High capacity

vectors
 Cloning vector :-

A cloning vector is a small piece of DNA,

taken from a virus, plasmid, or the cell of
high organism that can be stably maintain
in an organism and into which a foreign
DNA fragment can be inserted for cloning
purpose.
 Characteristics :-

 Cloning vectors were developed in 1970’s

 It should be replicate autonomously
 Cloning capacity less then 10kb
 Use for genome library construction and cDNA library
construction
 Itconsist of origin of replication selectable marker,
restriction sites
 High capacity vector :-

 They were developed to reduce the number of clones

that needed to b handle
 Increase the cloning capacity of the vector
 Use for physical mapping of genome
 To maintain the genome to be sequence in a library
 Cloning capacity is above 40 kb
 Types :-

 Cosmid vector
 Fosmid vector
 Bacteriophage p1 derived vector
 P1 derived artificial chromosome (PAC)
 Bacterial artificial chromosome (BAC)
 Yeast artificial chromosome (YAC)
 Cosmid vector :-

Combine parts of the lambda chromosome with

part of plasmid
Medium size cloning vector
Cloning capacity 35-45 kbp
Firstly it was described by collines in 1978
Use for construction of genome library
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colE1 origin origin of replication

Ampr ampicillin resistant gene
MCS multiple cloning sites
Cos site 200 base pair long
lambda phage sequence and essential
for packaging
 how
insertion
takes
place
 Fosmid vectors :-

Fosmid is a low copy number plasmid

constructed by combining the features of
F-factor plasmid and the cos sites
 Characteristics :-

 E-colibased cloning vector

 Cloning capacity is up to 100 kbp
 Size of fosmid vector is 8 kbp
 Use for detecting deletion, insertion and re-arrangments
 Able to maintain foreign DNA stably over generations
 Low copy number of vector
 Kanamycin- resistant gene or ampicillin resistant gene
use as a selectable marker
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parA, parB for maintaining

low copy number and avoid two
F-plasmid in a single cell
Cmr chloramphenicol
resistance
OriV origin of replication
lacZ gene encoding beta-
galactosidase (selectable marker)
 Bacteriophage p1 derived
vector :-

 High capacity cloning vector based on p1

bacteriophage
 cloning capacity is up to 70kbp-100 kbp
 Maintain one copy number per cell
 Foreign DNA is maintained stably
 Introduce into the host by transduction
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p1lytic replicon use for DNA

replication in lytic cycle
Pac sequence p1 packaging site
SacB selectable marker
Loxp site locus of cross over in p1
 P1 derived artificial chromosome :-

 PACs constructed by combining feature of p1 bacteriophage

vector and BAC
 E-coli based vector
 Low copy number
 Stable maintenance of foreign DNA
 Cloning capacity is upto 150 kbp
 It is introduced by electroporation use for genome maping
 Continue…..

p1lytic replicon use for DNA replication in

lytic cycle
Pac sequence p1 packaging site
Loxp site locus of cross over in p1
colE1 origin origin of replication
Kanr kanamycin resistant gene
 Bacteria artificial chromosome :-

 E-coli based highest capacity cloning vector

 Single copy plasmid
 Most widely used vector for genome sequencing projects
 Constructed based on F factor plasmid
 Introduced into the host by electroporation
 Cloning capacity upto 300 kbp
 Size of BAC vector is 7.4 kbp
 Continue……

Ampr ampicillin resistant gene

MCS multiple cloning sites
parA, parB for maintaining low copy
number and avoid two F-plasmid in a
single cell
Loxp site locus of cross over in p1
repE for plasmid replication
How insertion take place :-
 Yeast artificial chromosome :-

 Yeast based cloning vector

 Possesses highest cloning capacity (3000 kbp)
 Maintained as linear DNA like chromosome
 Introduced into the yeast cells by electroporation
 First discovered by Murray and Szostak in 1983
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 TEL telomers
 CEN centromeres
 ARS autonomously replicating
sequence
 URA3 involved in uracil
biosynthesis (selectable marker)
 TRP1 involved in traptophan
biosynthesis (selectable marker)
How
insertion
takes
place :-