AMB8 Flashcards
(15 cards)
what is primer extention used for why is this neccessary
exact mapping of 5’ end of RNA terminus as 3’ end is used for cdna synthesis often constructed using dT priming of poly A tail
whereas 5’ end isnt always incorperated and studying sequnece will not tell us if goes all the way to 5’ end
Describe the 3 steps of primer extetionq
- isolate RNA containing gene of interest, design radiolabelled primer specific to interested RNA sequence near 3’ end of mRNA and add reverse transcriptase and deoxynucleotides. - polymerise to 5’ end
- dentature hybrid and use extended primer cDNA as a marker on electrophoretic gel.
- transcriptional start site can be determined by comparing location on gel with the DNA sequence eg. sanger sequencing, by using the same primer on DNA template strand
cdna synthesis protocol but a gene specigfic primer is designed to anneal to sequence of interest.
what is a nuclease protection assay used for?
to determine the start or end of transciption. RNA or DNA protection assays
describe the stages of a nuclease protection assay
- radiolablled/fluorescent probes made that specifically hybridise with the gene at either start or end of transcript.
- nucleases that only degrade ss nucleic acid are added leaving RNA hybriised with primer and cutting away the rest of the RNA and part of the primer which was part of terminating or promoting region.
- products are then analysed by gel electrophoresis and size can be used to determine the exact start or end of transcription.
Why wouldnt protection assays or primer extention be used anymore?
as it is cheaper and easier to sequence all RNA that is extracted and then use bioinformatic processes to analyse sequences and see where polymerase falls off.
how can we define the 3’ end of promoter seq?
the base prior to the start of transcription
how can we define 5’ end of promoter seq?
more difficult to define, in theory promoter ends when bases have no more ingluence on transcriptional activity.
what is a reporter assay?
promoter/enhancer ligated to a reporter gene (easily assayable gene such as GUS) and introduced into organism to be studied.
repeated with targeted mutations in promoter enhancer sequence and activity determined to show importance of regions in promoter.
what is dnase protection assay footprinting used for?
to identigy dna sequences bound by DNA binding proteins
describe dnase protection assay footprinting
- dna labelled with a radioactive 32P
- DNA mixed with dna binding proteins
- mix treated with DNAse which will cut the DNA at random places not protected by protein
- Digested DNA is purified and denatured
- DNA analysed by gel electrophoresis and cleavage patterns give info about where proteins bind.
what does a gel shift assay do?
detect proteins bound to DNA. EMSA - electrophoretic mobility shift assay
describe a gel shift assay?
- label target dna with radioactivity
- effect of protein on mobility of dna fragment is analysed by page then autoradiography
- free dna fragments migrate rapidly to the bottom of the gel, while those bound to proteins are retarded
- number of retarded bands = to number of sequence specific dna binding proteins that bind to this sequence.
- to find if dna binding is specific to that sequence or not add a different non radioactive sequence.
- if non specific - band dissapears in presence of non radiolabelled competative sequence.
What is an open reading frame?
a sequence that when translated forms a potentially functional protein.
how can an open reading frame be identified
sequence of dna translated onto a computer
each dna can have 3 frames
in eukaryotes first protein start with methionine so scan for methionnie - any could in theory start translation.
short genes and presence of introns could complicate analysis.
how can introns be identified in eukaryotes?
by GT start and AG finish.
- programmes can be coupled with ORF analysis to predict where introns might be.
