Cloning Vectors and their Features

Vectors:-

A vector is a DNA molecule which is used for transporting exogenous DNA

into the host cell. A vector is capable of self-replication and stable integration
inside the host cell
A vector is a DNA molecule which is used for transporting exogenous DNA
into the host cell. A vector is capable of self-replication and stable integration
inside the host cell
 A vector is a DNA molecule that have the ability to replicate autonomously in the host cell and into which
DNA fragment to be cloned.
 It is a biological tool in rDNA technology.
 It is used for the delivery of desired foreign DNA into the host cell.

Characteristics of ideal vectors:-

 it must be small in size
 It must be self-replicating inside host cell
 It must possess restriction site for Restriction Endonuclease enzymes
 Introduction of donor DNA fragment must not interfere with replication property of the vector
 It must possess some marker gene such that it can be used for later identification of recombinant cell
(usually an antibiotic resistance gene that is absent in the host cell).
 it must possess multiple cloning site
Cloning Vectors
 Cloning vectors are small piece of DNA which have the ability and used to introduce foreign gene of
interest into the host cell.
 They can be stably maintained insides the host cell.
 Cloning vector are generally used to obtain multiple copies of desired foreign gene.
 Example- Plasmid, Cosmid and Phages, BACs, YACs.
 These type of vectors generally contains selectable marker, origin of replication and a restriction site.
Types of cloning vectors:-
1. Plasmids :-
 A plasmid is a naturally occurring extrachromosomal double stranded DNA, circular DNA.
 It replicates autonomously within bacterial cell.
 Plasmid carries an origin of replication.
 Plasmid vectors are the simplest cloning vectors.
 It is most widely used for gene cloning.
Characteristics of Plasmid vector:-
 It contains an ori site or origin of replication.
 It also Contain selective marker such as antibiotic resistance, blue white screening).
 Small in size (1.0 to 250kb)
 Contains multiple cloning site.
 Easily isolated from the host cell.
E.g.- = P- plasmid, B- Boliver, R- Rodriguez; 322- Differentiate it from the other plasmid produced in the same
laboratory E.g. – pBR325, pBR327, etc. It is 4363 base pair long. Cloning limit: 0.1-10 kb

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2. Phage Vector Or Bacteriophage Vector: -
 The viruses that infect bacteria are called bacteriophage. These are intracellular obligate parasites that
multiply inside bacterial cell by making use of some or all of the host enzymes.
 Bacteriophages have a very high significant mechanism for delivering its genome into bacterial cell. Hence
it can be used as a cloning vector to deliver larger DNA segments.
 Most of the bacteriophage genome is non-essential and can be replaced with foreign DNA.
 Using bacteriophage as a vector, a DNA fragment of size up to 20 kb can be transformed.
 It consists of DNA molecules having several gene for replication which is surrounded by Capsid.
 Example: lambda genome or lambda bacteriophage.
 It is a phase (Virus) genome.
 size: 48502 bp.
 1/3rd of the bacteriophase genome is non-essential, so that it can be cut, removed and replaced
by donor DNA fragment during cloning.
 It can recombinant only 4-5 Kbp of donor DNA fragment

3.Cosmid:-
 Collins and Hohn in 1978 was first to described cosmids.
 It is a type of hybrid plasmid.
 It contains lambda phage cos sequence.
 Cosmids = cos sites + plasmid.
 Genomic size of cosmids is about 30 to 52 kb.
 If they have suitable origin of replication than they can replicate as Plasmid within the host cells, E.g.-
SV40 Ori, ColE1 ori.
 It also contains selectable marker such as Ampicillin resistance gene.
 Example: super COS1. size: 7900 bp.It has combined feature of both phase and plasmid.Cloning limit: 30-
50 kb

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 4. Phagemid Vector

Phagemid vectors are prepared artificially like cosmid by combining the features of phages with plasmids. A, in
addition to that of the plasmid, is termed as Phagemids. pBlue script SK (+/-) is a type of phagemid vector of 2,958
bp derived from the pUC19.

It consists of the following:

 ColE1 ori
 plasmid vector which contains an origin of replication from a phage
 Phage f1 (M13) origin of replication.
 A small portion of lacZ gene.
 MCS within lacZ Gene from Lac promoter.
 Phage T7 and T3 promoter sequence.
 Ampicillin gene for Ampicillin resistance.

5. Bacterial artificial chromosomes (BAC):-

 Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to clone very large DNA
fragments ranging in size from 75 to 300 kb.
 BACs basically have marker like sights such as antibiotic resistance genes and a very stable origin of
replication (ori) that promotes the distribution of plasmid after bacterial cell division and maintaining the
plasmid copy number to one or two per cell.
 BACs are basically used in sequencing the genome of organisms in genome projects (example: BACs were
used in human genome project).
 Several hundred thousand base pair DNA fragments can be cloned using BACs.
 Example: pUvBBAC
 It is artificially synthesized plasmid
 size: 11827 bp
 It is modification of bacterial F-plasmid
 Cloning limit: 35-300 kb
 Marker gene: chloramphenicol resistant gene and lactose metabolizing gene (LacZ)

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6. Yeast artificial chromosome (YAC):
Construction of yeast artificial chromomes (YACs) provides more advantages over cloning in bacterial
cells. It can clone very large DNA fragments.
A YAC has the following features:
 A yeast telomere (TEL) at each end.
 A yeast centromere sequence (CEN) allowing regulated segregation during mitosis.

 A selectable marker on each arm for detecting the YAC in yeast (for example, TRP1 and URA3 for
tryptophan and uracil independence in trp1 and ura3 mutant strains respectively).
 An origin of replication ARS (autonomously replicating sequence) allows the vector to replicate in
a yeast cell,
 Restriction sites unique to the YAC that can be used for inserting foreign DNA (Fig. 9.18). For
cloning experiments a circular YAC is cut with one restriction enzyme that cuts in the multiple
cloning sites and another restriction enzyme that cuts between the two TELs. In this way, the left
and right arms are produced. High molecular weight DNA is ligated to the two arms.
 The capacity to clone large exogenous DNA fragments (up to 2 Mb) has made YACs a vital tool in
creating physical maps of large genomes such as the human genome.
 Example: pYAC3
 It is an artificial chromosome having yeast centromere isolated from Saccharomyces
cerevisiae and ligated to bacterial plasmid
 size: 11400 bp
 It has telomere sequence
 Marker: similar as for identification of yeast cell
 Cloning limit: 100-1000 kb

7. Human artificial chromosome (HAC):-

 A human artificial chromosome (HAC) is a mini-chromosome that is constructed artificially in human cells.
 That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6-10
mega-bases in size.
 able to carry new genes introduced by human researchers.
 Using its own self-replicating and segregating systems, a HAC can behave as a stable chromosome that is
independent of the chromosomes of host cells.
 They are useful in expression studies as gene transfer vectors and are tools for elucidating human
chromosome function. Grown in HT1080 cells, they are mitotically and cytogenetically stable for up to six
months.

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 8. Shuttle Vectors:
 Shuttle vectors are those which can multiply into two different unrelated species.
 Shuttle reactors are designed to replicate in the cells of two species, as they contain two origins of
replication, one appropriate for each species as well as genes that are required for replication and not
supplied by the host cell, i.e., it is self-sufficient with the process of its replication.
Common features of such shuttle vectors:-
 They are capable of replicating into two or more types of hosts including prokaryotic and eukaryotic cells.
 They replicate autonomously, or integrate into host genome and replicate when the host cell multiplies.
 These vectors are commonly used for transporting genes from one organism to another.
Disadvantage:
The presence of two replication origins some times poses special problems, one portion of replication origin of
one species is totally un related to another and interferes with the replication of other host. Hence, in a shuttle
vector various types of replication origins are to be inserted and checked before experimenting.
The shuttle vectors are of following types:
a. Eukaryotic – Prokaryotic Shuttle Vectors:
Vectors that can propagate in eukaryotes and prokaryotes. e.g., YEp vectors can be propagated in yeast (fungi) as
well as in E. coli (bacteria).
b. Prokaryotic – Prokaryotic Shuttle Vectors:
Vectors that can be propagated in two unrelated prokaryotic host cells, e.g., RSF1010 vectors can be propagated
both in bacteria as well in spirochetes.

9. Expression vectors:
 Vectors designed specifically for the expression of the transgene in the target cell are called expression
vectors, and generally have a promoter sequence that drives the expression of the transgene.
 Expression vectors produce proteins through the transcription of the vector’s insert followed by a
translation of the mRNA produced.
 It has a bacterial promoter, such as the lac promoter. The promoter precedes a restriction site where
foreign DNA is to be inserted, allowing transcription of foreign sequence to be regulated by adding
substances that induce the promoter.
 A DNA sequence that, when transcribed into RNA, produces a prokaryotic ribosome binding site.
 Prokaryotic transcription initiation and termination sequences. d. Sequences that control transcription
initiation, such as regulator genes and operators.
 In some types of expression vectors which are specifically used in association with the bacterial host (like
E. coli), multiple cloning site is not immediately adjacent to the ribosome binding sequence, but instead is
preceded by a special sequence coding for a bacterial polypeptide.
 The product of gene expression is therefore a hybrid protein, consisting of short bacterial polypeptide
fused into amino terminus of our target polypeptide sequence. This hybrid polypeptide chain consisting of
two different types of polypeptides is called a fusion protein.
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