Dr.

Peter John
M.Phil, PhD
Assistant Professor

Atta-ur-Rahman School of Applied Biosciences (ASAB)

National University of Sciences & Technology (NUST)
 Study of DNA Fragment
 To amplify the desired DNA fragment to generate
homogenous DNA population
 DNA Cloning: To study the structure & function of DNA
 Molecular Hybridization: The fragment of interest is not
amplified but is detected within a complex mixture of many
sequences.
 To determine its chromosomal location/expression in a
particular cell
 Cell Based DNA Cloning
 Cell based DNA cloning:
 Attaching DNA fragment to particle seq which are capable of
independent replication.
 Then recombinant DNA are transferred into host cell where
they can replicate
 Cell free DNA cloning:
 By Polymerase Chain Reaction (PCR)
 Principles of Cell based DNA cloning
 Construction of Recombinant DNA molecule

 Transformation in to host cell

 Selective propagation of cell clones

 Isolation of Recombinant DNA clones

 Replicon & Host Cell
 Replicon:
A DNA/RNA molecule or a region of DNA/RNA, that
replicate from a single origin of replication
 Vector: Replicon used for cloning

 Host Cell: Human, mammalian but mostly bacterial &

fungal cells
 Extrachromosomal replicons
 Plasmids: Small circular dsDNA molecules, contain very
few genes.
 Bacteriophages:
Viruses which infect bacterial cells. These are dsDNA viruses
which can be linear/circular
 Various modification are made in naturally occurring
replicons to be used as vector molecules
 Restriction endonuclease
 Enzymes which cleave DNA whenever a small specific

recognition seq, usually 4-8 bp

 Blunt ends: In a blunt-ended molecule both strands

terminate in a base pair.

 Overhang & Sticky ends: An overhang is a stretch of

unpaired nucleotides in the end of a DNA molecule.

Intera & Intermolecular Association
 When the fragments have same type of overhang different
types of associations can occur.
 Intramolecular: B/W two vector molecules/cyclization

 Intermolecular: B/W target DNA

Intera & Intermolecular Association
 How to avoid Intera & intermolecular association
 Cutting the vector with two different restriction
endonuclease

 Vector dephosphorylation: by alkaline phosphatase 5’

end phosphate can be removed, which avoid recircularization
 DNA Cloning in bacterial cells
 Transformation: Genetic alteration of a cell resulting from
the uptake, genomic incorporation, and expression of foreign
genetic material.
 Transduction: Genetic alterations resulting from
introduction of DNA by viruses
 Transfection: Uptake of DNA by eukaryotic cell
Plasmid Vectors Modifications
 Insertion of multiple cloning site polylinker

 Insertion of an antibiotic resistant gene

 Insertion of selection system for screening of recombinants

 Screening transformed cells
 Antibiotic resistant gene: Host cell is chosen which is
sensitive to certain particular antibiotic, but vector molecule
contain a gene so, transformed cell can survive
 β-galactoside gene complementation: The host cell &
vector contain fragments of β-galactoside gene (transformed
cell convert x-gal into blue color)
 Recombinant Screening
 Recombinant DNA molecule can be screened by insertional
inactivation of the marker gene

 Another screening system involve the use of suppressor

tRNA gene
 DNA libraries
 DNA library is a collection of cloned DNA fragments. There are

two types of DNA library

 The genomic library: contains DNA fragments representing the

entire genome of an organism

 The cDNA library: contains only complementary DNA

molecules synthesized from mRNA molecules in a cell.

Genomic DNA library
 cDNA library
 Extract total RNA from a specific tissue/development stage

 Convert RNA into cDNA

 Ligate to vector
cDNA library
Restriction Mapping for DNA study
 Restriction mapping of DNA involves cutting the DNA with
one or more of a series of different restriction nucleases and
separating the resulting fragments according to the size by gel
electrophoresis
Thanks