lecture 15 - functional genomics

Question 1

microarrays

Answer 1

design ssDNA probes to cover all your genes of interest
then your RNA hybridises to the probes
shows how much of eacg gene there is
has a flourescent marker

Question 2

RNA —> cDNA —> biotin labelled cDNA (at every uracil base) —> hybridises to genechip —> wash away non bound and scan array with laser and detect flourescense

Question 3

illumina sequencing

Answer 3

fragments of DNA bound to solid surface which is enabled by adaptors
PCR (solid phase bridge PCR) to form clone clusters
end product shows you what colour the cluster is to know what base it is and you can determine the sequence as each base binds to the DNA

Question 4

ChIP-Seq

Answer 4

cross link specific proteins to DNA
isolate DNA and shear
DNA is chopped up aprt from where protein is bound
immunoprecipitate protein of interest
reverse cross linking
purify DNA
sequence and shows where in the genome the protein was stuck to

Question 5

ATAC-Seq
shows you the openness of DNA

Answer 5

looks for regions of chromosome in open configuration (not wrapped around histones)
relies of Tn5 and it chops up exposed DNA
sticks an adapter to end so you can purify and sequence the fragments where the Tn5 has been able to access

Question 6

bisulphate sequencing

Answer 6

bisulphite treatment is used to determine the methylation state of DNA
methylated DNA is usually at cytosine
methylated cytosine protected from deamination (add sulphur then remove amine group so you get uracil)
if cytosine was methylated the methyl group sits where sulphur group is added so deammination doesnt work
so you can tell which were methylated and which werent
more methylation = less expression