RDT Brijesh presentation

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Recombinant DNA Technology:

Recombinant DNA Technology Presented By Brijesh Singh Yadav Department of Microbiology H.N.B.Garhwal, University [email protected]

Definition:

Definition A series of procedures used to recombine DNA segments Under certain conditions. A recombinant DNA molecule can enter a cell and replicate.

History:

History Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973.

Basic principle:

Basic principle The DNA is inserted into another DNA molecule called ‘vector ’ The recombinant vector is then introduced into a host cell where it replicates itself, the gene is then produced

The Tools Used for RDT:

The Tools Used for RDT The desired DNA to be cloned Restriction endonuclease (Molecular scissor) Vector (Capable of self replicating in host cell) DNA Ligase (Molecular glue) Host cell (for propagation of recombinant DNA)

STEPS IN GENE CLONING:

STEPS IN GENE CLONING Production and Isolation of the DNA to be cloned Insertion of the gene in to vector obtained recombinant DNA Introduction of recombinant DNA in to suitable host (generally E.coli ) Multiplication/ Expression of introduce gene in the host cells Transfer and expression of gene in to another organism if needed

CLONING DNA MOLECULES:

CLONING DNA MOLECULES Isolate and purify desired DNA from biological source Cut DNA with restriction endonuclease Join fragments to cloning vectors cleaved with compatible restriction endonuclease to create recombinant DNA molecules Transfer recombinant molecules to host cells Isolate DNA from individual clones of transformed host cells Do as you please with the isolated DNA segments…

RESTRICTION ENDONUCLEASES:

RESTRICTION ENDONUCLEASES Endonuclease an enzyme that produce internal cut, called cleavage in in DNA molecules A class of Endonuclease cleave DNA only within or near those site, which have specific base sequence, such endonuclease is known as Restriction endonuclease (RE) The site recognized by Restriction endonuclease are called recognition sequence or recognition site

DISCOVERY OF R.E.:

DISCOVERY OF R.E. 1971 paper by Kathleen Dana and Daniel Nathans described isolation of enzyme that cleaved DNA at specific sequences 1978 Nobel Prize to Nathans, Smith and Arber for restriction endonuclease discovery

TYPES:

TYPES Type I Type II Type III Complex Stable Intermediate Recognition Sequence about 15 Base pairs Mg+ ion require for cleavage Cleave the DNA, 100 basepairs away from recognition Sequence “TCA” Cleave the DNA, At or near the recognition Sequence Cleave the DNA, 24-26 basepairs to 3’ site of recognition Sequence Eg.EcoK , EcoB Eg . Hind II Eg . EcoPI , Eco15

RESTRICTION ENDONUCLEASES:

RESTRICTION ENDONUCLEASES

DNA CLEAVAGE BY ECORI:

DNA CLEAVAGE BY ECO RI

RECOMBINANT DNA MOLECULES:

RECOMBINANT DNA MOLECULES

CLONING VECTORS:

CLONING VECTORS Plasmids Phage Cosmids BACs YACs

PLASMIDS:

PLASMIDS Circular independent replicons, origin of replication ( ori ) Generally encode useful but not essential genes E.g. antibiotic resistance or catabolic pathways Allow cloning fragments up to about 10 kbp Selectable markers Multiple cloning sites

Plasmid Cloning Vectors:

Plasmid Cloning Vectors As small as possible with minimum restriction endonuclease cutting sites in genes ori Selectable marker(s) Multiple cloning site (MCS) Reporter function useful

Bacteriophage Vectors:

Bacteriophage Vectors Commonly based upon l phage Most internal genes deleted Insert DNA into middle region (up to10-15 kb) Package Infect host cells with integrated helper phage to provide missing protein-encoding genes

Cosmids:

Cosmids Plasmid with l phage packaging sequence ( cos ) Can clone up to 50 kb Packaged into l particles and injected into host cells Circularizes in cell and continues as a large plasmid

BACs:

BACs Bacterial artificial chromosomes Can clone up to 200+ kb DNA fragments Based upon F plasmid Origin, selectable marker, promoters to expressed cloned genes

YACs:

YACs Yeast artificial chromosomes Have centromere, telomeres and an origin of replication, plus selectable markers Cloned segments of 250 kb

Expression Vectors:

Expression Vectors Also include regulatable high level expression promoter T7 phage promoter lac operator lac repressor gene

Cloning into Plasmids:

Cloning into Plasmids

Expression of Recombinant Genes in Eukaryotes:

Expression of Recombinant Genes in Eukaryotes Expression is sometimes desirable in eukaryotic cells Especially if post-translational modifications are important or study simply requires it

Cloning into Plant Cells:

Cloning into Plant Cells Vectors based upon Ti plasmid Derived from Agrobacterium tumifaciens plasmid

HOW TO CREATE A GENETICALLY MODIFIED PLANT:

HOW TO CREATE A GENETICALLY MODIFIED PLANT 1.Create recombinant bacteria with desired gene. 2. Allow the bacteria to “infect" the plant cells. 3. Desired gene is inserted into plant chromosomes.

Applications of Recombinant DNA Technology:

Applications of Recombinant DNA Technology Large-scale production of human proteins by genetically engineered bacteria. Such as : insulin, Growth hormone, Interferons and Blood clotting factors (VIII & IX)

Production of Human Insulin:

Production of Human Insulin Obtaining the human insulin gene : Human insulin gene can be obtained by making a complementary DNA ( cDNA ) copy of the messenger RNA (mRNA) for human insulin.

JOINING THE HUMAN INSULIN GENE INTO A PLASMID:

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

Introducing the recombinant DNA plasmids into bacteria :

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.

Selecting the bacteria which have taken up the correct piece of DNA :

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

GOLDEN RICE- AGROBIOTECH:

GOLDEN RICE- AGROBIOTECH Golden rice is the result of an effort to develop rice varieties that produce provitamin -A (beta-carotene) as a means of alleviating vitamin A (retinol) deficiencies in the diets of poor and disadvantaged people in developing countries. Because traditional rice varieties do not produce provitamin -A, transgenic technologies were required.

SELECTIVE BREEDING:

SELECTIVE BREEDING Breed only those plants or animals with desirable traits People have been using selective breeding for 1000’s of years with farm crops and domesticated animals.

Vaccines:

Vaccines Bananas have potential to become the world's first edible vaccine due to Agrobacterium . An edible vaccine doesn't need sterile syringes, costly refrigeration, or multiple injections. According to the World Health Organization (WHO), more than 2 million children die worldwide each year from diarrhea that can be prevented easily with vaccines. Thus, researchers lead by Dr. Charles Arntzen are looking into making the food vaccines to prevent diarrhea caused by Escherichia coli and Vibrio cholara bacteria.

WHAT DO YOU THINK ABOUT EATING GENETICALLY MODIFIED FOODS?:

WHAT DO YOU THINK ABOUT EATING GENETICALLY MODIFIED FOODS?

Genetically modified organisms are called transgenic organisms:

Genetically modified organisms are called transgenic organisms TRANSGENIC ANIMALS Mice – used to study human immune system Chickens – more resistant to infections Cows – increase milk supply and leaner meat 4. Goats, sheep and pigs – produce human proteins in their milk

:

Human DNA in a Goat Cell This goat contains a human gene that codes for a blood clotting agent. The blood clotting agent can be harvested in the goat’s milk. . Transgenic Goat

Ha Ha Ha!:

Ha Ha Ha !

Genetic Engineering and Crime Scenes……:

Genetic Engineering and Crime Scenes……

Slide 40:

THANK YOU Email :[email protected]

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