Recombinant DNA Technology

Recombinant DNA Technology
2 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 1
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida
Abhijit Debnath
Asst. Professor
NIET (Pharmacy Institute)
Unit: 2
Subject Name: Biotechnology
(BP605T)
Course Details
(B. Pharm 6th Sem)

Recombinant
DNA Technology
 Recombinant DNA Technology
 Discovery of Recombinant DNA Technology
 Goals of Recombinant DNA Technology
 Procedure of Making rDNA
 Techniques Used In rDNA Technology
 Applications of rDNA Technology
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida

RECOMBINANT DNA (CO1.1)
Recombinant DNA technology is the joining together of
DNA molecules from two different species. The
recombined DNA molecule is inserted into a host organism
to produce new genetic combinations.
DNA molecules that are extracted from different sources
and chemically joined together; for example: DNA
comprising ananimal gene may be recombined with DNA
from a bacterium.

DISCOVERY OF RECOMBINANT DNA TECHNOLOGY (CO2.2)

 Toisolate and characterize a gene
 Tomake desired alterations in one or more isolated genes
 Toreturn altered genes to living cells
 Artificially synthesize new gene
 Alternating the genome of an organism
 Understanding the hereditary diseases and their cure
 Improving human genome
GOALS OF RECOMBINANT DNA TECHNOLOGY (CO2.2)

Isolating of DNA
Cutting of DNA
Joining of DNA
Amplifying of DNA
PROCEDURE OF MAKING rDNA (CO2.2)

ISOLATING OF DNA (CO2.2)

 DNA can be cut into large fragments by mechanical shearing.
 Restriction enzymes are the scissors of molecular genetics.
CUTTING OF DNA (CO2.2)

 A special class of sequence-specific enzyme
 Found in bacteria
 Site-specific-cleave DNA molecules only at
specific nucleotide sequence
 REases recognize DNA base sequence that are
palindrome
 REase make staggered cuts with
complementary base sequences for easy
circulization
RESTRICTION ENZYME (CO2.2)

JOINING DNA (CO2.2)

 Transforming the recombinant DNA into a bacterial host strain.
 The cells are treated with CaCl2
 DNA is added
 Cells are heat shocked at 42 C
 DNA goes into cell by a somewhat unknown mechanism.
 Once in a cell, the recombinant DNA will be replicated.
 When the cell divides, the replicated recombinant molecules go to both
daughter cells which themselves will divide later.
AMPLIFYING THE RECOMBINANT DNA (CO2.2)

• Bind to DNA molecules
DNA ligase
• Cleaves DNA at specific sites
Type II restriction endonuclease
• Make a DNA copy of RNA molecule
Reverse transcriptase
• Fill single stranded gapes of DNAduplex
DNA polymerase I
• Adds a phosephate to the 5'-OH end of a polynucleotide
Polynycleotide Kinase
• Adds homopolymer tails to the 3'-OH ends
Terminal transferase
• Removes nucleotide residues from the 3' ends
Exonuclease III
• removes nucleotides from the 5' ends
Bacteriophage {lamda} exonuclease
• Removes terminal phosphates
Alkaline phosphatase
ENZYMES USED IN RECOMBINANT DNATECHNOLOGY (CO2.2)

 Plasmids are small, circular DNA molecules that are separate from the
rest of the chromosome.
 They replicate independently of the bacterial chromosome.
 Useful for cloning DNA inserts less that 20 kb (kilobase pairs).
 Inserts larger than 20 kb are lost easily in the bacterial cell.
PLASMID VECTOR (CO2.2)

 Gel electrophoresis
 Cloning libraries
 Restriction enzyme mapping
 PCR
 Nucleic Acid Hybridization
 DNA Microarrays
TECHNIQUES USED IN RDNA TECHNOLOGY (CO2.2)

 Gel electrophoresis – DNA fragments of
different sizes can be separated by an
electrical field applied to a “gel”.
 The negatively charged DNA migrates away
from the negative electrode and to the
positive electrode.
 The smaller the fragment the faster it
migrates.
GEL ELECTROPHORESIS (CO2.2)

• Libraries are collection of DNA clones in a certain vector.
• The goal is to have each gene represented in the library at least once.
• Genomic- made from RE DNA fragments of total genomic DNA.
• cDNA (complementary DNA) – made from DNA synthesized from mRNA.
CLONING LIBRARIES (CO2.2)

 Agriculture: growing crops of your choice (GM food), pesticide resistant
crops, fruits with attractive colors, all being grown in artificial conditions.
 Pharmacology: artificial insulin production, drug delivery to target sites
 Medicine: gene therapy, antiviral therapy, vaccination, synthesizing
clotting factors.
 Other uses:fluorescent fishes, glowing plants etc..
 Technology Wide spectrum in improving health.
 Treat defected gene or introduce new one.
 Applications, laboratory test and parental diagnosis of genetic disease.
APPLICATIONS OF rDNA TECHNOLOGY (CO2.2)

 Introduce live attenuated .
 Acquired immunity.
 Rdna technology can be use to clone gene for protective antigen protein.
 Hepatitis B vaccine (rDNA) , influenza ,HIV and mouth and foot disease.
PRODUCTION OF VACCINE (CO2.2)

Insulin:
 Insulin(hormone) controls glucose level in humans.
 By rDna , done cloning of human insulin gene and put in E.coli.
 availability of insulin.
 devoid of getting by-products by animal slaughtering.
COMMERCIAL AND PHARMACEUTICAL PRODUCTS (CO2.2)

 HGH is homing polypeptide .
 121 amino acids, 2 to 115 Dalton molecular
weight.
 role in growth , regeneration or
differentiation.
 E.g: dwarfism treating by injecting these.
HUMAN GROWTH HORMONE (CO2.2)

 Interferon are group of proteins that interfere with viral
multiplication or replication.
 By rDna,capable of making interferon.
 Alpha component of which have role in curing lymphoma and
myelogenous leukemia.
INTERFERON (CO2.2)

 Antibodies are specific proteins produced by the immune system in response
to presence of a specific antigen.
 Monoclonal antibodies produce from singleclone of
antigen. That’s why are monospecific in nature.
 Production through hybridoma technology.
 Applications:
 mAb are used for diagnosis of disease, Pregnancy and Treatment of cancer.
.
MONOCLONAL ANTIBODIES (CO2.2)

 Infectious diseases diagnosis mainly depends upon
isolation and identification of pathogens, which may take several
days.
 Development of diagnostic kits to identify pathogenic organisms by
knowing the organism-specific DNA sequence has provided rapid,
specific and correct diagnosis.
 Various diagnostic kits have been developed for AIDS, cancer, foot and
mouth diseases, tuberculosis, etc.
MOLECULAR DIAGNOSIS OF DISEASES (CO2.2)

Dr. Alec Jeffreys developed DNA fingerprinting technique.
 Every person have its unique finger patterns that differs from other individual.
 There is possibility to alter these patterns but specific principle is unknown.
 Finger prints are detected on the basis of number of highly polymorphic genes i.e.
VNTR’s.
Applications:
 Used in criminal identification.
 For child parentage establishment.
 Helpful for deduction of racial group.
DNA FINGERPRINTING (CO2.2)

injects functional genes into a cell to replace missing or defective genes in order to
correct genetic disorders.
 A gene that is inserted directly into a cell usually does not function. Instead, a
carrier called a vector is genetically engineered to deliver the gene.
 Gene therapy may be donein-vivo or e-vivo.
Health Risks:
toxicity, inflammation, and cancer.
GENE THERAPY (CO2.2)

 We can use recombinant DNA technology in environment to cleanup
the environment.
 Measure the presence of hazardous compounds
IMPORTANCE (CO2.2)

• Molecular Biology: Gene Mapping
• Genetic Disorder
• Monoclonal Ab product
• Gene Therapy
• DNA Fingerprinting
• Vaccines
• Pharma Products
APPLICATION OF GENETIC ENGINEERING (CO2.2)

 A genetic disorder is a disease that is
caused by an abnormality in an individual's
DNA. Abnormalities can be as small as a
single-base mutation in just one gene, or
they can involve the addition or
subtraction of entire chromosomes.
 Most common disorders: Cystic
Fibrosis, Down syndrome, Duchenne
muscular dystrophy
GENETIC DISORDER (CO2.2)

 Medical Definition of DNAfingerprinting.
:a technique used especially for
identification (as for forensic purposes) by
extracting and identifying the base-pair
pattern of an individual's DNA—called
also DNA typing, genetic fingerprinting.
 DNA fingerprinting. Image caption: In
DNA fingerprinting, scientists
collectsamples of DNA from different
sources — for example, from a hair left
behind at the crime scene and from the
blood of victims and suspects. They then
narrow in on the stretches of
repetitive DNA scattered throughout
these samples.
DNA FINGERPRINTING (CO2.2)

Recombinant DNA Technology

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