Quarter 3 Recombinant DNA Technology PDF

Quarter 3 Recombinant DNA Technology (Background Information) The Basics of Recombinant DNA Recombinant DNA is the general name for taking a piece of one DNA, and combining it with another strand of DNA. The name Recombinant DNA is also sometimes referred to as "chimera." (By co...

Quarter 3 Recombinant DNA Technology (Background Information) The Basics of Recombinant DNA Recombinant DNA is the general name for taking a piece of one DNA, and combining it with another strand of DNA. The name Recombinant DNA is also sometimes referred to as "chimera." (By combining two or more different strands of DNA, scientists can create a new strand of DNA). The most common recombinant process involves combining the DNA of two different  How is Recombinant DNA made? There are three different methods by which Recombinant DNA is made: 1. Transformation, 2. Phage Introduction, and 3. Non-Bacterial Transformation 1.Transformation 1.1 Select a piece of DNA to be inserted into a vector. 1.2 Cut that piece of DNA with a restriction enzyme. 1.3 Ligate the DNA insert into the vector The inserted DNA contains a selectable marker that allows for the identification of recombinant molecules. An antibiotic marker is often used so a host cell without a vector dies when exposed to a certain antibiotic, and the host with the vector will live because it is resistant. The vector is inserted into a host cell, which is a process called transformation. One example of a possible host cell is E. 2. Non-Bacterial Transformation 2.1 In microinjection, the DNA is injected directly into the nucleus of the cell being transformed. 2.2 In Biolistic, the host cells are bombarded with high-velocity micro- projectiles, such as particles of gold or tungsten that have been coated with  YEAR SCIENTISTS CONTRIBUTIONS Werner Arber Restriction Enzyme 1968 (Swiss microbiologist) 1969 Hamilton O. Type II Restriction Smith Enzymes 1970– Daniel Advance the technique of DNA recombination 71 Nathans demonstrated that type (Molecular II enzymes could be Biologist) useful in genetic studies  YEAR SCIENTISTS CONTRIBUTIONS Paul Berg Developed methods for (American splitting DNA molecules at selected sites and Biochemist) attaching segments of the molecule to the DNA of a virus or plasmid 1973 Stanley N. First to insert Cohen and recombined genes into bacterial cells Herbert W. Boyer (American Biochemist) Historical Background: Restriction Enzyme in 1968 (Swiss microbiologist Werner Arber) Type II restriction enzymes -1969 ( Hamilton O. Smith- purified the so-called Type II – which are needed in Genetic engineering) (Type II enzymes comprise three domains: one for cleavage, one for methylation, and another for Historical Background: DNA methylation is the process of adding a methyl group to one of the bases of your DNA. It can affect gene expression and health in many ways. Advance the technique of DNA recombination (Molecular Biologist Daniel Nathans - in 1970–71 and demonstrated that type II enzymes could be useful in genetic studies)  American Biochemist Paul Berg developed methods for splitting DNA molecules at selected sites and attaching segments of the molecule to the DNA of a virus or plasmid American Biochemist Stanley N. Cohen and Herbert W. Boyer in 1973) First to insert recombined genes into bacterial cells, which then reproduced.  Quarter 3 Steps in Recombinant DNA Technology Steps in Recombinant DNA Technology 1. Isolation of Genetic Material The first step in rDNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules. Since DNA exists within the cell membrane along with other macromolecules such as RNA, polysaccharides, proteins, and lipids, it must be separated and purified which involves enzymes such as lysozymes, cellulose, chitins, ribonuclease, proteases etc. Other macromolecules are removable with other enzymes or treatments. Ultimately, the addition of ethanol causes the DNA to precipitate out as fine 2. Restriction Enzyme Digestion Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions are called ‘restriction enzyme digestions’. They involve the incubation of the purified DNA with the selected restriction enzyme, at conditions optimal for that specific enzyme. The technique ‘Agarose Gel Electrophoresis’ This technique involves running out the DNA on an agarose gel. On the application of current, the negatively charged DNA travels to the positive electrode and is separated out based on size. This allows separating and cutting out the digested DNA fragments. 3. Amplification Using PCR Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA sequence using the enzyme – DNA polymerase in vitro. It helps to amplify a single copy or a few copies of DNA into thousands to millions of copies. PCR reactions are run on ‘thermal cyclers’ using the following components: a. Template – DNA to be amplified b. Primers – small, chemically synthesized oligonucleotides that are complementary to a region of the DNA. c. Enzyme – DNA polymerase d. Nucleotides – needed to extend the primers by the enzyme. 4. Ligation of DNA Molecule The purified DNA and the vector of interest are cut with the same restriction enzyme. This gives us the cut fragment of DNA and the cut vector, that is now open. The process of joining these two pieces together using the enzyme “DNA ligase” is “ligation”. The result­ing DNA molecule is a hybrid of two DNA molecules – the interest molecule and the vector. In the ter­minology of genetics this intermixing of dif­ferent DNA strands is called recombination. Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is 5. Insertion of Recombinant DNA into Host In this step, the recombinant DNA is introduced into a recipient host cell mostly, a bacterial cell. This process is ‘Transformation’. Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make them ‘competent’ to accept new DNA. The processes used may be thermal shock, Ca++ ion treatment, electroporation etc. 6. Isolation of Recombinant Cells The transformation process generates a mixed population of transformed and non-trans- formed host cells. The selection process involves filtering the transformed host cells only. For isolation of recombinant cell from non- recombinant cell, marker gene of plasmid vector is employed. For examples, (PBR322) plasmid vector contains different marker gene (Ampicillin resistant gene and Tetracycline resistant gene. When (pst1 RE) is used it