Recombinant DNA

Technology
SPEAKER:
PRINCE JACOB
Contents
Introduction & Steps Involved.
Tools Used
a) Enzymes.
b) Vectors.
Transformation
Selection- DNA hybridisation.
Applications
References.
 Introduction
It is a technique involved in genetic engineering that involves the
Identification, isolation and insertion of gene of interest into a
Vector to form a Recombinant DNA Molecule and production of
large quantities of that gene fragment or product encoded by
that gene.

Steps Involved??
 Identification & Isolation of Gene of Interest.
Insertion of this Isolated Gene in a Suitable Vector.
Introduction of this Vector into a Suitable Organism
or host Cell(Transformation).
Selection of the Transformed Host Cell.
Multiplication or expression of the Introduced Gene
in the Host.
 1. Gene of Interest
From Where Do we Get this Gene of Interest??
DNA LIBRARY
Genomic Library.
cDNA Library.
 Enzymes
1) Restriction Endonucleases.
2) DNA Ligases.
Types
a)Type I RE.
b) Type II RE.
c) Type III RE.
 Type II Recognition
Restriction Sequences
Endonuclease

EcoRI (5)GAATTC(3)
CTTAAG
BamHI
(5)GGATCC(3)
CCTAGG

EcoRV (5)GATATC(3)
CTATAG

HaeIII
(5)GGCC(3)
CCGG
 Vectors
Any DNA Molecule that has the ability to replicate inside the
host to which the desired gene has integrated for Cloning.
Vectors include Plasmids, Bacteriophages, BAC, YACs and
Shuttle Vectors, etc.
Characteristics Feature-
a)Origin of Replication.
b)Small Size.
c)Suitable Marker Gene.
d)Unique Recognition Sequences.
 Plasmids
Plasmids are extrachromosomal Circular DNA
molecules that replicates separately from the host
Chromosome.
Range 4,000 to 40,000bp.
Examples E.coli plasmid pBR322, pUC18, pUC19.
Drawback DNA Segments >15,000 bps.
 Transformation
Mainly by three methods
Calcium chloride method.
Electroporation.
Phage mediated transduction.

Others Methods?
 Selection
1. By using probes.
(Colony hybridization)
2. Without using Probes.
(By using antibiotic resistance property)
 References
Lehninger, Principles of Biochemistry,6th Edition.
Biochemistry,7th Edition, Stryer.
Textbook of biochemistry by DM Vasudevan, seventh
edition.
TextBook of Biochemistry, Rafi MD,2nd Edition.
Principles of biochemistry, by Voet, fourth edition.