozygous mutant or homozygous wild type.
Figure 1. Standard and PCR treated DNA samples with either the wild type or mutant type primers on a 1.5% agarose gel. A DNA molecular weight marker (lane 1), and my PCR treated DNA samples with the wild type primer (lane 2) and mutant type primer (lane 3) were loaded onto a 1.5% agarose gel, separated by electrophoresis, and photographed after being illuminated using UV light.
Analysis of all of the PCR products loaded onto agarose gels from all 5 lab sections after electrophoresis can be seen in table 1. Based on the gels from all 5 lab sections 42.9% of the test subjects are homozygous mutant, 38.6% are heterozygous and 18.6% are homozygous wild type (table 1). From the data collected from all lab sections 81.4% of test subjects were predicted to have the lactose tolerant phenotype while 18.6% were predicted to have the lactose intolerant phenotype. When asked to report their phenotype 74.2% of test subjects reported they were lactose tolerant, whereas 25.8% reported to be lactose intolerant (table 1). The percent of predicted genotypes that matched self-reported phenotype was 65.2% (table 1)
Table 1. Summary of the genotype and phenotype data for the tested lactase gene
Lab Section % Homozygous Wild type % heterozygote % Homozygous Mutant % predicted lactose tolerant (based on genotype) % predicted lactose intolerant (based on genotype) % self- reported lactose tolerant % self- reported lactose intolerant % matching
Monday 6.25 25 68.8 93.75 6.25 75 25 68.8
Tuesday 21.4 57.1 21.4 78.6 21.4 91.7 8.3 83.3
Wednesday 26.7 26.7 46.7 73.3 26.7 80 20 53.3
Thursday 33.3 20 46.7 66.7 33.3 61.5 38.5 66.7
Friday 0 80 20 100 0 60 40 60
All Sections 18.6 38.6 42.9 81.4 18.6 74.2 25.8 65.2
Discussion The objective of this experiment was to determine the predicted genotype of lactase persistence or lactase non-persistence of all biology 225 lab students. The gel in figure 1 with my PCR products produced a band in lane 3 with the mutant allele specific primer and no band was produced in lane 2 with the wild type allele specific primer. This result indicates that I am homozygous mutant because only the mutant type primer binded to my genomic DNA during the PCR reactions. Thus, my predicted phenotype is lactose tolerance. This result corresponds to my observed phenotype of lactose tolerance and is consistent with my ethnic and demographic background as I am partially of Northern European decent and am an American white two ethnic backgrounds where lactase persistence is most prominent. From analyzing the PCR products on the agarose gels from all lab sections it can be inferred that the majority of the biology 225 students have the predicted genotype of lactose tolerance, with 81.4% predicted to be lactase persistant.
The percent of students who self- reported to be lactose tolerant was 74.02%, and the percent who self-reported to be lactose intolerant was 25.8%. The percent of subjects whose predicted phenotype matched their self-reported phenotype of either lactose tolerance or intolerance was 65%. Variations in predicted phenotype and self-reported phenotype could result from other mutations in the DNA that could cause lactase persistence or non-persistence other than the single nucleotide polymorphism tested for in this experiment or another chemical component in dairy that they cannot digest could cause them to exhibit symptoms of lactose
intolerance.
The results of this experiment were not expected since lactase persistence is the mutant allele and lactase non-persistence is the wild type allele most commonly found in the world population. Thus, this result is not consistent with the hypothesis that the majority of students should be lactose intolerant because it is the wild type allele. Lactase persistence exhibits an interesting characteristic of being the dominant allele even though it is mutant type, while lactase non-persistence is the recessive allele and wild type (Biology 225 lab manual, S2017). This unique characteristic of lactase persistence being dominant could explain why lactase persistence was so prominent in biology 225 lab students since 38.6% of students where heterozygous and produced both mutant type and wild type primer binding. A factor not investigated during this experiment but that could be investigated to explain the experimental results being inconsistent with the hypothesis is the demographics of the lab students. If the majority of the lab students are of Northern European, African or Asian from areas where people have been consuming milk for millennia then this could explain why the results varied from the hypothesis. The majority of bio 225 lab students were predicted to be lactose tolerant and retained their ability to produce lactase after weaning.
to consume lactose after weaning, the reduction in lactase production has no significant effect. Humans continue to ingest lactose even after weaning, yet the majority of human’s experience this reduction in lactase post weaning resulting in lactose being needing to be broken down by bacteria in the large intestine. When the bacteria in the large intestine metabolize lactose a mixture of hydrogen, methane, and carbon dioxide gas is produced from lactose fermentation. When this fermentation occurs after dairy is consumed symptoms of abdominal cramps, bloating, flatulence, and diarrhea would be experienced (Biology 225 lab manual, S2017). This person would demonstrate the phenotype of lactose intolerance due to the fact that they are lactase non-persistent, meaning they no longer produce the enzyme lactase. Someone who is lactose tolerant and does not exhibit these symptoms after consuming dairy are lactase persistent, meaning they still have active lactase in their gut to break down lactose.
The gene that codes the enzyme lactase is called the LCT gene.
The mutation investigated in this experiment that is associated with lactase persistence is the located upstream of the LCT gene. In this mutation a single nucleotide polymorphism changes a cytosine into a thymine that then can be detected using the polymerase chain reaction technique (Biology 225 lab manual, S2017). In this experiment amplification Refractory Mutation System PCR was used to detect this nucleotide change. Two primers were used in two different PCR reactions, one to detect the wild type allele and the other to detect the mutant allele. These primers are allele specific so the mutant type primer has the reverse complement of the single nucleotide polymorphism responsible for lactase persistence and the wild type primer has the reverse compliment of the gene responsible for lactase non-persistence (Biology 225 lab manual, S2017). Lactase persistence is a dominant mutation, but only about 30% of the world’s population is lactase persistent, these people are typically from Northern Europe, and parts of Africa and Asia where people have been consuming dairy for millennia. The other 70% of the world’s population is lactase non-persistent which is the recessive wild-type allele (Biology 225 lab manual, S2017). The objective of this experiment is to determine the predicted phenotype of lactase persistence or lactase non-persistence of all biology 225 lab students and compare their predicted phenotype to …show more content…
their self-reported phenotype. My self-reported phenotype is lactase tolerance, therefore my genotype would correspond to the phenotype of lactase persistence. Since lactase persistence is the mutant allele and lactase non-persistence is the wild type allele the majority of the lab students should be lactase non-persistent.
Materials and Methods The genomic DNA used for this experiment to test for lactase persistence and lactase non-persistence was crudely isolated from cheek cells using buccal wash protocol. Then following the procedure outlined in the lab manual, the sample was centrifuged at a low speed, the supernatant was removed, the cells were vortexed to be resuspended, and transferred into a microfuge tube to be microfuged at a high speed. Next most of the remaining supernatant was removed and the cells were resuspended. Chelex beads were then added to the sample and the sample was heated on a heat block. Once removed and let cool the sample was spun on the microfuge to pellet the cell debris and chelex (Biology 225 lab manual, S2017). The supernatant which now contained the DNA was transferred to a new tube to be used for PCR. Two PCR reactions were done one with the wild type primer and the other with the mutant primer. In two separate tubes with their respective primers, the primers were combined with the genomic DNA sample, and mix containing water, buffer, deoxy ribonucleotides, and Taq polymerase, following the procedure outlined in the lab manual (Biology 225 lab manual, S2017). The samples were then placed on the cool rack and in the PCR for a “hot-start” PCR experiment. After the genomic DNA was amplified by PCR the amplified samples were electrophoresed to identify their products based on molecular weight. The wild type and mutant primers were combined with loading die and loaded onto 1.5% agarose gels along with a molecular weight marker. Once the power supply was turned on and the gels ran they were illuminate and photographed for analysis.
Results
The predicted genotypes all of the biology 225 lab students were determined by analyzing the results of the argose electrophoesis of the PCR products. The resulting gel with my DNA sample can be seen in figure 1. Lane 1 on both rows contained the molecular weight marker. My PCR product containing the wild type primer was in lane 2, and my PCR product with mutant type primer was in lane 3 (figure 1). In the lane with my PCR product and wild type primer there was no band and in the lane with my PCR product and mutant type primer there was a band present (Figure 1). The results of the electrophrese on agarose gels of all the students PCR products were used in determining their predicted genotype and whether they were heterzyogous, homozygous mutant or homozygous wild type.
Figure 1. Standard and PCR treated DNA samples with either the wild type or mutant type primers on a 1.5% agarose gel. A DNA molecular weight marker (lane 1), and my PCR treated DNA samples with the wild type primer (lane 2) and mutant type primer (lane 3) were loaded onto a 1.5% agarose gel, separated by electrophoresis, and photographed after being illuminated using UV light.
Analysis of all of the PCR products loaded onto agarose gels from all 5 lab sections after electrophoresis can be seen in table 1. Based on the gels from all 5 lab sections 42.9% of the test subjects are homozygous mutant, 38.6% are heterozygous and 18.6% are homozygous wild type (table 1). From the data collected from all lab sections 81.4% of test subjects were predicted to have the lactose tolerant phenotype while 18.6% were predicted to have the lactose intolerant phenotype. When asked to report their phenotype 74.2% of test subjects reported they were lactose tolerant, whereas 25.8% reported to be lactose intolerant (table 1). The percent of predicted genotypes that matched self-reported phenotype was 65.2% (table 1)
Table 1. Summary of the genotype and phenotype data for the tested lactase gene
Lab Section % Homozygous Wild type % heterozygote % Homozygous Mutant % predicted lactose tolerant (based on genotype) % predicted lactose intolerant (based on genotype) % self- reported lactose tolerant % self- reported lactose intolerant % matching
Monday 6.25 25 68.8 93.75 6.25 75 25 68.8
Tuesday 21.4 57.1 21.4 78.6 21.4 91.7 8.3 83.3
Wednesday 26.7 26.7 46.7 73.3 26.7 80 20 53.3
Thursday 33.3 20 46.7 66.7 33.3 61.5 38.5 66.7
Friday 0 80 20 100 0 60 40 60
All Sections 18.6 38.6 42.9 81.4 18.6 74.2 25.8 65.2
Discussion The objective of this experiment was to determine the predicted genotype of lactase persistence or lactase non-persistence of all biology 225 lab students. The gel in figure 1 with my PCR products produced a band in lane 3 with the mutant allele specific primer and no band was produced in lane 2 with the wild type allele specific primer. This result indicates that I am homozygous mutant because only the mutant type primer binded to my genomic DNA during the PCR reactions. Thus, my predicted phenotype is lactose tolerance. This result corresponds to my observed phenotype of lactose tolerance and is consistent with my ethnic and demographic background as I am partially of Northern European decent and am an American white two ethnic backgrounds where lactase persistence is most prominent. From analyzing the PCR products on the agarose gels from all lab sections it can be inferred that the majority of the biology 225 students have the predicted genotype of lactose tolerance, with 81.4% predicted to be lactase persistant.
The percent of students who self- reported to be lactose tolerant was 74.02%, and the percent who self-reported to be lactose intolerant was 25.8%. The percent of subjects whose predicted phenotype matched their self-reported phenotype of either lactose tolerance or intolerance was 65%. Variations in predicted phenotype and self-reported phenotype could result from other mutations in the DNA that could cause lactase persistence or non-persistence other than the single nucleotide polymorphism tested for in this experiment or another chemical component in dairy that they cannot digest could cause them to exhibit symptoms of lactose
intolerance.
The results of this experiment were not expected since lactase persistence is the mutant allele and lactase non-persistence is the wild type allele most commonly found in the world population. Thus, this result is not consistent with the hypothesis that the majority of students should be lactose intolerant because it is the wild type allele. Lactase persistence exhibits an interesting characteristic of being the dominant allele even though it is mutant type, while lactase non-persistence is the recessive allele and wild type (Biology 225 lab manual, S2017). This unique characteristic of lactase persistence being dominant could explain why lactase persistence was so prominent in biology 225 lab students since 38.6% of students where heterozygous and produced both mutant type and wild type primer binding. A factor not investigated during this experiment but that could be investigated to explain the experimental results being inconsistent with the hypothesis is the demographics of the lab students. If the majority of the lab students are of Northern European, African or Asian from areas where people have been consuming milk for millennia then this could explain why the results varied from the hypothesis. The majority of bio 225 lab students were predicted to be lactose tolerant and retained their ability to produce lactase after weaning.