Recombinant DNA Technology PDF

RECOMBINANT DNA TECHNOLOGY presented by Dr. Adebayo Ismail Abiola (PhD USM Penang)  Recombinant DNA technology is the technology that involves the joining or combination of DNA molecules from different species, which are then inserted in to a viable host to produce new...

RECOMBINANT DNA TECHNOLOGY presented by Dr. Adebayo Ismail Abiola (PhD USM Penang)  Recombinant DNA technology is the technology that involves the joining or combination of DNA molecules from different species, which are then inserted in to a viable host to produce new genetic combinations that are of great importance to medicine, science, agriculture, and industry. Genetic engineering is a lab-based research process that involves the application of technologies to alter or modify the DNA make up of an organism. The alteration or modification may involve change in base pair (C – G or A – T), addition of a new segment of DNA or deletion of a segment or region of DNA.  Paul Berg is known as the ‘father of genetic engineering’ because he was the first person, who successfully inserted the DNA of an organism into the genetic make up of another organism. In 1972, Paul Berg successfully inserted the DNA of a bacterium into the DNA of a virus. The resulting DNA from such insertion is called ‘recombinant DNA or hybrid DNA’. The discovery or research finding of Paul Berg was advanced by Herbert Boyer and Stanley Cohen in 1973. The two researchers constructed organisms that could combine/modify and replicate genetic information from different species.  The two basic principles of Recombinant DNA technology are: 1. The gene of interest (DNA) is inserted in to the DNA (genetic make up) of a vector (plasmid, bacteriophages among others). 2. The recombinant vector gained entrance into suitable host, where in it replicates itself and produce multiple copies of gene of interest. Essential tools and substances needed for Recombinant DNA production (recombinant DNA technology/genetic engineering) DNA fragment (gene of interest) Restriction enzymes Ligase Cloning vector Suitable host Restriction Enzyme (RE) Restriction endonucleases are naturally produced by bacteria. The natural function of endonucleases is to destroy the DNA of bacteriophages in bacterial cells. Endonucleases can not digest host DNA with methylated cytosine (C). Endonucleases are enzymes that do internal cleavage in DNA molecule. A group of endonucleases that cleaves DNA only within or close to the sites which have specific base sequences are called restriction enzymes or restriction endonucleases and sites recognized by them are called recognition sites or recognition sequences. There are 3 types of RE: Type I, type II and type III REs.  ligase The DNA ligase is the enzyme that can link together DNA strands that have double strand breaks (a break in both complementary strands of DNA) DNA ligase is regularly used during DNA replication and DNA repair. The process of linking the broken strands requires chemical energy in form of ATP DNA ligase is extensively used in recombinant DNA technology and genetic engineering research. Cloning vector A cloning vector (vector) is a DNA molecule that can replicate independently in a suitable host, and it acts as a vehicle that carries the DNA fragment to be cloned. Hence, a vector must have an origin of replication (ori) that can function in the host. Bacterial plasmid, phage (bacteriophage), virus and any other extra- chromosomal small genome can serve as a vector. Elements of a vector Characteristics of a good vector It should be capable of replicating autonomously in the host It should have a relatively small size It should be easy to isolate and purify The transformation of the vector to the host cell should be easy. It should possess a selectable marker to indicate which host cells received the recombinant DNA molecules. It should have convenient and suitable RE sites for inserting the DNA (gene of interest). It should have multiple cloning sites. It should have good ability of gene transfer (integration of the DNA insert into the host genome) It should have good control ability of the expression of the gene of interest (DNA) Classification of vectors based on functions Cloning vector is a vector that is used for propagation of DNA insert in a suitable host. Expression vector is a vector that is used for expression of DNA insert (production of proteins that the gene codes for or the proteins the gene are specified for). examples of vectors are bacterial plasmid pUC18, bacteriophage lambda, cosmid (hybrid of phage and plasmid), bacterial artificial chromosomes (BACS), and yeast artificial chromosomes (YACS). Suitable host Mammalian cells, fish cell lines, plant cells and whole plants, yeasts and bacteria can be used as hosts for production of Recombinant DNA. Mammalian cells 1. They are very relevant for medical application 2. They are hard to grow 3. They may easily express eukaryotic genes  Plant cells and whole plants 1. Plants are easily grown, and they can be genetically modified with ease. 2. They can easily express eukaryotic genes Yeasts (Saccharomyces cerevisiae) 1. Saccharomyces cerevisiae is usually used because its genomics are well- known, and it can be easily grown and cultivated. 2. It can also express eukaryotic genes easily. 3. It can be easily collected (isolated) and purified Bacteria (E. coli) 1. E. coli is easily cultured and cultivated 2. Its genomics are well known 3. The gene product that is synthesized can be easily isolated and purified from the host. Sequential processes of producing a recombinant DNA Isolation of the DNA (gene of interest) Isolation of viable and adequate cloning vector Cutting of the region (DNA fragment) of interest from the DNA using the restriction enzyme Ligation of the isolated DNA fragments with the cloning vector’s DNA using the enzyme ligase Transformation of the recombinant DNA molecule into host cells Multiplication or amplification of the DNA (gene of interest) in the host cells during cell division and replication. Isolation of the DNA (gene of interest) The DNA is isolated from the cells Detergent are used to disrupt the lipid membranes of the cells Proteases and phenol are used to denature and destroy proteins RNAs are degraded by RNase The remaining intact macromolecule after the above-mentioned processes is DNA. Isolation of viable and adequate cloning vector (Bacterial plasmid as a case study) Alkaline lysis or boiling method can be used to remove and separate bacterial chromosomal DNA from plasmid DNA The plasmid DNA will serve as the cloning vector for the production of the recombinant DNA Purification of the isolated DNA and the vector’s DNA The DNAs isolated (DNA fragment to be cloned and the vector’s DNA) need to be purified. The purification is achieved using the following process; 1. Fractionation of the sample on a CsCl2 gradient 2. Precipitation of the fractionated samples with ethanol 3. The precipitated DNA sample is poured over a resin column that specifically binds DNA Cutting of the region (DNA fragment) of interest from the DNA using the restriction enzyme DNAs are cut into large fragments by mechanical shearing Restriction enzyme (RE) recognizes specific nucleotide sequences in the DNA and breaks the DNA strand at those points. Restriction enzymes are endonucleases, and they are the scissors of molecular genetics. Most endonucleases cut the DNA strand at specific palindromic sites in the DNA (a palindromic DNA fragment has same nucleotide sequences on both antiparallel DNA strands). The cutting of the DNA strand by the RE can be staggered, which generate ‘sticky or overhanging ends. Alternatively, the cutting can be blunt, which generate flush ends. Ligation of the isolated DNA fragments with the cloning vector’s DNA using the enzyme ligase The cut donor and vector DNAs must be joined together The DNAs are mixed in a tube If both DNAs were cut with the same RE, the ends will match up because they are sticky. DNA ligase seals the gaps between the DNA fragments and holds the DNA molecules together. DNA ligase forms the phosphodiester bond between two DNA ends. Transformation of the recombinant DNA molecule into host cells The host cells are treated with CaCl2 Then, the DNAs are added Cells are heated shocked at 42 0C DNA enters the cells. Multiplication or amplification of the DNA (gene of interest) in the host cells during cell division and replication Once the DNA is in the host cells, it will be multiplied or replicated as the cells divides. When the cell divides, the replicated recombinant molecules go to both daughter cells The daughter cells will also divide later Thus, the DNA is amplified. Application of Recombinant DNA Technology