A cloning vector is a genome that can accept the target DNA and increase the number of copies through its own autonomous replication. An adapter or adaptor, or a linker in genetic engineering is a short, chemically synthesized, single-stranded or double-stranded oligonucleotide that can be ligated to the ends of other DNA or RNA molecules. It may be used to add sticky ends to cDNA allowing it to be ligated into the plasmid much more efficiently.

1. ❖ Cloning vector : Definitations &
Properties
❖ Use of Linkers and Adaptors
Vanshika Varshney
18081564015 (4621)
BSc. (H) Microbiology
6th Semester (3rd Year)
Recombinant DNA
Technology

2. Why Cloning Vector?
01 Cloning Vector is used as a vehicle to artificial carry foreign genetic material
into another cell, where it can be replicated and expressed.
02 It is used to amplify a single molecule of DNA into many copes.
03 Cloning vectors are DNA molecules that are used to “transport” cloned
sequences between biological hosts and the test tube.

3. TOC
What is Cloning Vector?
Why Cloning Vector?
Properties of Cloning Vector
Four common properties share by Cloning Vectors
Types of Cloning vector
Linkers
Adaptors
Linkers and Adaptors

4. What is Cloning Vector?
“A cloning vector is a small piece of DNA into which a foreign DNA can be inserted for cloning
purposes.”
● For the cloning of any molecule of DNA it is necessary for that DNA to be incorporated
into a cloning vector.
● A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a
higher organism, that can be stably maintained in an organism and into which a foreign
DNA fragment can be inserted for cloning purposes.
● Most vectors are genetically engineered.
● A typical host organism is a bacterium, such as Escherichia coli, that grows and divided
rapidly.

5. ● Any vector with a replication origin in E.Coli will replicate efficiently. Thus, any DNA
cloned into a vector will enable the amplification of the inserted foreign DNA fragment
and also allows any subsequent analysis to be undertaken. In this way the cloning
process resembles the PCR, although there are some major differences.
● By cloning, it is possible not only to store a copy of any particular fragment of DNA but
also to produce unlimited amounts of it.
● The cloning vector chosen according to the size and type of DNA to be cloned.

7. Properties of Cloning Vector
1. Capability of autonomous replication: Vector
should contain an origin of replication (ori) to
enable independent replication using the host cell’s
machinery.
1. Small size: A cloning vehicle needs to be reasonably
small in size and manageable. Large molecules tend
to degrade during purification and are difficult to
manipulate.

8. 3. Presence of selectable marker genes: A cloning vector should have
selectable marker gene. This gene permits the selection of host cells
which bear recombinant DNA from those which don’t bear rDNA.
Eg. AmpR TetR, NeoR, LacZ genes.

9. 4. Presence of unique restriction sites: It should have restriction sites, to
allow cleavage of specific sequence by specific Restriction endonuclease.
Unique restriction should be present on the vector DNA molecule either
individually or as cluster (MCS - Multiple Cloning Sites or Polylinker).

10. 5. Ease of purification: It should be easy to purify and handle.
6. Minimum amount of nonessential DNA
7. It should be capable of working under the Prokaryotic and Eukaryotic
system
8. It should able to express itself utilizing the host machinery

11. Cloning vectors share four common properties:
1. Ability to replicate.
2. Contain a genetic marker for selection.
3. Unique restriction sites to facilltate cloning of insert DNA.
4. Minimum amount of nonessential DNA to optimie cloning.

12. Types of Cloning Vector
● Different types of cloning vectors are used for different types
of Cloning experiments (amplification/ expression).
● The vector is chosen according to the size and type of DNA to
be cloned (smaller/ larger fragments).
● Also depends on the host cell type used (Prokaryotic/
Eukaryotic).

13. Cont……
● Plasmid
1) Small circular piece of extrachromosomal DNA.
2) Must be a self-replicating genetic unit.
3) Plasmid DNA must replicate every time host cell divides or it will be
lost
a. DNA replication
b. partitioning .
1) Replication requires host cell functions.

14. Cont……
● Bacteriophage lambda & M13 vectors
1) They are one of the types of virus that attacks the bacteria and infects it.
2) It was first reported by Frederick Twort, a british biologist and later by
Felix d’Herelle, a French microbiologist.
3) They are classified by the International Committee on Taxonomy of
Viruses (ICTV) according to the morphology and characteristics.
4) There are two stages of bacteriophage replication
1. lytic cycle
2. lysogenic cycle

15. Cont……
● Cosmid
1) Cosmid vectors are hybrids between plasmid and phage λ vectors.
2) The classic example of cosmid vector is c2RB, which carries an origin of
replication and a cloning site and has antibiotic-resistant genes.
3) As with the phage λ vector, the cosmid vector encodes the cos sequences
required for packaging of DNA into λ capsid.
4) Cosmid vectors are designed to clone large fragments of DNA and to grow
their DNA as a virus or as a plasmid.
5) Cosmid vectors are used in homologous recombination between two
different plasmids in the same cell and grown in both bacteria and animal
cells.

16. Cont……
● BAC (Bacterial Artificial Chromosome)
1) BAC vectors are plasmids constructed with the replication origin of E. coli
F factor, and so can be maintained in a single copy per cell.
2) These vectors can hold DNA fragments of up to 300 kb.
3) Since they are present in low copies, recombination between the high copy
plasmids.

17. Cont……
● YAC (Yeast Artificial Chromosome)
1) YAC vectors contain all the elements needed to maintain a eukaryotic
chromosome in the yeast nucleus: a yeast origin of replication, two
selectable markers, and specialized sequences (derived from the
telomeres and centromere, regions of the chromosome needed for
stability and proper segregation of the chromosomes at cell divisions.
2) Before being used in cloning, the vector is propagated as a circular
bacterial plasmid.
3) Cleavage with a restriction endonuclease remove a length of DNA
between two telomere sequences leaving the telomeres at the ends of the
linearized DNA.
4) Cleavage at another internal site divides the vector into two DNA
segments, referred to as vector arms, each with a different selectable
marker

18. Linkers
● Linkers are the chemically synthesized short stretches of double stranded
DNA oligonucleotide containing on it one or more restriction sites for
cleavage by restriction enzyme.
● Ligated to blunt ends of DNA by ligase. Because of the high concentration
of these small molecules present in the reaction, the ligation is efficient
when compared with blunt-end ligation of large molecules.
● The cohesive ends are generated by digesting DNA with appropriate RE
that generates cohesive ends by cleaving in the linkers.
● The problem with linkers is that the sites for the enzyme used to generate
cohesive ends may be present in the target DNA fragment. This drawback
limits the use of linkers for cloning.

20. Adapters
● Adapters are linkers with cohesive ends or a linker digested with RE,
before ligation.
● It already has one sticky end.
● The most widely used definition is cut linkers also called as Adapters.
● They are not perfectly double stranded non single stranded. By adding
adaptors to the ends of a DNA, sequences that are blunt can be converted
into cohesive ends and used for cloning.
● Sticky ends are desirable for DNA cloning, but sometimes blunt ends are
present. Blunt ends will binds to the blunt ends of target to form sticky
ends.

22. LINKERS AND ADAPTERS
● Sticky ends are desirable for
DNA cloning experiments.
● Provided by treating the
target and vectors with same
RE or with different but
producing the sticky ends.
● But some time target DNA
are blunt ended.
● So, therefore we will have to
use Linkers and Adaptors.

24. REFERENCES :-
● Chapter - 2 (Vectors for gene cloning: Plasmids and Bacteriophages) Page - 13 - 23
Brown TA. “Gene cloning and DNA analysis” 6th edition. Blackwell Publishing, Oxford,
U.K.
● Chapter - 4 (Basic biology of Plasmid and Phage Vectors) Page - 55 - 74 Primrose SB
& Twyman RM “Principles of Gene manipulation and Genomics” 7th edition. Blackwell
Publishing, Oxford, U.K.
● http://www.unife.it/scienze/biologia/Insegnamenti/tecnologie-
ricombinanti/tecnologie-ricombinanti-2017/4-plasmidi.pdf
● https://bioinfo2010.wordpress.com/2009/07/08/vector-bacteriophage-lambda-and-
m13-7th-april/
● https://www.sciencedirect.com/topics/medicine-and-dentistry/cosmid-
vector#:~:text=Cosmid%20vectors%20are%20hybrids%20between,of%20DNA%20in
to%20%CE%BB%20capsid.
● http://www.csun.edu/~ll656883/lectures/lecture18.pdf

25. Thank you!