Cloning

Cloning is the process of producing genetically identical

individuals of an organism either naturally or artificially. In
nature, many organisms produce clones through asexual
reproduction. Cloning in biotechnology refers to the process
of creating clones of organisms or copies
of cells or DNA fragments (molecular cloning).
 Different types of cloning

1. Recombinant DNA technology or DNA cloning

2. Reproductive cloning and
3. Therapeutic cloning
DNA/ Gene Cloning:
The insertion of a fragment of DNA carrying a gene
into a cloning vector and subsequent propagation of
recombinant DNA molecules into many copies is
known as gene cloning.
Gene Cloning Technique
BASIC STEPS OF GENE CLONING

 Construction of recombinant DNA

molecule
 Transport of the recombinant DNA to the
host cell
 Multiplication of recombinant DNA
molecule
 Division of the host cell
 Numerous cell division resulting in a clone
 Reproductive Cloning
Dolly was created by reproductive cloning technology, in a process
called “somatic cell nuclear transfer” (SCNT).
 Therapeutic cloning
Therapeutic cloning, also called “embryo cloning”, is
the production of human embryos for use in research.

The goal of this process is not to create clone human

beings, but rather to harvest stem cells that can be
used to study human development and to treat
disease.

Stem cells are important to biomedical researchers

because they can be used to generate virtually any
type of specialized cell in the human body.
 DNA Cloning/ Gene Cloning
The first step of gene cloning is –
Identification and isolation of the desired gene or DNA
fragment. The desired DNA inserts can be obtained from
the following:

1. Genomic DNA library

2. Complementary DNA (cDNA) library
3. Chemical synthesis: Only for simple polypeptide chain
whose primary structure is clear.
4. Amplification through PCR
 Isolating a gene from gene library

Gene library: A collection of different DNA sequence from

an organism, each of which has been cloned into a vector
for ease of purification, storage and analysis.

Genomic DNA libraries

(made from genomic DNA)
Gene library
cDNA libraries

(made from cDNA- copy of mRNA)

 Genomic DNA library

The genomic DNA library is a

collection of the comprehensive

DNA fragments representing the

entire genome of a species.

 Genomic DNA libraries

1. Purify genomic DNA： prokaryotes or eukaryotes

2. Fragmentation of isolated and purified DNA by

physical shearing and restriction enzyme digestion

3. Clone the fragments into vectors

4. Transfer the constructed vectors into recipient cells

5. Culture and amplify the clones

6. Screen the clone of target

 Genomic DNA libraries: Purify genomic DNA

To make a representative genomic libraries, genomic DNA

must be purified and then broken randomly into fragments
that are correct in size for cloning into the chosen vector.
Purification of genomic DNA :

Eukaryotes :

1. prepare cell nuclei

2. remove protein, lipids and other unwanted macro-
molecules by protease digestion and phase extraction.

Prokaryotes :
Extracted DNA directly from cells
Genomic libraries
Genomic DNA libraries: Fragmentation of purified DNA

Break DNA into fragments randomly:

Physical shearing :
pipeting, mixing or sonicaion

Restriction enzyme digestion: Partial digestion is preferred

to get a greater lengths of DNA fragments.

 Genomic DNA libraries: Fragmentation of purified DNA

Selection of restriction enzyme

1. Ends produced (sticky or blunt) & The cleaved ends of

the vector to be cloned

2. Whether the enzyme is inhibited by DNA modifications

3. Time of digestion and ratio of restriction enzyme to

DNA is dependent on the desired insert size range.

 Genomic DNA libraries: Selection of Vector
According to genome’s size,we can select a proper vector
to construct a library .

Vectors Plasmid λ phage Cosmid YAC

insert (kb) 5 23 45 1000

The most commonly chosen genomic cloning vectors

are λ replacement vectors which must be digested with

restriction enzymes to produce two λ arms between

which the genomic DNA will be inserted.

 Genomic DNA libraries: Selection of Vector
λ phage vector in cloning
Long (left) short (right)
arm arm
cos Exogenous DNA cos
(~20-23 kb)

Long (left) short (right)

arm arm
cos cos
Exogenous DNA
(~20-23 kb)
 λ replacement
vector cloning
0.preparation of 2.Packing with a mixture
arm and genomic of the phage coat
inserts
proteins and phage
DNA-processing enzymes

1. Ligation
3.Infection and
formation of plaques

Library constructed
 Screening a genomic
library using DNA
hybridization to a
(radio-)labeled DNA
probe

Note: a cDNA is commonly

(radio-)labeled and used as
a DNA probe to screen a
genomic library
How many recombinant DNA molecules are required
in a library to get complete coverage of a genome?

p = probability of getting a specific

piece of DNA
ln(1-p)
N=
ln(1-f) f = fractional size of clone DNA relative
to genome

N = number of clones needed

 cDNA libraries
cDNA contains only the expressed genetic information
which allows us to study the amino acid sequence directly
from the DNA.

1. Generally cDNA library are made from eukaryotic

mRNA.
• Prokaryotic mRNA is very unstable
• Genomic libraries of prokaryotes are easier to make
and contain all the genome sequences.
 Generating cDNA Library

• The first step in creating a

cDNA library is to isolate
mRNA from the cell.

• All mRNA have a poly A tail.

By using a column that
contains a short poly T
sequence it is possible to
isolate the mRNA.
 Generating cDNA Library
• Once the mRNA is isolated, it is converted into
(ss)cDNA by the action of an enzyme named reverse
transcriptase. This enzyme will create a (ss)cDNA
intermediate from the mRNA.

• Based on the poly A of the mRNA, a primer is created

with oligo dT’ bases. Reverse transcriptase recognizes
this template and will add bases to 3’ end.
 Generating cDNA Library
• At this point the (ss)cDNA needs to be converted to the
double strand cDNA.

• The mRNA cDNA complex is treated with an alkali which

hydrolyzes the mRNA, but not the cDNA.

• Then by using terminal transferase which is a DNA

polymerase that adds deoxynucleotides to free 3 ends
without the need of template (this will add poly G).

• A synthetic poly C is hybridized with poly G which is used as

primer for the synthesis of the complementary strand of the
cDNA.
 Packing the cDNA
• The first step is to ligate each end of the cDNA with a short
restriction-site linkers such as EcoRI.

• Now it is necessary to protect the cDNA from unwanted

digestion by restriction enzymes. Therefore the cDNA is
treated with methylase enzyme.

• The next step is to treat the cDNA with restriction enzymes

that will result sticky ends.

• The final step is to ligate the sticky ends of the cDNA with the
λ-phage arms that have complementary sticky ends, thereby
inserting the Double strand cDNA into the vector.
 Transfer to nitrocellulose
or nylon membrane

Keep master Select positive

Denature DNA(NaOH)
plate from master plate Bake onto membrane

Probe with 32p-labled DNA

complementary to
gene of interest

Expose to film

Screening by plaque hybridization