Association of Gene Polymorphisms and Development of Neurological Conditions
Using Restriction Fragment Length Polymorphism
Samir Nacer
Lab Partners: Krystine Sora, Sierra Sandler, Ali, Alfredo Lam, Natalia Figueroa
Biology 3600 Genetics I with Lab
Instructor: Dr. Robert Smith
Laboratory Assistant: Eileen
Date of Experiment: November 7th, 2016
Abstract: A migraine is a neurological condition in which blood vessels dilate causing an extreme aching pain on a specific side of the human skull. There may be a link between pathogenesis of Migraines and having what is known as the ACE Gene Polymorphism. A method used for the identification of this polymorphism is the use of a technique known as: Restriction Fragment Length Polymorphism (RFLP). …show more content…
This important because Migraines is a cognitive disorder affecting more than 28 million women in the US alone (Migraine Research Foundation, 2016). To find SNPs, the control and mitochondrial region of DNA was isolated, followed by Polymerase Chain Reaction (PCR) amplification and gel electrophoresis to see the isolated DNA. For the PCR segment, base pair (bp) approximations of 500 (Figure 1) and 1100 were found for PCR Products A and B respectively. RFLP did not result in significant sites which provide suitable evidence of SNPs. Colleagues Natalia, Emily, Alfredo, Krystine and Sierra, all had bands of 300bp, 200bp and 100bp with 4 bands and 3 cut sites from using the restriction enzyme MseI. For RsaI, Natalia had band lengths of 920bp, 300bp, 200bp and 100bp with 3 cut sites and 4 bands. Alfredo also had a similar 3 cut site and 4 band regions with 900bp, 300bp, 200bp and 100bp. Sources of error could have been Star Activity, defective …show more content…
Annealing occurred at 58 °C for 1 min, and extension occurred at 72°C for 2 min (30 cycles); the final extension happened at 72 °C for 5 min (1cycle). PCR products were then stored at 4°C for 18-24 hours. Gel Electrophoresis was performed using 100 volts for 30 minutes and the usage of 5 uL Sybr-safe, 1% Agarose in Tris-Acetate EDTA (TAE) respectively. Two tubes were grabbed and filled with 15 uL of PCR A and PCR B. Each tube required 5 uL of loading dye (xylene cyanol or bromophenol blue) to clearly analyze the separation of our DNA. RFLP was then conducted by first purifying PCR B using the QiaQuick PCR Purification Kit. Using Table 2, all reagents for both tubes can be detailed. Cut Smart ® Buffer restriction enzymes (MseI and RsaI) were then used on our left-over purified amplicons of PCR B to make fragment patterns which could then be compared to colleagues’ genomic ancestry. Before use, all restriction enzymes were incubated overnight at 37 ˚C because this is their optimal temperature. All gels were analyzed using bio spectrum imaging. The second gel had conditions like the original gel with a 1% agarose gel (in TAE), and electrophoresed our PCR products again at 100 volts for 30 minutes to separate them from the genomic
Using Restriction Fragment Length Polymorphism
Samir Nacer
Lab Partners: Krystine Sora, Sierra Sandler, Ali, Alfredo Lam, Natalia Figueroa
Biology 3600 Genetics I with Lab
Instructor: Dr. Robert Smith
Laboratory Assistant: Eileen
Date of Experiment: November 7th, 2016
Abstract: A migraine is a neurological condition in which blood vessels dilate causing an extreme aching pain on a specific side of the human skull. There may be a link between pathogenesis of Migraines and having what is known as the ACE Gene Polymorphism. A method used for the identification of this polymorphism is the use of a technique known as: Restriction Fragment Length Polymorphism (RFLP). …show more content…
This important because Migraines is a cognitive disorder affecting more than 28 million women in the US alone (Migraine Research Foundation, 2016). To find SNPs, the control and mitochondrial region of DNA was isolated, followed by Polymerase Chain Reaction (PCR) amplification and gel electrophoresis to see the isolated DNA. For the PCR segment, base pair (bp) approximations of 500 (Figure 1) and 1100 were found for PCR Products A and B respectively. RFLP did not result in significant sites which provide suitable evidence of SNPs. Colleagues Natalia, Emily, Alfredo, Krystine and Sierra, all had bands of 300bp, 200bp and 100bp with 4 bands and 3 cut sites from using the restriction enzyme MseI. For RsaI, Natalia had band lengths of 920bp, 300bp, 200bp and 100bp with 3 cut sites and 4 bands. Alfredo also had a similar 3 cut site and 4 band regions with 900bp, 300bp, 200bp and 100bp. Sources of error could have been Star Activity, defective …show more content…
Annealing occurred at 58 °C for 1 min, and extension occurred at 72°C for 2 min (30 cycles); the final extension happened at 72 °C for 5 min (1cycle). PCR products were then stored at 4°C for 18-24 hours. Gel Electrophoresis was performed using 100 volts for 30 minutes and the usage of 5 uL Sybr-safe, 1% Agarose in Tris-Acetate EDTA (TAE) respectively. Two tubes were grabbed and filled with 15 uL of PCR A and PCR B. Each tube required 5 uL of loading dye (xylene cyanol or bromophenol blue) to clearly analyze the separation of our DNA. RFLP was then conducted by first purifying PCR B using the QiaQuick PCR Purification Kit. Using Table 2, all reagents for both tubes can be detailed. Cut Smart ® Buffer restriction enzymes (MseI and RsaI) were then used on our left-over purified amplicons of PCR B to make fragment patterns which could then be compared to colleagues’ genomic ancestry. Before use, all restriction enzymes were incubated overnight at 37 ˚C because this is their optimal temperature. All gels were analyzed using bio spectrum imaging. The second gel had conditions like the original gel with a 1% agarose gel (in TAE), and electrophoresed our PCR products again at 100 volts for 30 minutes to separate them from the genomic