Recombinant DNA Technology Dr. Kirti Singh: School of Science Maharishi University of Information Technology
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Kirti SinghRecombinant DNA Technology Dr. Kirti Singh: School of Science Maharishi University of Information Technology
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Kirti SinghMAHARISHI UNIVERSITY OF INFORMATION TECHNOLOGY
Lecture-1
Recombinant DNA Technology
By
Dr. Kirti Singh
School of Science
Maharishi University of Information technology
Recombinant DNA Technology
Production of a unique DNA molecule by joining
together two or more DNA fragments not normally
associated with each other, which can replicate in the
living cell.
Recombinant DNA is also called Chimeric DNA
First r DNA Developed by Boyer and Cohen in 1973
3 different methods of DNA recombination
• Transformation
• Non-bacterial Transformation
• Phage induction
DNA- the genetic secret!!
The molecule that carries genetic
information for the development
and functioning of an organism
Nucleotides are the basic building
block.
Nucleotide= Sugar + phosphate
+ Nitrogen bases.
Nucleoside=Sugar+Nitrogenous
Bases
Nitrogen bases
Anti-parallel strands
Nitrogen bases
• Adenine (A)
purines
• Guanine (G)
• Thymine (T) pyrimidin
• Cytosine( C) es
found in pairs, with A & T and G & C Double helix
sequence and number of bases creates the diversity
DNA mRNA Proteins
What is Gene???
• A gene is a stretch of DNA that
codes for a type of protein that
has a function in the organism.
• It is a unit of heredity in a living
organism.. All living things
depend on genes
• Genes hold the information to
build and maintain an organism's
cells and pass genetic traits to
offspring.
Recombinant DNA Technology
Basic steps involved in recombinant DNA technology
Isolation of the gene of interest
Insertion of this isolated gene in a suitable expression vector
Introduction of this vector into a suitable host
Selection of transformed host cell
Amplification of the recombinant DNA molecule in host cell.
Purification of Protein
Overview of rDNA technology
Bacterial cell
DNA containing
gene of interest
Bacterial
chromosome Plasmid
Gene of interest
Isolate Plasmid
Enzymatically cleave
DNA into fragments.
Isolate fragment with the
gene of interest.
Insert gene into plasmid.
Insert plasmid and gene
into bacterium.
Culture bacteria.
Isolation of gene
DNA molecule is extracted from the cell by using cell lysing method
Homogenization
Centrifugation
Gene of interest is isolated using probes and electrophoresis
DNA which is to be cloned have to be inserted in to a vector
molecule which act as a carrier of the DNA to the host cell.
The choice of a vector depends on the design of the experimental
system and how the cloned gene will be screened or utilized
subsequently.
Commonly used vectors are Plasmid, bacteriophage, cosmid,
bacterial artificial chromosome (BAC), yeast artificial chromosome
(YAC), yeast 2 micron plasmid, retrovirus, baculovirus vector
Plasmid vector
Covalently closed, circular, double stranded DNA
molecules that occur naturally and replicate extra
chromosomally in bacteria and in some fungi.
Eg: pBR 322 and pUC-18
Characteristic of an ideal plasmid
(antibiotic resistance gene, such as ampr and tetr
(i) An Ori (Origin of Replication sequesnce)
(ii) Recognition sites for restriction endonuclease
(iii)Presence of at least two markers with recognition
site being present.
(iv) Antibiotics resistance gene
A plasmid containing resistance to an antibiotic (usually
ampicillin) or Tetracycline, is used as a vector.
Restriction Endonucleases
Important tool for rDNA technology is the Restriction Enzymes
Bacterial enzymes that cut DNA molecules only at restriction sites
Molecular scissors
Palindromic sequences are the recognition sites
eg: EcoRI with recognition site GAATTC
5´ GAATTC 3´
3´ CTTAAG 5
Categorized into two groups based on type of cut
• Cuts with sticky ends
• Cuts with blunt ends
if one strand extends beyond the complementary region, then the DNA is
said to possess an overhang and it will have sticky ends.
Commonly used restriction enzymes
• EcoRI – Escherichia coli strain R, 1st enzyme
• HindIII – Haemophilus influenzae, strain D, 3rd enzyme
• Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme
• KpnI – Klebsiella pneumoniae, 1st enzyme
Restriction Endonucleases
Enzymes with staggered cuts complementary ends
• HindIII - leaves 5´ overhangs (“sticky”)
5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’
3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’
• KpnI leaves 3´ overhangs (“sticky”)
5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’
3’--CCATGG-- 5’ 3’ –C CATGG-- 5’
• Enzymes that cut at same position on both
strands leave “blunt” ends
SmaI
5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’
3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’
Actions of restriction enzymes-overview
Ligation of DNA
DNA Ligases close nicks in the phosphodiester backbone of
DNA
DNA ligase is a enzyme that can link together DNA strands
that have double-strand breaks (a break in both
complementary strands of DNA).
Needs ATP
ATP
Cloning-Transformation
• It is introduced into host cell by adding it into
culture of plasmid free bacteria or animal
cells.
• Heating and adding calcium chloride favors
the transformation
• Once inside the host cell, the recombinant
DNA begins to multiply and form the desired
product.
Selection of recombinant cells
Selection of recombinant cells
• Only bacteria which
have taken up plasmid
grow on ampicillin.
• Blue-white selection:
– White colonies have
insert
– Blue colonies have no
insert
Growing successfully….
• The transformed cell are cultured and multiplied.
• Colony of cell each containing the copy of the
recombinant plasmid is obtained.
Transformation
• There is three types of transformation
i) Non Bacterial Transformation
ii) Bacterial Transformation
iii) Phase Induction
Non-Bacterial transformation
Microinjection, using
micropipette.
The host cells are
bombarded with high
velocity micro-
projectiles, such as
particles of gold or
tungsten that have
been coated with DNA.
Phage Introduction
• Phage is used instead of bacteria.
• In vitro packaging of a vector is used.
• lambda or MI3 phages to produce phage
plaques which contain recombinants.
Electroporation
• It involves applying a brief (milliseconds)
pulse high voltage electricity to create tiny
holes in the bacterial cell wall that allows
DNA to enter.
Applications…
Pharmaceutical and Therapeutic Applications
Gene therapy
Medical diagnosis
Agricultural Applications
