Gene Cloning 1 Flashcards
(35 cards)
how many genes does the human genome encode?
20000-25000 genes
most of these genes encode proteins
at first they thought there would be far more genes however a single gene can produce several variants
how many base pairs are present in the human genome ?
3.4x10 to the power of 9
who wrote the paper “weve discovered the secret of life”?
francis crick in 1953
what is the central dogma ?
DNA to RNA by transcription
RNA to protein by translation
it flows in one direction
DNA transcribed into mRNA
mRNA exported into cytoplasm and read by ribosomes
3 bases encode 1 amino acid
mRNA is the reading block for the translation of the protein
what is the main difference between RNA and DNA?
DNA has thymine whereas RNA has a Uracil
why are viruses useful when you want to manipulate nucleic acids?
RNA is very hard to work with
viruses have RNA genomes and therefore they contain the enzyme reverse transcriptase which converts RNA into DNA which is easier to work with
why is it useful to determine where genes are expressed ?
its useful because it may help to determine function
how many genes are expressed in an average neuron ?
10000 genes - brain expresses more genes than other cells in the body
how can we find out where genes/proteins are expressed ?
- if you have a toxin or ligand that labels a protein you can see where it is expressed
- if you know the sequence of a gene you can make a nucleic acid probe to identify where the mRNA is found
- if you know the sequence of amino acids you can make an antibody to see where the protein is found
what tests use a nucleic acid probe to detect genes/proteins ?
northern blot
in situ hybridization
RT-PCR
what tests use antibodies to detect protein ?
western blot
immunohistochemistry
immunocytochemistry
what does alpha-bungarotoxin bind to ?
alpha-7 subunit of nAChR
where are alpha-3beta-4 subunits of nAChR present ?
autonomic ganglia
post synaptic exxcitation
where are alpha-7 subunits of nAChR present ?
brain
important for cognition and attention
mostly presynaptic
calcium permeable enhances glutamate signalling
where are alpha4beta2 subunits of nAChR present ?
brain
pre and post synaptically
how can we understand what is the function of individual proteins (genes) ?
- express a protein in a cell where it is not normally found and look for a new function resulting from it - need to clone the gene and need a cell that does not normally express that protein
- remove a protein from a cell/organism and look for the function that has been lost - need to clone the gene and a cell that does normally express that protein = this is generally a bit better because you are looking at the endogenous system
how much of the human DN encodes proteins ?
DNA is about 2m long and therefore about 0.02m encodes protein because only about 1% encodes protein
what happens in the processing process of mRNA after transcription ?
splicing- cuts out introns and splices together the exons
cap addition at the 5’ end to help protect the mRNA and help it leave the nucleus
poly a addition
poly a tail and cap are not encoding proteins
what are the problems with isolating RNA ?
RNases
RNA are associated with proteins
what happens when RNA is isolated and fractionalised?
strong denaturants remove proteins and inactivate RNases (Phenol chloroform )
RNase inhbitors are added and the apparatus is also treated to remove any exogenous RNases
how is rRNA and tRNA purified ?
by fractionalisation
rRNA (80%)
tRNA (15%)
how is mRNA purified ?
by affinity chromotography on oligodT to select for the poly A tail
mRNA (2-5%0
why is there are lot more tRNA ?
because lots is needed for translation
why do you want to extract RNA ?
because it is the 1% of DNA which we want to know about because it is encoding
