Over the first five months of my M/Phill program I worked on two major projects: SPTLC1 and UNC13a genes.
SPTLC1 Project
The goal of SPTLC1 project was to show that mutations in SPTLC1 gene are the cause of amyotrophic lateral sclerosis(ALS). The following project was done in three steps: exome sequencing of a trio, exome sequencing of a large ALS cohort and matched control samples, and Sanger sequencing of SPTLC1 gene. The projects began with the discovery of a family that consisted of unaffected parents, one severely affected by ALS child and an unaffected sibling. Our group exome sequenced DNA from each member of this family. Exome data analysis led to the discovery of de novo mutations in the affected child but not in the parents or sibling suggesting that the discovered mutations could be the cause of ALS. To further examine SPTLC1 gene, our group exome sequenced a large number of ALS cases and controls. Analysis of the …show more content…
Looking at the structure of the UNC13a gene we observed that it is rich in short repeats. Based on this observation we hypothesized the hit detected during GWAS analysis could be due to an expansion of one or more of these repeats.
To determine if there is a repeat expansion on UNC13a gene I selected 26 previously reported short repeats on UNC13a gene and tested each one on 86 sporadic and 10 familial ALS samples using the following three methods: repeat-primed PCR, fluorescent PCR, and Sanger sequencing. I chose repeat-primed (RT-PCR) because it is a robust method to detect repeat expansions. Fluorescent PCR helps to determine if both alleles are amplified by PCR. Finally, Sanger sequencing helps to determine sample heterozygosity, to look for monoallilic amplification and for sample drop-outs due to an expanded allele.
SPTLC1 Project
The goal of SPTLC1 project was to show that mutations in SPTLC1 gene are the cause of amyotrophic lateral sclerosis(ALS). The following project was done in three steps: exome sequencing of a trio, exome sequencing of a large ALS cohort and matched control samples, and Sanger sequencing of SPTLC1 gene. The projects began with the discovery of a family that consisted of unaffected parents, one severely affected by ALS child and an unaffected sibling. Our group exome sequenced DNA from each member of this family. Exome data analysis led to the discovery of de novo mutations in the affected child but not in the parents or sibling suggesting that the discovered mutations could be the cause of ALS. To further examine SPTLC1 gene, our group exome sequenced a large number of ALS cases and controls. Analysis of the …show more content…
Looking at the structure of the UNC13a gene we observed that it is rich in short repeats. Based on this observation we hypothesized the hit detected during GWAS analysis could be due to an expansion of one or more of these repeats.
To determine if there is a repeat expansion on UNC13a gene I selected 26 previously reported short repeats on UNC13a gene and tested each one on 86 sporadic and 10 familial ALS samples using the following three methods: repeat-primed PCR, fluorescent PCR, and Sanger sequencing. I chose repeat-primed (RT-PCR) because it is a robust method to detect repeat expansions. Fluorescent PCR helps to determine if both alleles are amplified by PCR. Finally, Sanger sequencing helps to determine sample heterozygosity, to look for monoallilic amplification and for sample drop-outs due to an expanded allele.