Mod 10 - Genetic engineering Flashcards

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1
Q

basic principles of molecular cloning

A

cutting
joining
and propagating recombinant DNA

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2
Q

one way that molecular cloning is useful

A

reduces complexity of DNA
since u can choose specific sequences and propagate them
to allow large scale production and analysis of purified single sequences

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3
Q

basic steps of molecular cloning

A

isolate DNA
cut DNA
join to vector (DNA plasmid) = makes recombinant
put recombinant vector into bacteria
and let bacteria grow to amplify recombinant DNA

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4
Q

what is used to cut DNA

A

restriction endonuclease
cuts palindromic sequence
- this gives sticky ends (5’ overhang bits, see onenote) that can be rejoined
this is temporarily rejoined until ligase comes to covalently join the plasmid and the DNA fragment

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5
Q

what are the three things that can form after an attempt at this molecular cloning technique

A

desired product = recombinant plasmid

undesired:
unmodified plasmid (very common since the cut ends of the fragment are in close proximity)
or
circularised DNA fragment (usually not a problem cuz it very rarely acc replicates)

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6
Q

how do we prevent the cut ends of the plasmid from just joining back to itself

A

treat it with phosphatase
cuts of the phosphate ends

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7
Q

how do you make sure that during proliferation of the plasmid, that only the recombinant one is replicated

A

grown in antibiotic
and the recombinant one holds antibiotic resistance
so all other die
but recombo lives and replicates

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8
Q

two types of DNA libraries

A

nuclear DNA - (genomic library)
- contains all genes

cDNA library (from mRNA - ie c stands for complementary DNA)
- only expressed genes
- hence why it is cell type specific
- must be converted to DNA to be clone

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9
Q

how to make cDNA library

A

isolate the mRNA from cytoplasm of a cell

convert it to cDNA via reverse transriptase enzyme

insert it into vector (plasmid) and put it into carrier bacteria

each bacteria can oly take up one recombinant plasmid

so when u grow colonies, each colony represents 1 mRNA sequence

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10
Q

how to make genomic DNA

A

isolate DNA from nucleus

cut the DNA snip snip via restriction ednonuclease

this is gonna make large fragments (generally 50 kb each)

then put into vector (plasmid)

put into bacteria
and each colony once again, represents a small fragment of the genome

can be identified by DNA hybridisation

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11
Q

see onenote for slide on key differences between cDNA and genomic

A

:)

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12
Q

Why did we need to use recombo dna in treatment rather than just getting proteins and using those

A

hard to obtain
Usually from other organisms
So cause immunological problems
And hard to isolate these problems

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13
Q

how do we ensure we have control over the gene’s protein production when making a recombo gene (specificlly for treatments)

A

ligate the gene downstream from a strong promoter
e.g. lac promoter
something we can easily manipulate

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14
Q

advantages and disadvantages of using bacteria as a host system

A

adv:
- cheap
- fast
- easy to maintain
-stable

disadv:
- proteins may be insoluble/denatured
- no post tranlational modification like some proteins usually need

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15
Q

advantages and disadvantages of using animal cells as a host system

A

adv:
- post-translational modification
- soluble, properly folded

disadv:
- expensive
- unstable

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16
Q

whats a case study for use of this stuff as treatment

A

insulin production
used to be derived from animals
but this caused immulonological problems
and potential for cross-species infection

so they produced human insulin in bacteria

17
Q

what was the problem they came across when making insulin in bacteria

A

insulin needs to be process post translation
during trans, 2 chains are main seperately and then joined post trans via disulphide bridges

and bacteria cant do this

18
Q

how did we overcome the problem of making insulin

A

synthesising 2 chains seperately
extracting them
and then joining them