Laboratory Lectures Flashcards
what is the oestrogen receptor?
the protein responsible for proliferation/growth of breast cancer cells
is a nuclear receptor (not found on cell surface)
75% of breast cancers express this receptor in their nucleus i.e. ER+ve cancers dependent on oestrogen for growth
outline the oestrogen signalling pathway?
signalling is activated by the presence of oestrodiol (biologically active form of oestrogen)
oestrodiol causes oestrogen receptor to dimerise and translocate to nucleus along with a range of co-factors
oestrogen receptor binds to oestrogen response elements present in genome causing transcription of genes driving proliferation e.g. myc, cyclin
how can ER+ve breast cancer be treated?
anti-oestrogen therapy which blocks oestrogen production or action of oestrogen
tamoxifen is a breast cancer drug which is a selective oestrogen receptor modulator
aromatase inhibitors block aromatase which is the enzyme producing oestrodiol i.e. blocking production and stopping cascade leading to growth/prolif
what do we denature the DNA during PCR?
makes the dsDNA ssDNA
why do we have forward and reverse primers?
DNA polymerase go from 5’–>3’ for their extensions and we want the primers sitting on opposite strands of DNA
why is annealing temperature lower than denaturing?
in the same way we need high temp for melting we need a low temp for base-pairing to occur
what does extension temperature depend on?
what enzyme we have i.e. what temperature it is functional at
what are some key considerations for PCR?
target sequence
primer design (specificity, flexibility)
DNApol used (accuracy)
cycling conditions (specificity)
why is it important to consider primer length when designing primers?
shorter primers have a lower annealing temperature cause don’t need as many hydrogen bonds but have a lower melting temperature cause less hydrogen bonds
longer primers have more specificity (easier to find a unique site on template) but higher annealing temp
how can you tag primers with other shit like restriction sites, fluorescent markers etc.?
the whole primer doesn’t actually have to match the target sequence, only enough of the 3’ end of primer to bind
extra part of the sequence at 5’ end can be anything you want
why is it important to consider the speed of the DNA polymerase used?
some are slow (e.g. 1 minute per 1000bp) and some are fast (e.g. 10 seconds per 1000bp)
consider because it affects how long you have to do the shit for but also if u know the rate of speed the enzyme operating at u can use this to increase specificity
how can the fidelity of DNA polymerase used for PCR affect results?
DNA polymerases aren’t perfect and have varying error rates (e.g. 1 in 50,000bp incorrect)
important to know this so you can guess how frequent you should see those errors and not think they genuine mutations in template
some will have proof reading capacity (3’-5’ exonuclease activity) allowing it to backtrack and fix incorrect bases however can also degrade your primers
what is hotstart PCR?
the polymerase is inactive to begin with and needs certain temp (about 98 degs for 2 mins) in order to be active
useful to stop primers being degraded and for controlling when reaction starts
what are the basics of DNA cloning?
treat with enzymes
ligate with plasmid cloning vector
transform into E. coli
get clones (your DNA fragment joined to plasmid vector)
outline the treatment with enzymes step of DNA cloning?
restriction sites in template (mayb you put them in on 5’ end of primer) get cut by restriction enzymes leaving sticky (unbasepaired) ends
this can then be ligated with plasmid cloning vector which has been treated with same restriction enzyme to give compatible ends
