B’ PHARMACY

SUBJECT: Pharmaceutical Biotechnology

Subject Code: BP605T
MODULE- 2

GENE CLONING VECTORS

PRESENTED BY Chamanpreet Kaur

Assistant Professor
Pharmaceutics
ASBASJSMCOP , BELA
 Objective of course
Genetic Engineering applications relation to production of pharmaceutical

• Learning Outcome

• Students will learn about cloning vectors. They will also learn
about the application of genetic Enginnering and various steps
of Polymerase Chain Reaction.
 INTRODUCTION

• A cloning vector is a DNA molecule in which

foreign DNA can be inserted or integrated and
which is further capable of replicating within
host cell to produce multiple clones of
recombinant DNA.

• Examples: Plasmids,phage or virus

 Characteristics

It should be able to replicate autonomously.

Origin of replication.
Selectable markers.
Restriction sites.
 Types

• Plasmid as vector .

• Bacteriophage as vector.

• Cosmid as vector

• Phagemid as vector.
 P lasmid Vector: pB R 3 2 2

• Contains:
• Selectable Markers:
•Ampicillin resistance gene.
•Tetracycline resistance gene.
4362 bp
• Col E I replication origin. Eco RI site.

Structure of E.Coli plasmid cloning vector pBR322

Screening procedure of cloning vector

Blue/White selection.

replica plating technique.

 B lue/ W hite Selection
Bacteria with
plasmid plus insert

IPTG + X-Gal

Overnight growth

Only colonies
Colonies with insert - white
from bacteria that
Colonies w/o insert - blue
have plasmid
Replica Plating Technique
 BACTERIOPHAGE VECTORS
• It infects bacteria.
• Follow either lytic or lysogenic cycle.
• Lambda phage vector
• high transformation efficiency, about 1000
times more efficient than the plasmid vector.
• Origin of replication.
• genes for head and tail protein.
• single- stranded protruding cohesive ends.
• Size is 48,502 bp.
 Cosmid vector
• Combine parts of the lambda chromosome
with parts of plasmids.
• an origin of replication (ori).
• a cos site(a sequence yield cohesive end) .
• an ampicillin resistance gene (amp),
• restriction sites for cloning
• Cosmids can carry up to 50 kb of inserted
DNA.
 APPLICATION
• A particular gene can be isolated and its
nucleotide sequence determined
• Control sequences of DNA can be identified&
analyzed
• Protein/enzyme/RNA function can be
investigated
• Mutations can be identified, e.g. gene defects
related to specificdiseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production.
PRODUCTION OF
HEPATITIS
PRODUCTION OF INSULIN
Polymerase Chain Reaction (PCR)
 DNA
DNA is a nucleic acid that is composed of
two complementary nucleotide building
block chains.

The nucleotides are made up of a phosphate

group, a five carbon sugar, and a nitrogen
base.
 DNA
 DNA Sugar
– Deoxyribonucleic acid

 RNA Sugar
– Ribonucleic acid
 DNA
DNA has four nitrogen bases.

 Two are purines ( 2 ringed base )

– Adenine ( A ), Guanine ( G )

 Two are pyrimidines ( 1 ringed base )

– Cytosine ( C ), Thymine ( T )
 DNA
These four bases are linked in a repeated
pattern by hydrogen bonding between the
nitrogen bases.

The linking of the two complementary

strands is called hybridization.
 DNA
Example of bonding pattern.

Primary strand
CCGAATGGGATGC
GGCTTACCCTACG
Complementary strand
 DNA
A purine always links with a pyrimidine base
to maintain the structure of DNA.

Adenine ( A ) binds to Thymine ( T ), with

two hydrogen bonds between them.

Guanine ( G ) binds to Cytosine ( C ), with

three hydrogen bonds between them.
 WHY WE NEED PCR?
• Template (the DNA you are exploring)
• Sequence-specific primers flanking the target
sequence, Forward & Reverse.
• Polymerases
• Nucleotides (dATP, dCTP, dGTP, dTTP)
• Magnesium chloride (enzyme cofactor)
• Buffer
• Water, mineral oil
 PCR REQUIREMENTS
 Magnesium chloride: .5-2.5mM
 Buffer: pH 8.3-8.8
 dNTPs: 20-200µM
 Primers: 0.1-0.5µM
 DNA Polymerase: 1-2.5 units
 Target DNA:  1 µg
• Denaturation 93 to 95°C 1min

• Annealing 50 to 55°C 45 sec.

• Elongation 70-75°C 1-2 min.

 PCR CYCLE REVIEW
 Denaturation: 94°- 95°C
 Primer Annealing: 55°- 65°C
 Elongation of DNA: 72°
 Number of Cycles: 25-40
 No target products are made until the
third cycle.
 At 30 cycles there are 1,073,741,764
target copies (~1×109).
 ADVANTAGES OF PCR
 Speed
 Ease of use
 Sensitivity
 Robustness
 APPLICATION OF PCR
 Screening human DNA samples for
mutations associated with genetic
diseases such as thalassemia and cystic
fibrosis.
 Can detect the presence of viral DNA before it
turns in to a killer.
 PCR enables rapid amplification of template DNA
for screening of uncharacterized mutations
 Can obtain sequences from hair, blood stain,
bones, other forensic specimens, other remains
preserved at archaeological sites.