Basic principles and applications by shubham marbade

1. RECOMBINAT DNA
TECHNOLOGY
by
Shubham Marbade
Department of Pharmacology

2. Recombinant DNA Technology
• Recombinant DNA technology procedures by which
DNA from different species can be isolated, cut and
spliced together --new "recombinant "molecules are
then multiplied in quantity in populations of rapidly
dividing cells. for example DNA comprising
ananimal gene may be recombined with DNA from a
bacterium.
• Exa – Humulin.

3. Discovery of recombinant DNA
technology
Discovery of DNA structure Watson & Crick in 1953
Isolation of DNA ligase in 1967
Isolation of REase in 1970
Paul Berg generated rDNA technology in 1972
Cohen & Boyer in 1973 produced first plasmid vector
capable of being replicated within a bacterial host

4. Basic principles of rDNA technology

5. Continued…
Expression of the gene to produce the desired product.
Multiplication & selection of clones containing the recombinant molecules.
Introduction of the recombinant vectors into host cells.
Insertion of the selected DNA into a cloning vector to create a rDNA or
chimeric DNA.
Generation of DNA fragments & selection of the desired piece of DNA.

6. Host cell
• Living systems or cells in which the rDNA molecule or
vector can be propogated.
• Micro- organisms are preferred because they multiply
faster .
• Exa- E. coli

7. Restriction enzymes/molecular Scissors
• Key tool in rDNA technology.
• Cut the DNA at specific locations.
• Protect the host bacterial DNA from DNA of foreign
organism.

9. 1. Cleavage Pattern
• Some restriction endonucleases make staggered cuts
on the two DNA strands, leaving two to four
nucleotides of one strand unpaired at each resulting
end. These unpaired strands are referred to as sticky
ends.
• Other restriction endonucleases cleave both strands of
DNA at the opposing phosphodiester bonds, leaving
no unpaired bases on the ends, often called blunt
ends.
• DNA fragments with sticky ends are particularly
useful for rDNA experiments.

10. 2.Recognition Sequence
• Recognition sequence is the site where the DNA is cut
by a restriction enzmye.
• Restriction endonucleases can specifically recognize
DNA with a particular sequence of 4-8 nucleotides and
cleave.
3. Nomenclature
• Restriction enzymes are named after the bacterium from
which they are isolated. For example- EcoRI from E.
coli and
BamHI from B. amyloliquefaciens

11. Continued…
• The first three letters in the name of enzymes consist of
first letter of genus (E) stands for Escherichia first two
letter of species (co) stands for coli.
• This is followed by the strain (R) which means RY13
and Roman numeral (I) to indicate the order of the
discovery.

12. Types of Restriction Endonucleases
• Type I
1) These cleave DNA at random sites that can be more than
1,000 base pairs from the recognition sequence.
2) Requires ATP
• Type II
1) Cleave the DNA within the recognition sequence itself.
2) No requirement of ATP
• Type III
1) Cleave the DNA about 25 bp from the recognition
sequence.
2) Requires ATP

14. Vectors
• A vector is an area of DNA that can join another
DNA part without losing the limit for self-replication.
• It should be capable of replicating in host cell
• It should have convenient RE sites for inserting DNA
of interest.
• It Should have a selectable marker to indicate which
host cells received recombinant DNA molecule.
• It should be small and easy to isolate.

15. VECTORS
Plasmid
Bacteri
ophage
YACs
BACs
Cosmid

16. Plasmid
• Small, circular, double stranded, extra chromosomal
forms of DNA.
• Replicate independently.
• 5000-4,00,000 bp.
• Contains a Multiple Cloning Site.
• Easy to be isolated from the host cell.
• Exa- pBR322, pUC19.
• Smaller the plasmid vectors, higher the effeciency of
transformation .
• The yield of foreign DNA is reduced with larger
plasmids because these plasmids replicate to lower copy
numbers.

17. Bacteriophage
• It is a virus that infects and
replicates within bacteria. The
term was derived from"bacteria"
and the Greek (phagein),
meaning "to devour".
• Two types
1. λ phage
2. M13 phage

18. λ phage
• Infect E.coli.
• Size is 48,502 bp.
• High transformation efficiency about 1000 times more
efficient than the plasmid vector.
• About one-third of the genome is non essential and can be
replaced with foreign DNA.
• These can be readily cleaved into three pieces, two of which
contain essential genes.
• The third piece, “filler” DNA, is discarded when the vector is
to be used for cloning, and additional DNA is inserted
between the two essential segments to generate ligated DNA
molecules long enough to produce viable phage particles.

19. Bacterial artificial chromosomes
• Plasmids designed for the cloning of very long segments
(typically 100,000 to 300,000 bp) of DNA.
• They generally include selectable markers such as
resistance to the antibiotic chloramphenicol (CmR).
• Stable origin of replication (ori) that maintains the
plasmid at one or two copies per cell.
• The large circular DNAs are then introduced into host
bacteria by electroporation.

20. Yeast Artificial Chromosomes
• Also called as shuttle vectors.
• The genome of the most commonly used yeast,
Saccharomyces cerevisiae, contains only 4x106bp and its
entire sequence is known.
• A YAC can be considered as a functional artificial
chromosome since it includes three specific DNA sequences:
1) TEL: Telomere located at each chromosome end, protects the
linear DNA from degradation by nucleases.
2) CEN: Centromere which is the attachment site for mitotic spindle
fibers, "pulls“ one copy of each duplicated chromosome into each
new daughter cell.
3) ORI: Replication origin sequences which are specific DNA
sequences.

21. Cosmid
• Hybrids between a phage DNA molecule (cos sequence)
and a bacterial plasmid.
• Contain 37-52 kbp.
• Cosmids are predominantly plasmids with a
bacterial oriV, an antibiotic selection marker and a
cloning site, two, cos sites derived from bacteriophage
lambda.
• Cosmids can be used to build libraries genomic
• Exa- SuperCos 1

22. DNA ligase
• Originally isolated from viruses. Also occur in
E.coli and eukaryotes.
• It facilitates the joining of DNA strands together
by catalyzing the formation of a phosphodiester
bond between the phosphate group of 5’- carbon
of one strand and hydroxyl group of 3’- carbon of
another strand of DNA.

23. Applications
1. Recombinant Pharmaceuticals
• Human Insulin
• Growth Hormone
• Blood clotting factors
• Vaccines
• Monoclonal Antibodies
• Interferons
• Antibiotics & other secondary metabolites

24. 2. Diagnosis of molecular diseases
sickel cell anaemia, thalassaemia, cystic fibrosis etc.
3. Forensic
DNA fingerprinting helps to identify criminals and to
seetle the dispute cases of parenthood of children.
4. Agriculture
Genetically engineered plants are developed to resist
draught and disease. Good quality of food and increased
yield crops are made available.
5. Production of transgenic animals
6. Production of gene libraries.