Recombinant DNA Technology Presented by Mona AL Boreikan
What is recombinant DNA?● Recombinant DNA is the formation of a novel DNA sequence by the formation of two DNA strands.● These are taken from two different organisms.● These recombinant DNA molecules can be made with recombinant DNA technology.
What is recombinant DNA technology? The procedure is to cut the DNA of the donor organism into pieces with restriction enzymes, and insert one of these fragments into the DNA of the host.
Recombinant DNA technology = Genetic engineering = Gene cloning
The History of Recombinant DNA Technology• 1970 Hamilton Smith, at Johns Hopkins Medical School, isolates the first restriction enzyme, an enzyme that cuts DNA at a very specific nucleotide sequence. Over the next few years, several more restriction enzymes will be isolated.• 1972 Stanley Cohen and Herbert Boyer combine their efforts to create recombinant DNA. This technology will be the beginning of the biotechnology industry.
The History of Recombinant DNA Technology• 1976 Herbert Boyer cofounds Genentech, the first firm founded in the United States to apply recombinant DNA technology.• 1978 Somatostatin, which regulates human growth hormones, is the first human protein made using recombinant technology.
What Required for Recombinant DNA TechnologyFirst ; Cloning Vectors A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted.
What are the kinds of Cloning Vectors1- Plasmid Cloning Vectors2- Bacteriophage Vectors3- Cosmids Vectors4- (BACs)5- YACs
1- Plasmid Cloning Vectors• A plasmid is a DNA molecule that is separate from the chromosomal DNA.• It can replicate independently of the chromosomal DNA.• It is double-stranded and, in many cases, circular.• Plasmid sizes vary from 1 to over 1,000 kilobase pairs (kbp), but it can only contain Illustration of a bacterium with plasmid inserts of about 1–10 kbp. enclosed showing chromosomal DNA and plasmids
1- Plasmid Cloning Vectors• Origin of replication (ORI).• Plasmid is used to multiply (make many copies of) or express particular genes.• Plasmid containing genes that make cells resistant to particular antibiotics, Selectable marker(s)• Plasmid containing a multiple cloning site (MCS, or polylinker).
1- Plasmid Cloning Vectors How are the plasmids inserted into bacteria?There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance; whereas episomes, the lower example, integrate into the host chromosome
1- Plasmid Cloning VectorsCloning DNA into a Plasmid to Produce Recombinant DNA
2- Bacteriophage Vectors● Bacteriophage is any one of a number of viruses that infect bacteria.● Commonly based upon l phage. Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The structure of a typical tailed bacteriophage
2- Bacteriophage Vectors● Most internal genes deleted.● Insert DNA into middle region (up to10-15 kb)● The phage genes expressed in lysogenic cycle code for proteins that repress expression of other phage genes.
2- Bacteriophage VectorsHow the DNA phage inserted into bacteria? The lysogenic and Lytic cycle.
2- Bacteriophage VectorsCloning DNA into Bacteriophage to Produce Recombinant DNA
3- Cosmids Vectors• Cosmid type of hybrid plasmid.• Plasmid with λ phage packaging sequence (cos)• Cosmids are able to contain 37 to 52 kb of DNA.• Packaged into λ particles and injected into host cells.• Circularizes in cell and continues as a large plasmid
3- Cosmids VectorsCloning DNA by cosmid to Produce Recombinant DNA
3- (BACs) Vectors• Bacterial artificial chromosome.• Can clone up to 200 kb DNA fragments.• Based upon F plasmid.• A similar cloning vector, called a PAC has also been produced from the bacterial P1-plasmid.• Origin, selectable marker, promoters to expressed cloned genes
3- (BACs) Vectors How the BAC insertedinto bacteria to ProduceRecombinant DNA?
4 - (YACs) Vectors• Yeast artificial chromosomes• Have centromere, telome res and an origin of replication, plus selectable markers• Cloned segments of 250 kb
4 - (YACs) Vectors How the YAC insertedinto yeast to ProduceRecombinant DNA?
Summary of vectors and what they can carry.The size of DNA that vector Vector can carry 0 – 10 kb Standard plasmid 0 – 23 Kb Lambda Bacteriophage 30 – 44 Kb Cosmid 70 – 100 Kb Bacteriophage P1 130 – 150 Kb P1 Artificial chromosome PAC Maximum 300 Kb Bacterial Artificial Chromosome BAC 0.2 – 2 Mb Yeast Artificial Chromosome YAC kb (= kbp) = kilo base pairs = 1,000 bp Mb = mega base pairs = 1,000,000 bp
5 - Expression Vectors• Also known as an expression construct• It include regulatable high level expression promoter – T7 phage promoter – lac operator – lac repressor gene
The three most important signals for Expression.
What Required for Recombinant DNA Technology Second; Enzymes A- Restriction enzyme• OR restriction endonuclease) is an enzyme that cuts double- stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites.
Summary of some kinds of Restriction enzymes and their cut sites.
Second; Enzymes B - A DNA ligase enzyme• Catalyse the joining or recombining of DNA fragments (ligation). DNA ligase Ligation by DNA ligase enzyme
What the steps for recombinant DNA technology?• Protocol; – Isolate target DNA – Cut with RE – Ligate to vector – Transform to host cells – Plate on antibiotic- containing medium – Identify recombinant plasmids – Identify/characterize specific clones
What the steps for recombinant DNA technology?
Examining the results of a restriction digest ( after recombinant )After treatment with a restriction endonuclease, the resulting DNA fragments can be examined by agarose gel electrophoresis to determinetheir sizes and to make An agarose gel plate. The hybridized DNA shore if recombinant strands show a pink light because of the DNA happened or not. binding of the fluorescent labeled probe.
What is Gel Electrophoresis• A technique used to separate DNA fragments by size• The gel (agarose or polyacrylamide) is subjected to an electrical field• The DNA, which is negatively-charged, migrates towards the positive pole.• The larger the DNA fragment, the slower it will move through the gel matrix.• DNA is visualized using fluorescent dyes.
Reproductive Cloning * Reproductive cloning is performed with the express intent of creating another organism.* This organism is the exactduplicate of one thatalready exists or has existedin the past.* Cloning ofplants, animals, andhumans falls into the classof reproductive cloning
How is Reproductive Cloning Performed?* It is performed using a technique called Somatic Cell Nuclear Transfer (SCNT).* The genetic material from a donor egg is removed, so that you are left with an empty egg.* Then, a cell is taken from the organism to be cloned and its nucleus is removed.* This nucleus is then transferred into the empty donor egg.
What Is Reproductive Cloning Used For?Reproductive cloning has only been used for research purposes.Reproductive cloning could be used effectively for repopulating endangered species or to help make breeding of specific animals easier.Reproductive cloning uses could also include the production of organisms with specific characteristics, such as drug- producing animals or genetically "unique" animals.
Dolly was created in a process called “Somatic Cell Nuclear Transfer”Professor Ian Wilmut is now a Sir for his work creating the world’s first cloned mammal in 1996 - 2003
Therapeutic Cloning• It is performed, not to produce another organism, but to harvest embryonic stem cells for use in medical treatments.• Embryonic stem cells are those cells found inside of developing embryos.• They can be used to produce a number of different cells including tissue, muscle, and organ cells.
How is Therapeutic Cloning Performed?* A cell is removed from the patient requiring medical treatment.* The nucleus of this cell is removed and inserted into an empty donor egg.* Division is encouraged through the use of special chemicals or an electric current.* The resulting embryonic stem cells are then removed from this embryo and used to treat the patient.
What is Therapeutic Cloning Used For?• Therapeutic cloning is intended for medical use.• The embryonic stem cells that this type of cloning produces can be used to create skin for burn victims, organs for transplant patients, or cells for those with spinal cord injuries.
What is Therapeutic Cloning Used For?• And because the cells come from the patient herself, there are no issues of cell rejection.• Therapeutic cloning may also help those suffering from heart disease, Alzheimers Disease, or Parkinsons Disease.
Can organs be cloned for use in transplants? Scientists think that therapeutic cloning can be used to make tissues and organs for transplants. To do this, DNA would be taken from the person who needs the transplant and put in a egg. After the egg with the patients DNA starts to divide, embryonic stem cells that can be transformed into any type of tissue would be harvested.. Stem cells would generate an organ or tissue that is a genetic match to the recipient, as a result organ donation would reduce significantly.
Risks of Cloning More than 90% of cloning attempts fail to produce viable offspring Cloned animals tend to have higher rates of infection tumor growth, and other disorders. Clones have been known to die mysteriously.
Because of the lack of understanding about reproductive cloning scientists believe it is unethical to attempt to clone humans. About 30% of clones born alive are affected with “large offspring syndrome”
• Several cloned animals have died prematurely from infections or other complications, the same problems would be expected in human cloning• The attempt to clone humans at this time is considered potentially dangerous, and ethically irresponsible.