Diagnosis and Therapy of Genetic Disease Flashcards
integrated genome viewer
When looking at an IGV, be wary of. . .
Heterozygous deletions
You cannot see a read for a gene that isn’t there, and if it is heterozygous (which may cause haploinsufficiency-dependent disease) it may be hard to tell.
In this case, the total # of reads for this site would be about half of what one would expect. But, with current technology, this is bound to happen by chance for some areas on the genome/exome anyway.
In a clinical setting, the most common sequencing orders are. . .
- Targeted sequencing of one or a small panel of genes
- Whole exome sequencing
multiplex ligation-dependent probe amplification
May be used in a clinical lab to look for deletions too small to show up on Array CGH.
Aggregators of genetic variant data
ClinGen
ClinVar
Rough Mechanism of CRISPR-Cas9
Cas9 is loaded with a guide RNA.
Cas9 scans the genome, looking for two consecutive guanines (GG). When it finds one, it opens the DNA and checks if its guide sequence matchers. If not, it continues scanning. If so, Cas9 will cut that strand out of the DNA.
Then, the cell takes over. It may 1) directly stick the ends together, often creating a small (1 nucleotide) insertion at the site of the break. This in turn usually creates a frameshift mutation, knocking the gene out. In other cases, it may 2) find another matching piece of DNA to repair the cut. This scenario may be exploited by introducing pieces of DNA which match only on the ends, but have additional insertions that may add new DNA to the genome.