Download Recombinant DNA Technology (b)

RECOMBINANT DNA
TECHNOLOGY
DNA- the genetic secret!!
Encodes the genetic
virusesinstructions of all known
living organisms and many .
Nucleotides are the basic
building block.
 Nucleotide= Sugar +
phosphate + Nitrogen bases.
4 Nitrogen bases
Anti-parallel strands
Nitrogen bases
• Adenine (A)
• Guanine (G)
• Thymine (T)
• Cytosine( C)
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
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
Developed by Boyer and Cohen in 1973
3 different methods of DNA recombination
• Transformation
• Non-bacterial Transformation
• Phage induction
Recombinant DNA Technology
Basic steps involved in recombinant DNA technology
Isolation of the gene of interest
Preparation of Vector DNA and DNA to be cloned
Insertion of the gene to the vector molecule and
ligation
Introduction of the vector DNA to the appropriate
host cell
Amplification of the recombinant DNA molecule in
host cell.
Overview of rDNA technology
Bacterial cell
Bacterial
chromosome
DNA containing
gene of interest
Plasmid
Isolate Plasmid
Gene of interest
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
(i)Presence
of minimum
amountgene,
of its own
DNA. as
(antibiotic
resistance
such
ampr and tetr
(ii) Recognition sites for restriction endonuclease
(iii)Presence of at least two markers with recognition site being present
in one of the two markers
(iv)Relaxed replication control so that the recombinant plasmid is
capable of forming several copies.
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 –
• BamHI –
• DpnI –
• HindIII –
• BglII –
• PstI –
• Sau3AI –
• KpnI –
Escherichia coli strain R, 1st enzyme
Bacillus amyloliquefaciens strain H, 1st enzyme
Diplococcus pneumoniae, 1st enzyme
Haemophilus influenzae, strain D, 3rd enzyme
Bacillus globigii, 2nd enzyme
Providencia stuartii 164, 1st enzyme
Staphylococcus aureus strain 3A, 1st enzyme
Klebsiella pneumoniae, 1st enzyme
Restriction Endonucleases
Enzymes with staggered cuts  complementary ends
• HindIII - leaves 5´ overhangs (“sticky”)
5’ --AAGCTT-- 3’
5’ --A
3’ --TTCGAA-- 5’
3’ –TTCGA
AGCTT--3’
A--5’
• KpnI leaves 3´ overhangs (“sticky”)
5’--GGTACC-- 3’
5’ –GGTAC
3’--CCATGG-- 5’
3’ –C
C--
3’
CATGG--
5’
• Enzymes that cut at same position on both
strands leave “blunt” ends
SmaI
5’ --CCCGGG-3’ --GGGCCC--
3’
5’
5’
3’
--CCC
--GGG
GGG-- 3’
CCC-- 5’
Actions of restriction enzymesoverview
Recombinant techniques
• DNA to be cloned and the vector molecule are treated with the same
restriction nuclease separately
• It produces complimentary sticky ends
• Sticky ends will self ligate through covalent bonding
• This results in recombinant DNA molecule
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.
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
Xenotransplants
Agricultural Applications
Production of transgenic organisms
Environmental applications
• Many waste products of agriculture/industry do not break down
naturally/break down slowly.
Many bacteria have been GE capable of breaking down oil and other
organic wastes in Cheese making industry : GE Saccharomyces cerevisiae
able to dispose of whey by converting lactose to alcohol.
Agricultural waste products, eg. corn husks, contain cellulose that
normally decomposes slowly, can be converted into sugar by cellulase.
Cellulase has been inserted in E.coli making it useful in waste
management/disposal programs..
THANK YOU
-PHARMA STREET