2. HISTORY
Herbert Boyer(1936) and Stanley N. Cohen(1935) devlop
recombinant DNATecnology,showing that ther genetically
engineered DNA molecules may be Cloned in foreign cells
THEY CONSTRUCTED 1ST RECOMBINENT DNA using
Salmonella typhimurim.
3. Recombinant DNA is term used to describe the combination of two DNA
Strands that are constructed artificially.Genetic scientists can do this to create
Unique DNA strand for diffrent perposes,using several type of techniques
DEFINITION:
PRINCIPLE:
Source-authorstream.com
4. 1. Restriction enzymes
2. Ligases
3. Miscellanious enzymes
RESRICTION ENZYMES:-
ENDONUCLEASES
Cuts DNA at ends
EXONUCLEASES
Cuts DNA at specific sites
Pallindromic sites (i.e the sequence of base pairs reads the same on both
DNA strands).
Ex- 5’-GAATTC-3’
3’-CTTAAG-5’
4.Vectors
5.Host organism/cell
Ex- Exonuclease III
TOOLS OF RECOMBINANT DNA TECHNOLOGY
5. 1.Firstly letter derived from genus name (italics)
2.Next,two letters derived from species name (italics)
3.The Roman number show order in which the enzyme
were iosolated From bacterial strain.
Ex- EcoRI is from Escherichia (E) coli(co),
strain RY13(R) and first
endonucleases(I) to be discovered.
HindIII is from Haemophillus(H) influenzae(in),
Strain Rd(d) and the third endonucleases(III) to be
discovered.
NOMENCLATURE OF RESTRICTION ENZYMES:
6. Cut DNA fragments joint covalently by DNA ligases
Originally isolated from viruses
Also occur in E.Coli and eukaryotic cells
Actively participate in cellular DNA repair process
Parmanently hold DNA pieces together......
Alkaline phosphatase : removes phosphate groups from
5’ends of single Or double stranded DNA /RNA.
Polyneucleotide kinase: involved in addition of phosphate
groups
Nucleases:degrades single stranded DNARNA.
polymerases: Synthesis of nucleic acid .
DNA LIGASES:
MISCELLANEOUS ENZYMES:
7. Microbes multiply faster compared to cells of higher organism
(plants/animals) thus preffered to as host cell.
Prokaryotic hosts include E.Coli ,Bacillus subtilis
Eukaryotic hosts include saccharomyces cerevisiae(fungi),
Mammalian cells ,plants(protoplast)......
Host cells:
VECTORS:
Vectors are DNA molecules which carry a forign DNA fragment
into the Host cells
TYPES:
Plasmid
Cosmid
Phasid
Bacteriophage
Artificial cromosomes
8. I. Should be small in size
II. Self replicating, circular, double stranded
III. Should contain 4 segments:
a) Origin of replication
b) Restiction endonuclease recognition site
c) Selectable marker
d) Tag gene
CHARECTERSTICS OF VECTOR:
Fig-plasmid vector
9. STEPS INVOLVED IN RECOMBINENT DNA TECHNOLOGY:
GENERATION of DNA fragments ,and SELECTION of desired
DNA pieces(eg:human gene)
AMPLIFICATION of gene of interest
LIGATION of the DNA fragment into a cloning vector
INSERTION of recombinant DNA into Host cell
CULTURING the Host cell in a suitable medium
GENE EXPRESSION done
DESIRED produt obtained
12. IN MOLECULAR BIOLOGY:
Used to ELUCIDATE MOLECULAR EVENTS in BIOLOGICAL PROCESSES like
CELL DIFFERENTIATION
AGENING
GENE MAPPING
MOLECULAR DIAGNOSIS OF DISEASES:
Eg-HIV;Hepatitis virus ,Food poisoniong
MONOCLONAL ANTIBODIES PRODUCTION
GENE THERAPY
DNA FINGER PRINTING
VACCINE PRODUCTION
PRODUCTION OF PHARMACEUTICALS PRODUCTS(insulin)
To increase CROP PRODUICTIVITY,NUTRITION VALUE and
to increase tolerance againt ABIOTIC STRESS (COLD,DROUGHT,HEAT etc)
APPLICATION:
