Position independent gene identification

Question 1

How could we identify a disease gene if we had the partial amino acid sequence of a protein known to be involved?

Answer 1

Work backwards from the AA sequence to get an mRNA sequence, then make degenerate probes from that and screen a cDNA library

Question 2

Why do we need to use degenerate probes when identifying a gene from an amino acid sequence?

Answer 2

Genetic code is degenerate

Question 3

Why don’t we need to have every single possible codon in our pool of degenerate probes when identifying a gene from an amino acid sequence?

Answer 3

Codon bias in an organism. Will usually favour one codon for an amino acid over the others, so we only have to use this codon and not the others in our probes

Question 4

Why is identifying a gene from an amino acid sequence pretty rare in practice?

Answer 4

Not often we know something about the protein without knowing the gene

Question 5

How could we identify a disease gene using antibody screening?

Answer 5

If we have small amounts of protein that is involved in the disease phenotype, we can generate an antibody against it. Then use that antibody to screen an expression cDNA library in E coli to find the disease gene

Question 6

How could we identify a disease gene using model organisms?

Answer 6

If we can create a model organism with a similar disease phenotype, we can study it there and identify a homolog in humans

Question 7

What are synteny blocks?

Answer 7

Regions of similar genome organization between mice and humans

Question 8

How do synteny blocks help us identify disease genes in humans?

Answer 8

If we can map a disease gene in a mouse, we can use the corresponding synteny block to find the corresponding region in humans

Question 9

What is exome sequencing?

Answer 9

Sequencing only the coding regions in the whole genome for an affected individual

Question 10

Why is exome sequencing an actually feasible approach?

Answer 10

Sequencing is cheap and exonic sequences only make up about 1-2% of the genome anyways

Question 11

How do you do exome sequencing?

Answer 11

1. Extract genomic DNA
2. Fragment it
3. Capture exonic sequences through hybridization to biotinylated mRNA or cDNA
4. Use strepavidin coated magnetic beads to pull out the hybrids
5. Elute the captured exons off the beads (change temperature or salt)
6. Sequence

Question 12

Why do we capture exons using mature mRNA or cDNA? What’s the advantages of either one?

Answer 12

Contains no introns since they’ve already been spliced. cDNA is more stable than mRNA but requires an extra step