Vectors Flashcards
How are restriction sites incorporated in primers?
Within 5’ tag with a few extra bases for endonuclease
How can restriction digests provide orientation for gene insertion?
Different forward and reverse sites
What action do alkaline phosphatases have?
Remove 5’ phosphate from vector to prevent religation
When are alkaline phosphatases inactivated?
When the gene insert is added
What type of DNA do restriction enzymes target?
Unmethylated- host must be methylation disabled
What types of sequence do type 2 restriction enzymes digest?
pallindromic to leave sticky or blunt
What is an alternative method to insert genes?
Transposition
What size are natural plamids?
1-100kb
How many natural copies of plamids do cells have?
Up to 1000
What size insert do plamids carry?
10kb
How many copies of a plasmid are transformed into a cell?
15-20 for natural levels
Why are synthetic plasmids used in cloning?
They replicate independently and no not undergo horizontal gene transfer
What characteristics do plasmids have?
OriC, MCS, drug resistance markers
How can cells be transformed?
Electroporation, CaCl2 and heat shock, CaPO3 precipitation, liposomes or mechanical bombardment
How can transformation be observed?
Antibiotic resistance, blue/white selection, colony PCR, hybridisation and restriction digest
What are key characteristics of host cells?
Restriction enzyme deficient
No mutation or rearrangement of DNA
Auxotrophic
What is the structure of bacteriophage lamda?
dsDNA linear genome of 49kb with 12bp cohesive ends separated by 37-40bp
Viral packaging
How do bacteriophages insert DNA into a cell?
Transfection
What are is the lytic cycle of phage lamda?
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
What is the lysogenic lamda phage cycle?
Genomic integration forms prophage
Repressor molecule for lytic cycle
Turbid plaques produced
Which type of lamda cycle produces phages resistant to reinfection?
Lysogens
What is the lamda packaging constraint?
78-105%
What are the 2 types of gene insertion into lamda vectors?
Replacement with 10-22kb stuffer fragment
Insertion of up to 1-10kb fragment
Which type of non essential DNA is removed from phages?
Between capsid and DNA synthesis genes, involved in lysogeny
What are cosmids?
Plasmids that contain a cos site for transfection as phage
What features does the cosmid genome contain?
OriC Cos Insert up to 40kb Restriction site Antibiotic resistance
What features are replaced by the insert in a cosmid?
Packaging and lysis genes
What size is the M13 phage?
6.4kb ssDNA genome with no packaging constraint
What size inserts does M13 accept?
10kb due to rearrangement
What is the M13 life cycle?
Targets male specific/ F pilus
Replicates at dsDNA
Produces ssDNA for release
No cell death
What is M13 used for?
Was used for sequencing but now used for phage display with gene3 replaced for expression of recombinant protein on capsid surface
What type of cells is mechanical bombardment used to transform?
Plant cells through cell wall
How efficient is liposome transformation?
90%
Which cells is Electroporation used for?
Native animal, treated yeast cells
How does calcium phosphate precipitation transform cell?
Endocytosis with 30-50% efficiency
