Recombinant DNA Technology

Presented byStiti Prangya Dash

Bs 12 416

Guided byDr. Puspashree puhan

Contents….• What is recombinant DNA technology.• Biological and chemical tools used in rDNA-

technology. • Procedure.• PCR technique. • Applications.• Conclusion.

Recombinant 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

HERBERT BOYER STANLEY COHEN

ENZYMES USED IN R-DNA TECH

• Enzymes used in R-DNA technology falls into 4 broad categories :

• 1) Template dependent DNA polymerase : those which are used in synthesis of new polynucleotide complementary to an existing DNA or RNA template. Example: DNA polymeraseI, DNA poly III, Reverse transcriptase, Taq polymerase etc .

• 2) Nucleases : these enzymes used to degrade DNA molecules by breaking phosphodiester bonds. Example : exonucleases and endonucleases . Endonucleases also have a broad category i.e restriction endonucleases, used to cut DNA at specific sequence.

Continued….• 3) End modification enzymes : these enzymes make

changes to the ends of DNA molecules. Example : Terminal deoxynucleotidyl transferase , Alkaline phosphatase , T4 polynucleotide kinase.

• 4) Ligases : DNA ligases join DNA molecules together by synthesizing phosphodiester bonds between nucleotides at the ends or between two different molecules. They can be Linkers and Adaptors.

Restriction EndonucleasesImportant tool for rDNA technology is the Restriction Enzyme

Bacterial enzymes that cut DNA molecules only at restriction sites Molecular scissors Palindromic sequences are the recognition siteseg: EcoRI with recognition site GAATTC

5´ GAATTC 3´3´ CTTAAG 5Categorized 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 enzymes

EcoRI – Escherichia coli strain R, 1st enzyme• 5´ GAATTC 3´ STICKY END

3´ CTTAAG 5

BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme 5‘GGATCC 3’ STICKY END

• 3’CCTAGG 5’ HindIII –Haemophilus influenzae, strain D, 3rd enzyme ‘

• 5’AAGCTT 3’ STICKY END• 3’ TTCGAA 5’

Taq I – Thermus aquaticus , • 5’TCGA3’ STICKY END• 3’AGCT5’

ECORV - Escherichia coli , 5’GATATC3’ BLUNT END

Isolation of gene DNA molecule is extracted from the cell by using cell lysing

methodHomogenization

Centrifugation Gene of interest is isolated using probes and electrophoresis

DNA , RNA and Plasmids can be isolated by different methods .

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.

Vectors

• Vector is an autonomously replicating genetic element used to carry a fragment of target DNA into a host cell for the purpose of clonning and expression.

• Vectors should be :• 1) able to replicate inside host • 2)should contain genetic marker to select for host

cell containing vector• 3) should have unique restriction enzyme sites for

insertional cloning.

Vectors commonly used

• Plasmid : (E.coli) 1-5 kb insert size • Example ; pBR322 , Puc19

• Cosmids : (E. coli) : 35-45 kb size

• Phagemids : 1-4 KB size • Ex : M13

• BACs : < 300 Kb (E.coli)

• YACs : 200-2000 kb (yeast)

• AGROBACTERIUM (PLANT)

• ANIMAL VIRUSES (ANIMAL)

HOST (in vivo gene amplification or expression)

• The vector carrying desired gene should be inserted into a suitable host.

• Host should be suitable enough to provide the vector a shelter, as well as freedom to replicate inside it.

• The host enzymes shouldn’t interfere with the external genetic material.

• Among prokaryotes E.coli is suitable host used in RDNA TECH.

• Among eukaryotes yeast is suitable.

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.

Overview of rDNA technologyBacterial cell

DNA containinggene of interest

Bacterialchromosome Plasmid

Isolate Plasmid Gene of interest

Enzymatically cleaveDNA into fragments.

Isolate fragment with the gene of interest.

Insert gene into plasmid.

Insert plasmid and gene into bacterium.

Culture bacteria.

Selection of recombinant cells

Selection of recombinant cells• Only bacteria which have

taken up plasmid grow on amphicillin.

• Blue-white selection (x-gal medium) :– 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 introduction-Phage is used instead of bacteria.

• In vitro packaging of a vector is used.• lambda or MI3 phages to produce phage plaques

which contain recombinantsIt • Electroporation-involves applying a brief

(milliseconds) pulse high voltage electricity to create tiny holes in the bacterial cell wall that allows DNA to enter.

PCR TECHNIQUE(polymerase chain reaction)

This technique used in desired gene amplification. This is a rapid and versatile in vitro method for amplification

of target DNA sequences. This technique was developed by Kerry mullis (1985). This method includes 1) primer design 2) degenerate primers 3) reaction cycle * denaturation (93-95 c) * primer annealing (50-70 c) * DNA synthesis (70-75 c)

MODIFICATIONS OF PCR

• Nested PCR • Asymmetric PCR • RT – PCR • Hot-start PCR • Touchdown PCR • Anchored PCR • Inverse PCR • RAPD • RACE

Applications of recombinant DNA technology

• Recombinant DNA is widely used in biotechnology, medicine and research. Today, recombinant proteins and other products that result from the use of rDNA technology

• The most common application of recombinant DNA is in basic research, in the biological and biomedical sciences, Recombinant DNA is used to identify, map and sequence genes, and to determine their function. rDNA probes are employed in analyzing gene expression within individual cells, and throughout the tissues of whole organisms. Recombinant proteins are widely used to generate antibody probes for examining protein synthesis within cells and organisms.

• Many additional practical applications of recombinant DNA are found in industry, food production, human and veterinary medicine, agriculture, and bioengineering. 

• Recombinant chymosinFound in rennet, is an enzyme required to manufacture cheese. It was the first genetically engineered food additive used commercially. Traditionally, processors obtained chymosin from rennet, a preparation derived from the fourth stomach of milk-fed calves. Scientists engineered a non-pathogenic strain (K-12) of E. coli bacteria for large-scale laboratory production of the enzyme. This microbiologically produced recombinant enzyme, identical structurally to the calf derived enzyme, costs less and is produced in abundant quantities. Today about 60% of U.S. hard cheese is made with genetically engineered chymosin.

• Recombinant human insulin Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. A variety of different recombinant insulin preparations are in widespread use. Recombinant insulin is synthesized by inserting the human insulin gene into E. coli, which then produces insulin for human use.Recombinant human growth hormone (HGH, somatotropin) Administered to patients whose pituitary glands generate insufficient quantities to support normal growth and development.

• Recombinant blood clotting factor VIII A blood-clotting protein that is administered to patients with forms of the bleeding disorder hemophilia, who are unable to produce factor VIII in quantities sufficient to support normal blood coagulation. Before the development of recombinant factor VIII, the protein was obtained by processing large quantities of human blood from multiple donors, which carried a very high risk of transmission of blood borne infectious diseases, for example HIV and hepatitis B. DrugBank entry Recombinant hepatitis B vaccine 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.

• The development of the recombinant subunit vaccine was an important and necessary development because hepatitis B virus, unlike other common viruses such as polio virus, cannot be grown in vitro. Vaccine information from Hepatitis B FoundationDiagnosis of infection with HIV Each of the three widely used methods for diagnosing HIV infection has been developed using recombinant DNA. 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. The DNA test looks for the presence of HIV genetic material using reverse transcriptase polymerase chain reaction (RT-PCR). Development of the RT-PCR test was made possible by the molecular cloning and sequence analysis of HIV genomes  .

• GOLDEN rice A recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis. This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population. Golden rice is not currently in use, pending the resolution of regulatory issues. Herbicide-resistant crops Commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed that incorporate a recombinant gene that results in resistance to the herbicide glyphosate (trade name Roundup), and simplifies weed control by glyphosate application.These crops are in common commercial use in several countries. Insect-resistant crops Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties.[The bacterium has been applied to crops as an insect-control strategy for many years, and this practice has been widely adopted in agriculture and gardening. Recently, plants have been developed that express a recombinant form of the bacterial protein, which may effectively control some insect predators.

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..

Conclusion…• R-DNA technology is the new era in field of

biology which has huge applications and possibilities.

• It is now a great topic for researching and exploring .

• It can take us to a new level of biological science , which is “biotechnology” – living organisms manipulated by technology .

Bibliography• www.nature.com• Wikipedia• Principles of gene manipulation

S.B.Primrose • B.D singh biotechnology

SPECIAL THANKS TO

Dr. Puspashree puhan

For your sincere support and guidance