To extract the DNA samples, we will ask 50 males and 50 females for a total of 100 voluntary Tibetans who are 18 and older from the same region to give a small sample of blood. The blood samples contain DNA and genes that we will use to analyze to find the EPAS1-TD gene. In order to analyze the genes from the blood samples, we need to perform the procedure of PCR, polymerase chain reaction, to amplify and clone the DNA. Performing PCR is necessary because it allows us to amplify a particular region of the DNA, the EPAS1 gene, set up by the primers in the annealing process. This is very quick and efficient for cloning the EPAS1 gene. After getting the PCR product containing the EPAS1-TD gene, we need to clean the PCR product through column chromatography. This will dissolve all the contaminants such as the excess primers and NTPS which will preserve our DNA specimen. To put the gene into the plasmid PGEM3Z, we will digest the DNA by using restriction enzymes such as Xba I to cut the DNA into smaller fragments containing the EPAS1-TD gene. The restriction enzymes will also cut at the designated restriction enzyme cleavage site in the plasmid where it will transfer the cut up EPAS1 gene into the plasmid PGEM3Z. The DNA Ligase will come and seal the plasmid shut with the EPAS1 gene DNA segment in it. Doing this will allow for the control of gene expression. In order to design the plasmid so it will be easy to screen and select bacteria that took up the plasmid, we can use the method of transformation between other bacteria. Before that happens though, we
To extract the DNA samples, we will ask 50 males and 50 females for a total of 100 voluntary Tibetans who are 18 and older from the same region to give a small sample of blood. The blood samples contain DNA and genes that we will use to analyze to find the EPAS1-TD gene. In order to analyze the genes from the blood samples, we need to perform the procedure of PCR, polymerase chain reaction, to amplify and clone the DNA. Performing PCR is necessary because it allows us to amplify a particular region of the DNA, the EPAS1 gene, set up by the primers in the annealing process. This is very quick and efficient for cloning the EPAS1 gene. After getting the PCR product containing the EPAS1-TD gene, we need to clean the PCR product through column chromatography. This will dissolve all the contaminants such as the excess primers and NTPS which will preserve our DNA specimen. To put the gene into the plasmid PGEM3Z, we will digest the DNA by using restriction enzymes such as Xba I to cut the DNA into smaller fragments containing the EPAS1-TD gene. The restriction enzymes will also cut at the designated restriction enzyme cleavage site in the plasmid where it will transfer the cut up EPAS1 gene into the plasmid PGEM3Z. The DNA Ligase will come and seal the plasmid shut with the EPAS1 gene DNA segment in it. Doing this will allow for the control of gene expression. In order to design the plasmid so it will be easy to screen and select bacteria that took up the plasmid, we can use the method of transformation between other bacteria. Before that happens though, we