Plasmids & Cloing

Question 1

What is the origin, insert size limit and major application of Plasmid vector?

Answer 1

Naturally occuring or genetically modified plasmids, <10Kb, subcloning and downstream manipulation, CDNA cloning, expression assays

Question 2

What is the origin, insert size limit and major application of Phage vector?

Answer 2

Bacteriophage lambda, 5-20Kb, genomic DNA cloning, cDNA cloning, expression libraries

Question 3

What is the origin, insert size limit and major application of Cosmid vector?

Answer 3

Recombinant plasmid with lambda cos site, 35-45Kb, genomic libraries

Question 4

What is the origin, insert size limit and major application of BAC vector?

Answer 4

E. coli F episome, 75-300Kb, analysis of large genomes

Question 5

What is the origin, insert size limit and major application of YAC vector?

Answer 5

S. cervisiae centromere, telomere and autonomously replicating sequence, 100-1000Kb, analysis of large genomes and YAC transgenic mice

Question 6

What is the origin, insert size limit and major application of MAC vector?

Answer 6

Mammalian centromere, telomere and origin of replication, 100Kb- >1Mb, animal biotechnology, human gene therapy

Question 7

What are the properties and cofactors of E. coli DNA ligase?

Answer 7

joins only cohesive ends, requires NAD+

Question 8

What are the properties and cofactors of T4 DNA ligase?

Answer 8

joins both cos ends and blunt ends, requires ATP

Question 9

What are the 4 steps of the ligation process?

Answer 9

1) DNA ends randomly collide with each other and DNA ligase, which is easier to achieve at low temps, when the kinetic energy of the molecules is low.
2) Self-adenylation of an active site lysine using ATP or NAD+ and release of PPi
3) Transfer of adenylyl group to the 5-phosphorylated DNA terminus and forming a pyrophosphate bond.
4) Nucleophilic attack of 3’-hydroxyl group (acceptor) on 5’-phosphate group (donor), loosing the AMP and formation of a phosphodiester bond.

Question 10

What determines the cos ends compatibility?

Answer 10

Some enzymes half an unspecified base in their cleavage site. Such cos ends will only be compatible with cos ends that contain the same basepair within.

Question 11

What are cos ends?

Answer 11

12nt-long 5’overhangs of linear lambda DNA, which are used to circularise the phage within the infected bacterium.