Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Molecular Neuroscience > Gene Cloning 1 > Flashcards

Gene Cloning 1 Flashcards

0
Q

how many genes does the human genome encode?

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
1
Q

how many base pairs are present in the human genome ?

A

3.4x10 to the power of 9

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

who wrote the paper “weve discovered the secret of life”?

A

francis crick in 1953

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what is the central dogma ?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what is the main difference between RNA and DNA?

A

DNA has thymine whereas RNA has a Uracil

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

why are viruses useful when you want to manipulate nucleic acids?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

why is it useful to determine where genes are expressed ?

A

its useful because it may help to determine function

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

how many genes are expressed in an average neuron ?

A

10000 genes - brain expresses more genes than other cells in the body

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

how can we find out where genes/proteins are expressed ?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

what tests use a nucleic acid probe to detect genes/proteins ?

A

northern blot
in situ hybridization
RT-PCR

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

what tests use antibodies to detect protein ?

A

western blot
immunohistochemistry
immunocytochemistry

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

what does alpha-bungarotoxin bind to ?

A

alpha-7 subunit of nAChR

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

where are alpha-3beta-4 subunits of nAChR present ?

A

autonomic ganglia

post synaptic exxcitation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

where are alpha-7 subunits of nAChR present ?

A

brain
important for cognition and attention
mostly presynaptic
calcium permeable enhances glutamate signalling

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

where are alpha4beta2 subunits of nAChR present ?

A

brain

pre and post synaptically

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

how can we understand what is the function of individual proteins (genes) ?

A
  • 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
16
Q

how much of the human DN encodes proteins ?

A

DNA is about 2m long and therefore about 0.02m encodes protein because only about 1% encodes protein

17
Q

what happens in the processing process of mRNA after transcription ?

A

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

18
Q

what are the problems with isolating RNA ?

A

RNases

RNA are associated with proteins

19
Q

what happens when RNA is isolated and fractionalised?

A

strong denaturants remove proteins and inactivate RNases (Phenol chloroform )
RNase inhbitors are added and the apparatus is also treated to remove any exogenous RNases

20
Q

how is rRNA and tRNA purified ?

A

by fractionalisation
rRNA (80%)
tRNA (15%)

21
Q

how is mRNA purified ?

A

by affinity chromotography on oligodT to select for the poly A tail
mRNA (2-5%0

22
Q

why is there are lot more tRNA ?

A

because lots is needed for translation

23
Q

why do you want to extract RNA ?

A

because it is the 1% of DNA which we want to know about because it is encoding

24
Q

how is cDNA produced for cloning ?

A

using oligo dT to anneal to the poly A tail of mRNA because hydrogen bonds can form
the reverse transcriptase is applied and dNTPs are added to synthesise the first strand of cDNA complementary to the mRNA strand
then DNA polymerase and dNTPs are added to synthesis the second strand - the mRNA strand is removed from the first strand of cDNA by heating to about 60 degrees

25
Q

if you have 10000 different mRNAs, how many different cDNAs do you have and how many different plasmids can be produced ?

A

10000 different cDNAs

10000 different plasmids

26
Q

what are the different functional aspects of a plasmid ?

A

origin of replication- allows replication in bacteria
antibiotic resistance- allows selection in bacteria
eukaryotic promoter- drives expression in eukaryotic cells
multiple cloning site - this is where cDNA is inserted
some have a fluorescent protein to allow detetion of the gene

27
Q

what are the advantages of expressing a gene in a xenopus oocytes ?

A

simple to learn to clamp

simple to inject with mRNA

28
Q

what are the disadvantages of inserting a gene into a xenopus oocyte?

A

no interacting proteins present
can be less responsive when looking at fast channels
no interacting protens

29
Q

how can you clone a gene ?

A
  • using cDNA libraries you can split this into pools and then inject this into oocytes and identify the pool containing the gene
  • isolate protein, determine its amino acid sequence then synthesise a DNA probe based upon the amino acid sequence
  • use a low stringency hybridisation of a related gene e.g GluR5 was cloned using the cDNA probe of GluR1
30
Q

how are northern blots carried out ?

A

electrophorese the sample through agarose gel to separate based on size
transfer it to a nitrocellulose membrane
hybridide with a labelled probe
identify labelled probe - expose to x ray film

31
Q

what can a northern blot tell you ?

A

size and relative abundance of mRNA, identify splice variants - although its the least sensitive technique

32
Q

what are southern blots used for ?

A

to identify specific sequences of DNA

33
Q

what are microarrays used for ?

A

to analyse all genes

  • contain 10000 spots
  • determine the intensity spot of fluorescence
  • each spot has a specific probe to bind to a sspecfic gene
34
Q

why is high throughput sequencing better than microarray analysis ?

A

its quicker

Molecular Neuroscience (12 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions