Download Recombinant DNA Technology

RECOMBINANT DNA TECHNOLOGY
DNA GENETIC SECRET
 Encodes the genetic instructions of all known living organisms and many viruses.
 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.
REOMBINANT 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
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 amount of its own DNA.
(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.
ESTRICTION 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 ENZYME
•
EcoRI –
Escherichia coli strain R, 1st enzyme
•
BamHI –
Bacillus amyloliquefaciens strain H, 1st enzyme
•
DpnI –
Diplococcus pneumoniae, 1st enzyme
•
HindIII –
Haemophilus influenzae, strain D, 3rd enzyme
•
BglII –
Bacillus globigii, 2nd enzyme
•
PstI – Providencia stuartii 164, 1st 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’
3’ --TTCGAA-- 5’
•
5’ --A
3’ –TTCGA
AGCTT--3’
A--5’
KpnI leaves 3´ overhangs (“sticky”)
5’--GGTACC-- 3’
3’--CCATGG-- 5’
5’ –GGTAC
C-- 3’
3’ –C
CATGG-- 5’
• Enzymes that cut at same position on both
strands leave “blunt” ends
SmaI
5’ --CCCGGG-- 3’
3’ --GGGCCC-- 5’
3’
5’ --CCC
GGG-- 3’
--GGG CCC-- 5’
Actions of restriction enzymes-overview
RCOMBINANT TCHNIQUES
•
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
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
•
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
•
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..