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Genetics > Vectors > Flashcards

Vectors Flashcards

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1
Q

How are restriction sites incorporated in primers?

A

Within 5’ tag with a few extra bases for endonuclease

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2
Q

How can restriction digests provide orientation for gene insertion?

A

Different forward and reverse sites

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3
Q

What action do alkaline phosphatases have?

A

Remove 5’ phosphate from vector to prevent religation

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4
Q

When are alkaline phosphatases inactivated?

A

When the gene insert is added

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5
Q

What type of DNA do restriction enzymes target?

A

Unmethylated- host must be methylation disabled

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6
Q

What types of sequence do type 2 restriction enzymes digest?

A

pallindromic to leave sticky or blunt

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7
Q

What is an alternative method to insert genes?

A

Transposition

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8
Q

What size are natural plamids?

A

1-100kb

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9
Q

How many natural copies of plamids do cells have?

A

Up to 1000

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10
Q

What size insert do plamids carry?

A

10kb

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11
Q

How many copies of a plasmid are transformed into a cell?

A

15-20 for natural levels

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12
Q

Why are synthetic plasmids used in cloning?

A

They replicate independently and no not undergo horizontal gene transfer

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13
Q

What characteristics do plasmids have?

A

OriC, MCS, drug resistance markers

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14
Q

How can cells be transformed?

A

Electroporation, CaCl2 and heat shock, CaPO3 precipitation, liposomes or mechanical bombardment

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15
Q

How can transformation be observed?

A

Antibiotic resistance, blue/white selection, colony PCR, hybridisation and restriction digest

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16
Q

What are key characteristics of host cells?

A

Restriction enzyme deficient
No mutation or rearrangement of DNA
Auxotrophic

17
Q

What is the structure of bacteriophage lamda?

A

dsDNA linear genome of 49kb with 12bp cohesive ends separated by 37-40bp
Viral packaging

18
Q

How do bacteriophages insert DNA into a cell?

A

Transfection

19
Q

What are is the lytic cycle of phage lamda?

A

Transfection,
Hijacks host machinery for rolling circular replication
Catenane is cleaved at cos sites and associates with preformed packaging heads and tails
Lysis of cell membrane

20
Q

What is the lysogenic lamda phage cycle?

A

Genomic integration forms prophage
Repressor molecule for lytic cycle
Turbid plaques produced

21
Q

Which type of lamda cycle produces phages resistant to reinfection?

A

Lysogens

22
Q

What is the lamda packaging constraint?

A

78-105%

23
Q

What are the 2 types of gene insertion into lamda vectors?

A

Replacement with 10-22kb stuffer fragment

Insertion of up to 1-10kb fragment

24
Q

Which type of non essential DNA is removed from phages?

A

Between capsid and DNA synthesis genes, involved in lysogeny

25
Q

What are cosmids?

A

Plasmids that contain a cos site for transfection as phage

26
Q

What features does the cosmid genome contain?

A 
OriC
Cos
Insert up to 40kb
Restriction site
Antibiotic resistance
27
Q

What features are replaced by the insert in a cosmid?

A

Packaging and lysis genes

28
Q

What size is the M13 phage?

A

6.4kb ssDNA genome with no packaging constraint

29
Q

What size inserts does M13 accept?

A

10kb due to rearrangement

30
Q

What is the M13 life cycle?

A

Targets male specific/ F pilus
Replicates at dsDNA
Produces ssDNA for release
No cell death

31
Q

What is M13 used for?

A

Was used for sequencing but now used for phage display with gene3 replaced for expression of recombinant protein on capsid surface

32
Q

What type of cells is mechanical bombardment used to transform?

A

Plant cells through cell wall

33
Q

How efficient is liposome transformation?

A

90%

34
Q

Which cells is Electroporation used for?

A

Native animal, treated yeast cells

35
Q

How does calcium phosphate precipitation transform cell?

A

Endocytosis with 30-50% efficiency

Genetics (4 decks)

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