POTENTIAL OF RECOMBINANT DNA

TECHNOLOGYSubmitted to: Dr. Irfana Iqbal Khan

Submitted by: Ayesha Tahir Roll. No. 38155233

HOW R-DNA FORMS AND BEING CLONEDSteps:• Obtain desired

fragment• Obtain a suitable vector• Cut both with same

restriction enzyme• Mix all the fragments

and vectors• Ligation—DNA ligase• Transformation• Individual colonies• Clones • Libraries

----Genomic----cDNA

http://www.britannica.com/science/recombinant-DNA-technology

ANALYSIS OF THE CLONED DNA

• can be performed at the stage when it is in the bacterial host

• reintroduce the cloned DNA back into cells of the original donor organism

---- to carry out specific manipulations of genome structure and function. http://www.ncbi.nlm.nih.gov/books/NBK21881/

RECOMBINANT DNA TECHNOLOGYPotential of r-DNA technology---various fields• Molecular biology---Gene mapping• Genetic disorders• Production of monoclonal antibodies• Gene therapy• Vaccine and pharma products formation• Crop improvement• Fingerprinting

MOLECULAR BIOLOGY---GENE MAPPING

RESTRICTION MAPPING:• the first stage of analysis

• provides a compilation of the number, order, and distance between restriction endonuclease cutting sites along a cloned DNA fragment

• plays an important role in characterizing DNA, mapping genes, and diagnostic tests for genetic diseases

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

RFLPs can serve as markers of genetic diseases• single base pair substitutions, insertion of genetic material

such as transposable elements, or by tandem duplications, deletions, translocations, or other chromosomal rearrangements

• In linkage analysis, families in which individuals are at risk for a genetic disease are identified

RFLP

• DNA samples from various family members are analyzed to determine the frequency with which specific RFLP markers segregate with the mutant allele causing the disease.

• This frequency is a measure of the distance between the markers and the mutation-defined locus.

GENE THERAPY

• TIL tissue infiltrating lymphocytes from patient tumors• Tagged with neo-resistance gene• Readminister in patient• Track the path of TIL cells in the body to determine the

actual level of TIL infiltration into the tumor• Hope to correct certain genetic diseases

PRODUCTION OF

MONOCLONAL ANTIBODY

https://en.wikipedia.org/wiki/Monoclonal_antibody

• Hybridoma

• monospecific antibodies

• monovalent affinity

R-DNA TECH AND MONOCLONAL AB• recombinant monoclonal Ab repertoire cloning or phage/yeast

display technologies• use of viruses or yeast, rather than mice. • These techniques rely on rapid cloning of immunoglobulin gene

segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected.• The phage antibody libraries are a variant of phage antigen libraries.• Fermentation chambers have been used for large scale antibody

production.

MONOCLONAL ANTIBODIES• Hybridoma technology and monoclonal Ab have made significant

impact in all disciplines of biology and medicine.

• Detect AIDS, meningitis, hepatitis, chlamydia, tuberculosis and many other diseases.

• enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy and their detectability in diagnostic applications

VACCINES AND OTHER PHARMA PRODUCTS FORMATION

Human proteins of Therapeutic Value:Product Therapeutic Use

Insulin DiabetesTissue plasminogen activator Thrombolysis

Growth hormone Dwarfism erythropoietin Anemia

interferons Virus infection, cancer therapy, AIDS

DNase Cystic fibrosisVaccines Hepatitis, malaria, Feline

leukemia, AIDS

• Human insulin treat diabetes mellitus first recombinant protein to be commercialized• Human growth hormone children with pituitary

dwarfism• Bovine growth hormone production of milk by dairy

cows• Interferons alpha-interferon substaintially inhibit in

vitro growth of renal cell carcinoma and hairy cell leukemia cells• Tissue plasminogen activator dissolve blood clots at

particular sites without causing bleeding elsewhere

• IL-2 induce remission of malignant melanoma

• Tumor necrosis factor TNF TNF binds to a specific plasma membrane receptor present on a wide variety of different cells, both normal and malignant, and causes a cytotoxic effect

• Coagulation Factors IX treat some hemophiliacs

VACCINESHBsAg• Isolate gene for the surface antigen of hepatitis B virus

HBsAg• Espressed in yeast or mammalian cell culture industry• R-HBsAg used for diagnostic purposes, potential vaccineRecombivax HB• Vaccine for hepatitis B virus• Using HBsAg intracellularly in yeast

CROP IMPROVEMENTR-DNA technology played a great role in crop improvement by:• Herbicide resistance• Pathogen resistance• Insecticide resistance• Frost damage• Preservation of fruit• Sterile male plants

HERBICIDE RESISTANCE

• Weeds and microbes resistance genes plants• Resistance to the herbicide sulfonylurea was transferred

into tobacco• Sulfonylurea inhibits the activity of enzyme

acetolactate synthase control the biosynthesis of branched chain amino acids• Genes that code for aceto-lactate synthase resistance

to sulfonylurea herbicides were isolated from bacteria and expressed in tobacco.

PATHOGEN RESISTANCE

• Bioengineered microbes combat soil born plant diseases

• Fusarium wilt and phytophthora root rot

• Microbes in rhizosphere can be selected and engineered to promote plant growth and kill plant pathogens

FROST DAMAGE

• Ice forming protein coded by bacteria living on the leaves of certain crop plants forst damage• Scientists deleted the gene • ICE MINUS BACTERIA• Allow the plant to survive colder temperature

FINGERPRINTING• Small pieces of DNA can be used in hybridization to identify in a mixture

of total genomic sequences the particular gene which contains that piece of DNA DNA probes• Specificity of DNA-DNA interactions

• Identify human pathogens, hereditary defects and plant diseases• Genetic defects in fetus• DNA probes and southern blotting forensic science

FINGERPRINTINGDNA can be isolated from human hair, blood and other body fluids and amplified using PCR Polymorphic tandem repetitive human DNA sequences minisatelliteProduce a fingerprint by detecting many hypervariable minisatellites• Fingerprints are specific to a given individual • Used in courts to settle crime, paternity etc

THANK YOU Any question??