Vikram Viswajit Mishra Branch-Pharma Tech Reg No.

1008268017

Content
Introduction History of recombinant DNA technology Creating recombinant DNA Steps In Preparation of Clone DNA Applications of rDNA technology Conclusion References

Introduction
y Recombinant DNA (rDNA) molecules

are DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms y Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure y Recombinant DNA molecules are sometimes called chimeric DNA

History of recombinant DNA technology

y The idea for recombinant DNA was first proposed by

Peter Lobban, a graduate student of Prof. Dale Kaiser in the Biochemistry Department at Stanford University Medical School. y Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973. y The first licensed drug generated using recombinant DNA technology was human insulin, developed by Genentech and Licensed by Eli Lilly and Company

Creating recombinant DNA

y Molecular cloning is the laboratory process used to

create recombinant DNA y It is used to direct the replication of any specific DNA sequence chosen by the experimentalist y Requirements for preparation of clone DNA 1. Passenger DNA 2. Vector DNA 3. DNA lygase 4. Restriction Endonucleus enzyme 5. Host cell

Passenger DNA
y Passenger DNA or foreign DNA is the segment of

DNA which is enzymatic ally isolated and cloned . E.g. Mammalian Clone y The Mammalian DNA is first isolated or extracted from body y The DNA is subjected to E.Coli y The E.Coli cuts the mammalian DNA into two distinct Ends a) Blunt end b) Stick end

Vector DNA
y The agents which helps in carrying the passenger DNA y

y y y y y

is called as vector DNA The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Types of Vector Plasmid Vector Cosmid Vector Phase Virus Viral DNA

DNA lygase enzyme

y DNA ligase is a specific type of enzyme, a ligase, that

repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break y The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor").

Restriction Endonucleus enzyme

y A restriction endonuclease is an enzyme that cuts

double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. y Restriction enzymes recognize a specific sequence of nucleotidesand produce a double-stranded cut in the DNA. y Different restriction enzymes that recognize the same sequence are known as neoschizomer.

Steps In Preparation of Clone DNA

y The passenger DNA is used for preparation of CDNA y The DNA is subjected to ECO R1 enzyme which is a

category of restriction endonucleases y It cuts the mammalian DNA into small fragments having blunt end and sticky end y The sticky end now combines with the passenger DNA by the help of DNA lygase so a hybrid DNA is formed y The sticky end of the passenger DNA binds with the stick ends of phase DNA and this process is called as annealing

y This Chimeric DNA now becomes ready to enter the

host cell y In host cell it replicates along with the host cell. y The cloned DNA by is extracted by lysis of cell wall y It is purified and then isolated

Applications of rDNA technology

1. Recombinant human insulin

Recombinant insulin has almost completely replaced insulin obtained from animal sources for the treatment of insulin-dependent diabetes.

y Joining the human insulin gene into a plasmid

The bacterial plasmids and the cDNA are mixed together. The human insulin gene (cDNA) is inserted into the plasmid through complementary base pairing at sticky ends.

y Introducing the recombinant DNA plasmids

into bacteria
The bacteria E.coli is used as the host cell. If E. coli and the recombinant plasmids are mixed together in a test-tube.

y Selecting the bacteria which have taken up

the correct piece of DNA

The bacteria are spread onto nutrient agar. The agar also contains substances such as an antibiotic which allows growth of only the transformed bacteria.

2) Recombinant human growth hormone y Growth hormone is administered to patients whose pituitary glands generate insufficient quantities to support normal growth and development. y Before recombinant HGH became available, HGH for therapeutic use was obtained from pituitary glands of cadavers. 3)Recombinant hepatitis B vaccine. y Prevention of hepatitis B infection is controlled through the use of a recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen that is produced in yeast cells.

4)Diagnosis of infection with HIV. y Each of the three widely-used methods for diagnosing HIV infection has been developed using recombinant DNA. y The antibody test (ELISA or western blot) uses a recombinant HIV protein to test for the presence of antibodies that the body has produced in response to an HIV infection

Conclusion
Recombinant DNA research is a challenging field, but it holds great promise for the future. In the future, rDNA technologies will play a key role in preventing genetic diseases, producing targeted medicines, and providing patients with less toxic pharmaceuticals.

References
y Pharmeaceutical Biotechnology-By S.P Vyash & V.K. y y y y

Dixit page no.340-400 Pharmaceutical Biotechnology- By S.S. Kori & M.A.Halki page no.42-60 http://www.fiercebiotech.com/topics/rDNA.asp http://www.rpi.edu/dept/chem-eng/BiotechEnviron/Projects00/rdna/rdna.html en.wikipedia.org/wiki/Recombinant_DNA