RDT

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BIOTECHNOLOGY

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Presentation on :RECOMBINANT DNA TECHNOLOGY : 

Presentation on :RECOMBINANT DNA TECHNOLOGY By: Arti Kesarwani M.Pharm(P’ceutics)

What is RDT? : 

What is RDT? Recombinant DNA (rDNA) technology can be defined as ‘changing the genes of an organism by using in vitro processes’. The fundamental knowledge that made RDT possible is based on the understanding of structure and function of DNA. It was Stanley Cohen and Herbett boyer who developed the first RDT in 1973. In the same year the rDNA molecules were produced by Paul Berg, H.Boyer,Annie Chang and S.Cohen. In 1980,the first rDNA product i.e. Humulin was produced by Genetech Co.(USA).

STRATEGIES OF RDT : 

STRATEGIES OF RDT There is no single method of RDT ,but it involves several steps- Isolation of DNA (also called insert DNA, target DNA, foreign DNA) of known function from organism. Enzymatic cleavage and joining of insert DNA to another DNA molecule (cloning vector) to form a rDNA (i.e. vector +insert DNA). Tranformation of host cell i.e. transfer and maintainence of this rDNA molecule into a host cell. Identification of transformed cells (i.e. cell carrying rDNA) and their selection from non-transformants.

Slide 4: 

Amplification of rDNA to get its multiple copies in a cell. Cell multiplication to get a clone i.e. a population of genetically identical cells.

BASIC TOOLS OF RDT : 

BASIC TOOLS OF RDT The techniques developed in the early 1970s for isolation,cutting and joining of DNA molecules are used for analysis and engineering of DNA and RNA molecules . The basic tools that are required to generate recombinant host cells are described below- Isolation and purification of DNA The host cell that contains genes of desired function are disrupted mechanically so that intracellular components including nucleic acid may be released. DNA is purified and recovered using techniques such as centrifugation,electrophoresis,precipitation.

2. Restriction enzymes : 

2. Restriction enzymes These are also called as ‘molecular scissors’. These enzymes are present in bacteria and provide a type of defence mechanism called the restriction- modification system’. Types of Restriction endonuclease: 3 main types of restriction endonuclease. These are designated as- Type 1- this enzyme cut DNA at random sites that can be than 1000 base pairs from the recognition sequence. These moves along the DNA in a reaction and require Mg,S-adenosyl methionine,ATP as co-factor.

Slide 9: 

Type 2- This type of restriction enzyme was first isolated by Hamilton Smith. These are simple and require no ATP for degradation of DNA. They cut DNA within the recognition sites. Type 3- This group of endonucleasecut the DNA about 25 base pairs from the recognition sites. It moves along the DNA and require ATP as a source of energy.

Some common Type 2 restriction enzymes- : 

Some common Type 2 restriction enzymes-

(b) Cleavage of recognition sites : 

(b) Cleavage of recognition sites The recognition sites are then cut into 4-8 base pairs long. (c) Construction of rDNA molecules A rDNA molecule can be generated by cutting two different DNA fragments with the same restriction enzymes and mixing the fragment together. The two different DNA fragments join together due to the presence of sticky ends(identicle single stranded nucleotides) on both different fragments.

Recombinant DNA Technique : 

Recombinant DNA Technique Restriction enzymes used to cut out insulin gene and to cut a bacterial (E. coli) plasmid at the same “sticky ends”

Cloning vectors or vehicle DNA : 

Cloning vectors or vehicle DNA Vectors which is used for delivery of desired foreign DNA into a host cell. It act as a vehicle or a carrier. The vectors must posses the following features- It should be easily isolated from the organisms. It should be small in size because larger vector DNA may often get broken during purification. It must have an origin of replication (ori) so that it may multiply within a host cell alongwith the foreign DNA. It must contain atleast a unique restriction site for restriction enzyme. It must have a selectable markers i.e. gene which help to select the host cell .

Cloning Vectors : 

Cloning Vectors A vector is used to amplify a single molecule of DNA into many copes. A DNA fragment must be inserted into a cloning vector. A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a bacterial cell. Most vectors are genetically engineered plasmids or phages. There are also cosmid vectors, bacterial artificial chromosomes, and yeast artificial chromosomes.

Plasmid Cloning Vectors : 

Plasmid Cloning Vectors Plasmids are circular, double-stranded DNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells. They can replicate independently of the host cell. The size of plasmids ranges from a few kb to near 100 kb Can hold up to 10 kb fragments Plasmids have an origin of replication, antibiotic resistance genes as markers, and several unique restriction sites. After culture growth, the clone fragment can be recovered easily. The cells are lysed and the DNA is isolated and purified. A DNA fragment can be kept indefinitely if mixed with glycerol in a –70 degrees C freezer. pBR322 is the basis of most engineered plasmids

pUC19 another plasmid cloning vector : 

pUC19 another plasmid cloning vector Characteristics: Interruption of b-lactamase gene gives rise to white colonies (cloned DNA) vs blue ones (empty)

Plasmid Polylinkers and Marker Genes for Blue-White screening : 

Plasmid Polylinkers and Marker Genes for Blue-White screening A vector usually contains a sequence (polylinker) which can recognize several restriction enzymes so that the vector can be used for cloning a variety of DNA samples. Colonies with recombinant plasmids are white, and colonies with nonrecombinant plasmids are blue. Example: pUC19 Resistant to ampicillin, has (ampr gene) Contains portion of the lac operon which codes for beta-galactosidase. X-gal is a substrate of beta-galactosidase and turns blue in the presence of functional beta-galactosidase is added to the medium. Insertion of foreign DNA into the polylinker disrupts the lac operon, beta-galactosidase becomes non-functional and the colonies fail to turn blue, but appear white.

Phage Cloning Vectors : 

Phage Cloning Vectors Fragments up to 23 kb can be may be accommodated by a phage vector Lambda is most common phage 60% of the genome is needed for lytic pathway. Segments of the Lambda DNA is removed and a stuffer fragment is put in. The stuffer fragment keeps the vector at a correct size and carries marker genes that are removed when foreign DNA is inserted into the vector. Example: Charon 4A Lambda When Charon 4A Lambda is intact, beta-galactosidase reacts with X-gal and the colonies turn blue. When the DNA segment replaces the stuffer region, the lac5 gene is missing, which codes for beta-galactosidase, no beta-galactosidase is formed, and the colonies are white.

Cosmid Cloning Vectors : 

Cosmid Cloning Vectors Fragments from 30 to 46 kb can be accommodated by a cosmid vector. Cosmids combine essential elements of a plasmid and Lambda systems. Cosmids are extracted from bacteria and mixed with restriction endonucleases. Cleaved cosmids are mixed with foreign DNA that has been cleaved with the same endonuclease. Recombinant cosmids are packaged into lambda caspids Recombinant cosmid is injected into the bacterial cell where the rcosmid arranges into a circle and replicates as a plasmid. It can be maintained and recovered just as plasmids. Shown above is a 50,000 base-pair long DNA molecule bound with six EcoRI molecules, and imaged using the atomic force microscope. This image clearly indicates the six EcoRI "sites" and allows an accurate restriction enzyme map of the cosmid to be generated.

Bacterial Artificial Chromosomes(BACs) and Yeast Artificial Chromosomes(YACs) : 

Bacterial Artificial Chromosomes(BACs) and Yeast Artificial Chromosomes(YACs) BACs can hold up to 300 kbs. The F factor of E.coli is capable of handling large segments of DNA. Recombinant BACs are introduced into E.coli by electroportation ( a brief high-voltage current). Once in the cell, the rBAC replicates like an F factor. Example: pBAC108L Has a set of regulatory genes, OriS, and repE which control F-factor replication, and parA and parB which limit the number of copies to one or two. A chloramphenicol resistance gene, and a cloning segment. YACs can hold up to 500 kbs. YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes. YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers. YAC plasmidYeast chromosome DNA is inserted to a unique restriction site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.

BACs: Bacterial Artificial Chromosomes : 

BACs: Bacterial Artificial Chromosomes

Host cells : 

Host cells For the multiplication of foreign DNA efficient host cells are required. Different types of host cells such E.coli, yeast, plant and animal cells are available for gene cloning. E.coli has been most successfully used because- It is easy to handle It doubles its cell no. in each 20 minutes. The rDNA also reproduce along with doubling of bacteria. Within hours 1000s of bacteria cells and the much no.of foreign genes are produced.

Slide 23: 

Yeast also have several advantages such as- Easy to grow and manipulate. Simplest unicellular eukaryote. Well characterized. Requirement of no complex medium. Their multiplication in laboratory in a small vessel or large sized fermenter.

Construction of rDNA molecule : 

Construction of rDNA molecule

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