Pv92 Lab Report
Presence of Alu element within PV92 on chromosome 18 in a mixed ethnical population and its connection with humans’ ancestral origins
By Shiyu Song
Biology 155Q
LAB-A: 14:30-17:30
Dr. Jacob
April 17th 2013
Abstract The Alu insertion (+) within PV92 on chromosome 16 usually reflects a person’s ancestral origin. This study aimed to find the correlation between the presences of Alu insertion (+) within PV92 on chromosome 16 within a population. We used PCR to detect the Alu insertions by amplifying the PV92 region on chromosome 16 in order to detect the presence or absence of Alu at this locus. The result shows that 78% of the population in this population had a genotype “+/-”, and rest of the 22% of the populations had a genotype …show more content…
“-/-”. The people originate from East Asian and American Indian usually obtains genotype “+/-”, which means their ancestors originated from East Asian and America Indian.
Introduction
The human genome composed of 46 chromosomes, and Alu element can be found within the PV92 section of chromosome 16. Alu elements comprise 10% of the mass of the human genome. (Batzer et al. 2013) East Asian and American Indians are most likely to obtain Alu insertions (‘+’ allele) in their chromosomes. (Mateus Pereira, et al. 2005) The idea of migration waves into the New World from northeastern Asia having a Caucasian component might explain the high frequencies insertion alleles in American Indians and East Asians. (Paphiha et al. 1999) The experiment was aimed to study the prevalence of a particular DNA fragment in a group of students. The alu insertion (+) and three different types of genotype represent the ethical background that individual students come from. It is hypothesized that there is a high frequency of Alu insertion within the population. If majority of the participants have accurately reported their country/ethnicity/tribe of ancestral origins, there should have high frequencies of genotype “+/-” and Alu insertion within PV92 on chromosome 16 in the population.
2. Materials and method
2.1 Obtaining genomic DNA from Cheek Cells Each individual used one sterile cotton-tipped applicator to swab on both cheeks, between the gum line and under the tongue and twirled in a tube containing 2 ml of 1X PBS to dislodge cheek cells, and each person repeated the step as stated once. After obtaining the cheek cells, PBS cell suspension was placed into a micro centrifuge with spinning rate of 10,000 rpm for one minute. After obtaining a pellet of cheek cells, supernatant was discarded without disturbing the pellet in the tube. A tube of chelating agent was placed on a vortex for a few second to mix the contents and mixed the cell pellet. The tube was placed in the boiling water bath for ten minutes and cooled down at room temperature for 2 minutes. After boiling, the tube was centrifuged for two minutes with spinning rate of 14,000 rpm. After centrifugation, the supernatant was transferred to a tube.
2.2 PCR After the completion of genomic DNA extracted, 20μL of PV92 primer solution was transferred to a white PCR bead. Genomic DNA was taken out of the ice and spin in a micro centrifuge for one minute with a spinning rate of 14,000 rpm. After centrifuging the DNA tube, 5μL of liquid of the genomic DNA was transferred to each PCR tube. The PCR tube was put in the Applied Bio systems DNA thermal cycler, which was programmed as follows:
Initial denaturation: 94ºC for 5 minutes
Thermal cycling 32 cycles: denature -94ºC, 30 sec, anneal -61ºC, 30 sec, extend -72ºC, 45 sec, final extension -72ºC, 4 min, soak -4ºC, 24 hours
After the program was settled down, the PCR program started.
2.3 Gel Electrophoreses After the PCR program completed, 2.7μL of 10X loading dye was transferred into each PCR tube. An electrophoresis gel chamber was completely immersed by 1X Tae buffer. Then, 10μL of DNA ladder was loaded in to the middle of the gel, and 20μL of each PCR sample were loaded into distinguish location in the gel. After all lanes are loaded, electrophoresis was turned on at a constant rate of 110-115V for one hour.
2.4 Gel imaging and analysis After the gel electrophoresis was completed, the gel was put into Kodak Gel Logic 100 Gel Documentation, and an image of the gel was captured. The DNA ladder fragments were used to locate 1000bp, which serves as an object of reference to locate Alu insertion. After the sample band was established, the captured image was used to decide the genotype by analyzing the locations of the bands compared to 1000bp. After the genotype was obtained, PV92 genotype data for the whole population, genotype frequency and allele frequency were analyzed. Genotype frequency was calculated by count total number of individual with specific genotype, such as “+/+” or “+/-” divided by the total number of individuals in the population. Allele frequency was calculated by the number of “+” or “-” alleles in the population divided by the total number of all alleles in the population, which is total number of individuals in the population multiples by …show more content…
2. 3. Results
The results were obtained from the captured image during Gel imaging and analysis. The presence of the frequency in each genotype and its connection with Country/Ethnicity/Tribe of ancestral origin was measured. The sample of the total population in this experiment was nineteen students because one PCR DNA result was not available.
Table 1. PV92 genotype data for the whole population and genotype frequency observed in a population. Genotype | Total number of individuals with this genotype | Frequency | +/+ | 0 | 0 | +/- | 14 | 78% | -/- | 4 | 22% | Majority of the class has ancestral origin from American Indian and East Asia (China, Japan and Korea). The participants who obtained a genotype “+/-” reported their country/ethnicity/tribe of ancestral origin as following: China, Nigeria, US/Welsh*, Burma/China*, India/Pakistan*, African American, and England/Germany/Netherland/Norway*. While other who obtained a genotype of “-/-” identified their country/ethnicity/tribe of ancestral origin as following: China, England/Scotland*, Hungary Czech Republic, Iran/Turkey/Azerbaijan/Ireland/France/Germany* (* Indicates the country/ethnicity/tribe of ancestry origin contained mixed ethnicities.)
The Table 1 shows that 78 percent of the population in class obtains a genotype of “+/-” while only 22 percent of the population have the genotype “-/-”. The statistics generally match population’s self report of their country/ethnicity/tribe of ancestral origin. However, as the figure 3 showed below, there are 25 percent of the population who report themselves originate from East Asian/American Indian does nor have Alu insertion while another 28.5 percent of the population who are not East Asian/African American obtain Alu insertion within the PV92 region on chromosome 16.
Table 2. Allele frequency observed in the population Allele | Number of alleles | Frequency | + | 14 | 39% | - | 22 | 61% | Table2 shows that no Alu insertion (-) within the PV92 location of chromosome 16 is still larger than the Alu fragment (+) within the PV92 location.
Figure 3.Comparison of frequencies of East Asian and American Indian and non East Asian and American Indians to have or not have Alu insertion 4. Discussion
The experiment should show that these is a high frequency of the presence of Alu insertion in the experiment. The result attained shows that 14 people in the sample were heterozygous for the Alu insertion, while only four people were homozygous for no insertion. This tells us that a majority of the population in the class might have an ancestral origin with East Asia or American India or both. The genotype distribution frequencies of the “+/+”,“+/-” and “-/-” allele among participants revealed that 0% “+/+”, 78% “+/-” and 28% “-/-”. We could draw a primarily observational conclusion that there is a frequency Alu insertion presence in the population, and the result generally matches the prediction.
Some East Asians and American Indian had no Alu Insertion, while some Non-East Asian and American Indian had Alu Insertion within PV92 on chromosome 16.
Even though Alu element on PV92 could use to trace the ethnicity origin of one person, there might be people who do not fit in expected trends because people report their ethnic identity might not clearly distinguish their ethnical background (Tripathi, et al. 2008). Any unknown ethical background might have an impact with the presence of Alu element within PV92 (Solovieva, et al. 2010). These statements explain why 28 percent of the populations who are non East Asian and American Indian obtain Alu insertion while 25 percent of the populations who have ancestral origins of East Asian and American Indian do not have Alu insertion within PV92. In this experiment, the information of country/ethnicity/tribe of ancestral origin was solely based on self-report. The participants’ unfamiliarity of their accurate ethnical backgrounds might explain the obtained
data. With the above mentioned, high frequencies of genotype “+/-” and Alu insertion within PV92 on chromosome 16 present in the population. The result of the experiment is consistent with the hypothesis and prediction.
References
Batzer, Mark A., and Prescott L. Deininger. "Alu Repeats and Human Genomic Diversity." Nature Reviews 3 (2012): 370-80. Print.
Mateus Pereira, L.H., A. Socorro, I. Fernandez, M. Masleh, D. Vidal, N.O. Bianchi, S.L. Bonatto, F.M. Salzano, and R.J. Herrera. "Phylogenetic Information in Polymorphic L1 AndAlu Insertions from East Asians and Native American Populations." American Journal of Physical Anthropology 128.1 (2005): 171-84. Print.
Papiha, Surinder Singh., Ranjan Deka, and Ranajit Chakraborty. "Worldwide Distribution of A Polymorphic Alu Insertion in The Progesteron Receptor Gene." Genomic Diversity: Applications in Human Population Genetics. New York: Kluwer Academic/Plenum, 1999. 215-22. Print.
Solovieva, D. S., E. V. Balanovska, M. A. Kuznetsova, O. A. Vasinskaya, S. A. Frolova, E. A. Pocheshkhova, I. V. Eveseeva, and O. O. Balanovsky. "The Russian Gene Pool: The Gene Geography of Alu Insertions (ACE, APOA1, B65, PV92, TPA25)." ResearchGate. Molecule Biology/Pleiades Publishing, Inc.,, 2010. Web. 01 Mar. 2013.
Tripathi, Manorama, U.K. Chauhan, Piyush Tripathi, and Suraksha Agrawal. "Role of Alu Element in Detecting Population Diversity." International Journal of Human Genetics 2nd ser. 8.1 (2008): 61-74. Print.
By Shiyu Song
Biology 155Q
LAB-A: 14:30-17:30
Dr. Jacob
April 17th 2013
Abstract The Alu insertion (+) within PV92 on chromosome 16 usually reflects a person’s ancestral origin. This study aimed to find the correlation between the presences of Alu insertion (+) within PV92 on chromosome 16 within a population. We used PCR to detect the Alu insertions by amplifying the PV92 region on chromosome 16 in order to detect the presence or absence of Alu at this locus. The result shows that 78% of the population in this population had a genotype “+/-”, and rest of the 22% of the populations had a genotype …show more content…
“-/-”. The people originate from East Asian and American Indian usually obtains genotype “+/-”, which means their ancestors originated from East Asian and America Indian.
Introduction
The human genome composed of 46 chromosomes, and Alu element can be found within the PV92 section of chromosome 16. Alu elements comprise 10% of the mass of the human genome. (Batzer et al. 2013) East Asian and American Indians are most likely to obtain Alu insertions (‘+’ allele) in their chromosomes. (Mateus Pereira, et al. 2005) The idea of migration waves into the New World from northeastern Asia having a Caucasian component might explain the high frequencies insertion alleles in American Indians and East Asians. (Paphiha et al. 1999) The experiment was aimed to study the prevalence of a particular DNA fragment in a group of students. The alu insertion (+) and three different types of genotype represent the ethical background that individual students come from. It is hypothesized that there is a high frequency of Alu insertion within the population. If majority of the participants have accurately reported their country/ethnicity/tribe of ancestral origins, there should have high frequencies of genotype “+/-” and Alu insertion within PV92 on chromosome 16 in the population.
2. Materials and method
2.1 Obtaining genomic DNA from Cheek Cells Each individual used one sterile cotton-tipped applicator to swab on both cheeks, between the gum line and under the tongue and twirled in a tube containing 2 ml of 1X PBS to dislodge cheek cells, and each person repeated the step as stated once. After obtaining the cheek cells, PBS cell suspension was placed into a micro centrifuge with spinning rate of 10,000 rpm for one minute. After obtaining a pellet of cheek cells, supernatant was discarded without disturbing the pellet in the tube. A tube of chelating agent was placed on a vortex for a few second to mix the contents and mixed the cell pellet. The tube was placed in the boiling water bath for ten minutes and cooled down at room temperature for 2 minutes. After boiling, the tube was centrifuged for two minutes with spinning rate of 14,000 rpm. After centrifugation, the supernatant was transferred to a tube.
2.2 PCR After the completion of genomic DNA extracted, 20μL of PV92 primer solution was transferred to a white PCR bead. Genomic DNA was taken out of the ice and spin in a micro centrifuge for one minute with a spinning rate of 14,000 rpm. After centrifuging the DNA tube, 5μL of liquid of the genomic DNA was transferred to each PCR tube. The PCR tube was put in the Applied Bio systems DNA thermal cycler, which was programmed as follows:
Initial denaturation: 94ºC for 5 minutes
Thermal cycling 32 cycles: denature -94ºC, 30 sec, anneal -61ºC, 30 sec, extend -72ºC, 45 sec, final extension -72ºC, 4 min, soak -4ºC, 24 hours
After the program was settled down, the PCR program started.
2.3 Gel Electrophoreses After the PCR program completed, 2.7μL of 10X loading dye was transferred into each PCR tube. An electrophoresis gel chamber was completely immersed by 1X Tae buffer. Then, 10μL of DNA ladder was loaded in to the middle of the gel, and 20μL of each PCR sample were loaded into distinguish location in the gel. After all lanes are loaded, electrophoresis was turned on at a constant rate of 110-115V for one hour.
2.4 Gel imaging and analysis After the gel electrophoresis was completed, the gel was put into Kodak Gel Logic 100 Gel Documentation, and an image of the gel was captured. The DNA ladder fragments were used to locate 1000bp, which serves as an object of reference to locate Alu insertion. After the sample band was established, the captured image was used to decide the genotype by analyzing the locations of the bands compared to 1000bp. After the genotype was obtained, PV92 genotype data for the whole population, genotype frequency and allele frequency were analyzed. Genotype frequency was calculated by count total number of individual with specific genotype, such as “+/+” or “+/-” divided by the total number of individuals in the population. Allele frequency was calculated by the number of “+” or “-” alleles in the population divided by the total number of all alleles in the population, which is total number of individuals in the population multiples by …show more content…
2. 3. Results
The results were obtained from the captured image during Gel imaging and analysis. The presence of the frequency in each genotype and its connection with Country/Ethnicity/Tribe of ancestral origin was measured. The sample of the total population in this experiment was nineteen students because one PCR DNA result was not available.
Table 1. PV92 genotype data for the whole population and genotype frequency observed in a population. Genotype | Total number of individuals with this genotype | Frequency | +/+ | 0 | 0 | +/- | 14 | 78% | -/- | 4 | 22% | Majority of the class has ancestral origin from American Indian and East Asia (China, Japan and Korea). The participants who obtained a genotype “+/-” reported their country/ethnicity/tribe of ancestral origin as following: China, Nigeria, US/Welsh*, Burma/China*, India/Pakistan*, African American, and England/Germany/Netherland/Norway*. While other who obtained a genotype of “-/-” identified their country/ethnicity/tribe of ancestral origin as following: China, England/Scotland*, Hungary Czech Republic, Iran/Turkey/Azerbaijan/Ireland/France/Germany* (* Indicates the country/ethnicity/tribe of ancestry origin contained mixed ethnicities.)
The Table 1 shows that 78 percent of the population in class obtains a genotype of “+/-” while only 22 percent of the population have the genotype “-/-”. The statistics generally match population’s self report of their country/ethnicity/tribe of ancestral origin. However, as the figure 3 showed below, there are 25 percent of the population who report themselves originate from East Asian/American Indian does nor have Alu insertion while another 28.5 percent of the population who are not East Asian/African American obtain Alu insertion within the PV92 region on chromosome 16.
Table 2. Allele frequency observed in the population Allele | Number of alleles | Frequency | + | 14 | 39% | - | 22 | 61% | Table2 shows that no Alu insertion (-) within the PV92 location of chromosome 16 is still larger than the Alu fragment (+) within the PV92 location.
Figure 3.Comparison of frequencies of East Asian and American Indian and non East Asian and American Indians to have or not have Alu insertion 4. Discussion
The experiment should show that these is a high frequency of the presence of Alu insertion in the experiment. The result attained shows that 14 people in the sample were heterozygous for the Alu insertion, while only four people were homozygous for no insertion. This tells us that a majority of the population in the class might have an ancestral origin with East Asia or American India or both. The genotype distribution frequencies of the “+/+”,“+/-” and “-/-” allele among participants revealed that 0% “+/+”, 78% “+/-” and 28% “-/-”. We could draw a primarily observational conclusion that there is a frequency Alu insertion presence in the population, and the result generally matches the prediction.
Some East Asians and American Indian had no Alu Insertion, while some Non-East Asian and American Indian had Alu Insertion within PV92 on chromosome 16.
Even though Alu element on PV92 could use to trace the ethnicity origin of one person, there might be people who do not fit in expected trends because people report their ethnic identity might not clearly distinguish their ethnical background (Tripathi, et al. 2008). Any unknown ethical background might have an impact with the presence of Alu element within PV92 (Solovieva, et al. 2010). These statements explain why 28 percent of the populations who are non East Asian and American Indian obtain Alu insertion while 25 percent of the populations who have ancestral origins of East Asian and American Indian do not have Alu insertion within PV92. In this experiment, the information of country/ethnicity/tribe of ancestral origin was solely based on self-report. The participants’ unfamiliarity of their accurate ethnical backgrounds might explain the obtained
data. With the above mentioned, high frequencies of genotype “+/-” and Alu insertion within PV92 on chromosome 16 present in the population. The result of the experiment is consistent with the hypothesis and prediction.
References
Batzer, Mark A., and Prescott L. Deininger. "Alu Repeats and Human Genomic Diversity." Nature Reviews 3 (2012): 370-80. Print.
Mateus Pereira, L.H., A. Socorro, I. Fernandez, M. Masleh, D. Vidal, N.O. Bianchi, S.L. Bonatto, F.M. Salzano, and R.J. Herrera. "Phylogenetic Information in Polymorphic L1 AndAlu Insertions from East Asians and Native American Populations." American Journal of Physical Anthropology 128.1 (2005): 171-84. Print.
Papiha, Surinder Singh., Ranjan Deka, and Ranajit Chakraborty. "Worldwide Distribution of A Polymorphic Alu Insertion in The Progesteron Receptor Gene." Genomic Diversity: Applications in Human Population Genetics. New York: Kluwer Academic/Plenum, 1999. 215-22. Print.
Solovieva, D. S., E. V. Balanovska, M. A. Kuznetsova, O. A. Vasinskaya, S. A. Frolova, E. A. Pocheshkhova, I. V. Eveseeva, and O. O. Balanovsky. "The Russian Gene Pool: The Gene Geography of Alu Insertions (ACE, APOA1, B65, PV92, TPA25)." ResearchGate. Molecule Biology/Pleiades Publishing, Inc.,, 2010. Web. 01 Mar. 2013.
Tripathi, Manorama, U.K. Chauhan, Piyush Tripathi, and Suraksha Agrawal. "Role of Alu Element in Detecting Population Diversity." International Journal of Human Genetics 2nd ser. 8.1 (2008): 61-74. Print.