Action of Restriction enzyme
The enzyme cuts both DNA EcoRI cuts the DNAbetween bases
strands at the same site G and A only when the sequence
GAATTCis present in the DNA
Vector DNA Foreign DNA
T AA
EcoRl
Sticky end
Sticky end
DNA fragments join at sticky ends
Recombinant DNA
Foreign DNA Vector
DNA
(plasmid)
Same restriction enzyme cutting both foreignb
DNA and vector DNA at specific point EcoR I-Cla I Hind II
Pvu I
Pst I -BamH I
Ligases join foreign
DNA to plasmid
amp tet
pBR322 Sal I
Recombinant DNA
Molecule
ori
rop
Transformation
E.coli
7/16
(Cloning Host) Cells divide Pvu II
Figure 9.2 Diagrammatic representation of recombinant DNA technology
Region to be amplified
5' 3
ds DNA
5'
Heat
Denaturation
Primers Annealtng
-5
DNA polymerase
(Taq polymerase)
+ deoxynucleotides
5
Extension
3
5'
30 cycles
Amplified
(~1 billion times)
IA ves several procesSeS. AIter naVing cl
source DNA as well as the vector DNA with a specific restriction enzy
Jie cut out 'gene of interest' fromt 1esir DiAan: thecTvctorw
space are mixed and ligase is added. This results in the preparation
recombinant DNA.
9.3.3 Amplification of Gene of Interest using PCR
PCR stands for Polymerase Chain Reaction, In this reaction, multi
copies of the gene (or DNA) of interest is synthesised rn vitro using
Region to be amplified
ds DNA
3 5
Heat
Denaturation
Primers Annealing
DNA
(Ta
+ deoxynucleotides
3
Extension
33 5
30 cycles
2
Amplified
(-1 billion times)
ure 9.6 Polymerase chain reaction (PCR) : Each cycle has three steps: (1) Denaturati
(1) Primer annealing; and (ii) Extension of primers
2024-25
INOLOGY: PRINCIPLES AND PROCESSES
primers (small chemically synthesised oligonucleotides that are
mentary to the regions of DNA) and the enzyme DNA polymerase.
zyme extends the primers using the nucleotides provided in the
n and the genomic DNA as template. If the process of replication
is repeated many times, the segment of DNA can be amplified
roximately billion times, i.e., 1billion copies are made. Such
ed amplification is achieved by the use of a thermostable DNA
rase (isolated from a bacterium, Thermus aquaticus), which
active during the high temperature induced denaturation of
stranded DNA. The amplified fragment if desired can now be
oligate with a vector for further cloning (Figure 9.6).
Insertion of Recombinant DNA into the Host
Cell/Organism
re several methods of introducing the ligated DNA into recipient
Recipient cells after making them 'competent' to receive, take up
esent in its surrounding.So, if a recombinant DNA bearing gene
stance to an antibiotic (e.g., ampicillin) is transferred into E. coli
ne host cells become transformed into ampicillin-resistant cells.If
ad the transformed cells on agar plates containing ampicilin, only
cells will die.
