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Biotechnology > Biotechnology 5 > Flashcards

Biotechnology 5 Flashcards

1
Q

Define recombinant protein

A

A protein produced through use of recombinant DNA techniques

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2
Q

What was the first form of recombinant protein?

A

Humulin, a recombinant form of insulin produced in 1976

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3
Q

What are the 2 main steps for making a recombinant protein?

A

Cloning a cDNA copy of a gene

Optimising expression of the cDNA

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4
Q

Why is optimisation of expression important?

A

You are moving the sequence from one organism into a bacteria, which has a different climate for transcription and translation

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5
Q

Define cDNA

A

Complementary DNA, a DNA copy of the mRNA which is made using reverse transcriptase

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6
Q

Why does producing recombinant proteins require cDNA?

A

Because mRNA cannot be cloned directly

Only dsDNA can be cloned

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7
Q

Why use cDNA and not extracted DNA?

A

Enrichment, Eukaryotic genomes are very large and contain mostly non-coding DNA whereas cDNA is mostly coding
Bacteria don’t have the ability to splice

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8
Q

Describe the synthesis of cDNA

A

The mRNA is collected from the cytoplasm
The mRNA undergoes polyadenylation, where are poly-a-tail is added to stabilise the molecule
The Oligo primer forms hydrogen pairs with poly-a-tail
The cDNA strand is synthesised from 5’ to 3’ using reverse transcriptase
The mRNA strand is degraded by RNAase G enzyme, Hairpin loop at 3’ end is likely to form
The second strand is synthesised using DNA polymerase from the hairpin loop, using it as a primer
S1 nuclease cuts the hairpin loop
A strand of only cytosine is added to the 3’ end of each cDNA strand
OligoG primer hydrogen pairs with the only cytosine section of cDNA

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9
Q

Define cDNA library

A

A combination of cloned cDNA fragments inserted into a collection of host cells and inserted into the transciptome, where they are stored as a library so that researcher can isolate and identify DNA fragments that they want to study further

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10
Q

What are the differences between cDNA libraries and genomic libraries?

A

Genomic libraries use mostly YACs and BACs as vectors whereas CDNA libraries use mostly plasmids
Genomic libraries contrain non coding regions, whereas cDNA libraries don’t
Genomic libraries are produced from genomic DNA whereas cDNA libraries are not

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11
Q

How can you construct a cDNA library?

A

Isolate and collect mRNA
Produce double stranded cDNA
The cDNA is ligated into the plasmid
The plasmids that contain one of the cDNA sequences are inserted into the bacteria transforming them
These bacterial cells are then cultured
You then have to figure out which of the clones you are invested in and purify them

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12
Q

What are the three techniques you can use to find your sequence of interest?

A

Hybridisation
Immunodetection
Complementation

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13
Q

What do immunodetection and complementation have in common?

A

They are both approaches that depend on the properties of the encoded protein, rather than the DNA sequences

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14
Q

What is the aim of hybridisation?

A

Locating the cDNA clone of interest using a nucleic acid probe

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15
Q

Describe the hybridisation probe.

A

DNA or RNA fragments
100-1000 bases
Specific to target cDNA sequence
Labelled

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16
Q

What are the two types of hybridisation probes?

A

Homology and degenerate

17
Q

Describe homology probes

A

The probe carries the corresponding DNA sequence from a related organism

18
Q

Describe degenerate oligonucleotide probes.

A

Different sequences will be able to make a known primary sequence due to the degenerate nature of the genetic code

19
Q

Describe radiolabelling

A

A radioactive phosphate is incorporated into the DNA via the nucleoside

20
Q

Why is the radiolabled at the alpha position of the NTP?

A

Because when the nucleotide is incorporated into the DNA it loses the beta and gamma phosphate groups

21
Q

What are the advantages of using radio labels?

A

They are robust (always work)

They are easy to detect, even at low levels

22
Q

Describe non-radiolabelling

A

Nucleotides coupled with DIG

23
Q

What are the advantages of non-radioactive labelling?

A

Safe

Stable when frozen

24
Q

How are the DIG labels detected?

A

DIG label recognised by specific antibodies that are coupled with alkaline phosphate enzyme
AMPPD is added, which when cleaved by the alkaline phosphate enzyme produces chemiluminescene

25
Q

What are disadvantages of non-radioactive labelling?

A

More complex detection methods

26
Q

How are radioactive and non radioactive labels incorporated into the DNA?

A

Using terminal transferase

Using taq polymerase

27
Q

Describe colony hybrisisation

A

A method of specific DNA sequences

28
Q

How does colony hybridization work?

A

A DNA stamp from each colony is made on a membrane that can then be screened with a hybridization probe to detect the clone(s) of interest.

The denaturation (separating) of both plasmidic DNA strands allows for binding of the probe to its complementary sequence.

The colonie(s) are tagged by the labelled probe if they carry the corresponding cDNA

29
Q

What is immunodetection?

A

Clone identification using antibodies

30
Q

Describe the method of imumunodetection

A

The cDNA is in the phage
The phage infects E Coli
The E Coli replicates and the cDNA produces the corresponding protein
The phage kills the E Coli which releases the proteins
The proteins are transferred to a nitrocellulose membrane
The proteins are probed with the antibodies specific to the protein
The sequence can be identified if the sequences used were labelled

31
Q

What is the principle of complementation?

A

It uses the properties of the protein to rescue a mutation in the host organism

32
Q

What are the requirements of complementation?

A

The loss of the protein must lead to an easily observable phenotype
The cDNA must be cloned in a specific mutant background

33
Q

What is a advantage of complementation?

A

Easy, fast and cheap

34
Q

What is a disadvantage of complementation?

A

Not always applicable

Biotechnology (10 decks)

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