LECTURE 3:
Recombinant DNA
Technology & Cloning
Biotechnology; 3 Credit hours
Atta-ur-Rahman School of Applied Biosciences (ASAB)
National University of Sciences and Technology (NUST)
What are clones?
• Clones
– Genetically identical molecules, cells, or organisms
all derived from a single ancestor
• Cloning
– The production of identical copies of molecules,
cells, or organisms from a single ancestor
What is DNA cloning?
• DNA cloning is a technique for reproducing
DNA fragments.
• It can be achieved by two different
approaches:
▪ cell based
▪ using polymerase chain reaction (PCR)
• a vector is required to carry the DNA fragment
of interest into the host cell.
What is Gene Cloning?
1. A fragment of DNA, containing the gene to be
cloned, is inserted into a circular DNA molecule
called a vector, to produce a recombinant DNA
molecule.
2. The vector transport the gene in the host cell, which
is usually a bacterium, although other type of cells
can also be used
3. Within the host cell the vector multiplies, producing
numerous identical copies not only itself but also
the gene it carries
What is Gene Cloning?
4. When the host cell divides, copies od
recombinant DNA molecule are passed to the
progeny and further vector replication take
place
5. After a large number of cell divisions, a clony or
clone, of identical host cell is produced. Each cell
in the clone contains one or more copies of the
recombinant DNA molecule; the gene carried by
recombinant DNA molecule is said to be cloned.
Gene Cloning
Selection of Recombinant Plasmid
• Insertion of a piece of DNA into the
plasmid cloning vector pUC19 to
produce a recombinant DNA molecule.
• The vector pUC19 contains several
unique restriction enzyme sites
localized in a polylinker that are
convenient for constructing
recombinant DNA molecules.
• The insertion of a DNA fragment into
the polylinker disrupts part of the galactosidase (lacZ+) gene, leading to
nonfunctional  -galactosidase in E.
coli.
• The blue–white color selection test can
be used to select for vectors with or
without inserts.
Selection of Recombinant Plasmid
LacZ, White Blue Selection
• Colonies with recombinant plasmids
are white, and colonies with
nonrecombinant plasmids are blue.
• Resistant to ampicillin, has (ampr
gene)
• Contains portion of the lac operon
which codes for beta-galactosidase.
• X-gal is a substrate of betagalactosidase and turns blue in the
presence of functional betagalactosidase is added to the medium.
• Insertion of foreign DNA into the
polylinker disrupts the lac operon,
beta-galactosidase becomes nonfunctional and the colonies fail to turn
blue, but appear white.
X-gal and Beta-galactosidase
• The presence of an active β-galactosidase can be
detected by X-gal, a colourless analog of lactose that
may be cleaved by β-galactosidase to form 5-bromo4-chloro-indoxyl, which then spontaneously
dimerizes and oxidizes to form a bright blue insoluble
pigment 5,5'-dibromo-4,4'-dichloro-indigo. This
results in a characteristic blue colour in cells
containing a functional β-galactosidase.
• Isopropyl β-D-1-thiogalactopyranoside (IPTG), which
functions as the inducer of the lac operon, may be
used in the media to enhance the production of
LacZ.
Transformation
• The process of transferring exogenous DNA
into cells is call “transformation”
• There are basically two general methods for
transforming bacteria.
• The first is a chemical method utilizing CaCl2
and heat shock to promote DNA entry into
cells.
• A second method is called electroporation
based on a short pulse of electric charge to
facilitate DNA uptake.
MoU Between NUST, MoST and PCST
• Establishment of “National Analytical
Laboratory for Substance of Abuse”
• By Mir Changez Khan Jamali & Dr Mudassir
Israr
• Dope Testing
• Drug Analysis
• Drug Abuse
• Diagnosis
Transformation
Transformation; Electroporation
To electroporate DNA into cells, washed E. coli are mixed with the DNA to be transformed
and then pipetted into a plastic cuvette containing electrodes. A short electric pulse, about
2400 volts/cm, is applied to the cells causing smalls holes in the membrane through which
the DNA enters.
Transformant Selection