Steps of DNA Cloning technique

2.  The word cloning means creating a perfect
replica or genetically identical copy.
 The technique of generating identical copies
of short stretches of DNA is referred to as
molecular cloning.
 DNA cloning refers to replication of a single
DNA molecule to create a large number of
identical DNA molecules.
 DNA Cloning are of two types:
A. Cell based DNA cloning.
B. Cell free DNA Cloning.

3. In molecular DNA Cloning, the cloning of any
DNA fragment can be divided into three main
steps:
1. Formation of recombinant DNA molecule
2. Transfer of recombinant DNA into host
organism.
3. Selection and screening for positive clones.

4. Formation of recombinant DNA molecule
which includes:
a) Selecting the host organism and cloning
vector.
b) Preparation of DNA to be cloned.
c) Preparation of vector DNA.
d) Preparation of recombinant DNA.

5.  Majority of the molecular cloning experiments use strains of the
bacterium Escherichia coli as the host organism.
 A vector is a molecule of DNA which can replicate inside the host
cell and is maintained in a stable and characteristic number of
copies inside the cell. Eg- Plasmids, bacteriophages, BACs, YACs,
etc.
 If the size of insert DNA is exceptionally large BAC or YAC vectors
are chosen.
 Most of the gene cloning experiments use plasmids as the cloning
vector as they can handle DNA insert size of up to 15 kb. Plasmids
are extrachromosomal DNA molecules naturally present in
bacterial populations.
 Four DNA segments which are important for vector-
i) Ori ii) MCS iii) Selectable marker gene
iv) Reporter gene

6. i) Origin of replication (ori) is necessary for replication of
recombinant vector inside the host organism.
ii) Multiple cloning site (MCS) contains restriction sites for
many enzymes. These restriction sites are the site where
foreign DNA can be integrated.
iii) Selectable marker genes that confer resistance
against selection agents allow selection of transformed
cells.
iv) A reporter gene that allows screening of positive
clones by easy visual identification.

7. Fig: pUC18 plasmid vector having an origin of replication
(ori), reporter gene (lacZ), selectable marker gene (ampR)
and MCS (polylinker).

8.  Polymerase chain
reaction (PCR) is often
used for amplification
of insert DNA.
 Sequence specific
primers are designed
to amplify gene of
interest from the
genomic DNA
 Using PCR, any
sequence present in
the genomic DNA can
be amplified in the
form of a pure linear
DNA fragment.

9.  The insert DNA obtained
through PCR is treated
with a restriction enzyme
to generate fragments
with blunt-end or sticky
end.
 Restriction enzymes are
naturally produced by
bacteria as a defense
mechanism against
foreign DNA.
 Restriction enzymes
recognize 4-8 nucleotide
long palindromic
sequences in DNA and
cut them in a specific
manner.

10.  Plasmid DNA is isolated from a bacterial culture
by alkaline lysis method.
 After isolation of plasmid DNA, it is treated with
restriction endonuclease to cleave the DNA at
the site where foreign DNA will be inserted.
 To suppress self-ligation of vector DNA, the
cleaved vector is treated with alkaline
phosphatase.
 The desired fragment of plasmid DNA generated
by various treatments is isolated by gel
electrophoresis and purified.

11.  The vector and
insert DNA are
mixed together at
appropriate
concentrations and
treated with DNA
ligase enzyme.
 Ligation process
creates a covalent
bond between the
5′-P and 3′-OH
ends of the vector
and insert DNA.

12.  The circularized
recombinant DNA
formed by ligation of
vector and insert
DNA is introduced
into a host by
transformation.
 Transformation is the
process where
microorganisms take
up DNA from their
local environment.

13.  Only a small fraction of competent cells will successfully
receive and replicate a plasmid and these transformed
cells are selected using selection markers.
 The selectable marker is usually a gene conferring
antibiotic resistance when bacterial cells are used as
host.
 Cells harboring the ligated vector DNA will survive in the
presence of antibiotic, while those cells which have not
taken up vector sequence will die.
 The end result is formation of multiple colonies on
antibiotic containing growth medium.

14. Photo: Plasmid DNA bound to the cell exterior by a competent
bacterial cell.