Functional Genomics Flashcards
What is functional genomics
Finding gene functions and interactions through whole genome at once
What do microarrays measure
Hybridisation between nucleic acids and exploits their comp base pairing mechanism
What are attached to microarray which bind complementary dna
Probes which fluoresce after binding
What would probes be designed for to test methylation using micro arrays
Cpg islands. Probes would bind and it would detect areas of methylation for computer analysis
How would you find a protein bs using microarrays
Via chip first then purify dna from the protein and probe the dna that was bound with protein and sequence it
What would be probed to test gene expression via microarrays
Mrna would be RT into cDNA and probes designed complementary to it
How can you find snps using microarrays
SNPs would reduce levels of hybridisation which can be measured
Why is next gen sequencing used instead of microarrays
Less noisy data (measures sequence directly not via hybridisation)
Single base pair resolution
Cheaper
Detect rarer mrnas
What is next gene sequencing / high throughput sequencing most used technique
Illumina sequencing
Explain illumina sequencing
A dna sample obtained and fragmented
and attached to adaptors. These adapters allow binding to a flow cell
Bridge PCR then causes amplification of the same dna sample many times across flow cell
A primer attaches to allow labelled nucleotides to be added to the comp dna fragment
The nucleotides fluoresce diff colours and terminate/block extension so you detect which one was added at which time point for sequencing
What is PCR of the same dna fragment called in illumina
Solid phase bridge PCR - uses adaptors
What are the same dna fragments amplified called in illumina
Clonal clusters
Is termination via labelled nucleotides reversible
Yes. After every cycle you can add another base for sequencing
How is rna sequencing done via illumina to detect gene expression
Mrna is pulled out of a sample using its poly A tail which attached to a dT magnetic bead which pulls it from the sample
Mrna is then fragmented
RT into cDNA and copied for ds dna
Fragmented ds dna attached to adaptors for solid phase bridge PCR on flow cell
How can you tell how much mrna was in sample at start of rna sequencing
It is proportional to the amount of sequence data present
