It involves bringing together DNA molecules from two different species which are inserted into a host organism to produce new genetic combinations useful for science, medicine, agriculture and industry. . Since the gene is at the center of all genetics, the fundamental goal of laboratory geneticists is to isolate, characterize and manipulate genes. Although it is relatively easy to isolate a DNA sample from a set of cells, finding a specific gene in that DNA sample can be compared to finding a needle in a haystack . Consider the fact that each human cell contains about 2 meters (6 feet) of DNA. Therefore, a small sample of tissue will contain several kilometers of DNA. However, recombinant DNA technology has made it possible to isolate a gene or any other segment of DNA, allowing researchers to determine its nucleotide sequence, study its transcripts, mutate it in a very specific way and reinsert the modified sequence in a living organism. no to plagiarism. Get a tailor-made essay on “Why Violent Video Games Should Not Be Banned”? Get the original essay Definition: A series of procedures used to assemble (recombine) segments of DNA. A recombinant DNA molecule is constructed from segments of two or more different DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate there, either alone or after being integrated into a chromosome. Requirements to produce rDNA: I. Gene of interest that must be cloned.II. Molecular scissors to cut out the gene of interest.III. Molecular support or vector, on which the gene of interest could be placed.IV. The gene of interest together with the vector are then introduced into an expression system, following which a specific product is produced. How to obtain a gene?I. Isolate it from chromosome ;II. Synthesize it chemically; andIII. Make it from mRNA. Genes can be isolated from chromosomes by cutting the chromosomes at adjacent sites on the gene using special enzymes called restriction endonucleases. (2) rDNA Basics: So, what is rDNA? Before we get to the “r” part of DNA, we need to understand DNA. All DNA is made up of ribose sugar, nitrogen bases and phosphate. There are four nitrogen bases, adenine (A), thymine (T), guanine (G) and cytosine (C). These nitrogenous bases are found in pairs, with A&T and G&C being paired. These bases can be arranged in an infinite manner which gives rise to the formation of the famous “double helix” structure as shown in the figure: The sugar used in DNA is deoxyribose (oxygen removed from the 2nd carbon of the sugar). These four bases are the same in all organisms, but the variety of their arrangement and sequence in DNA leads to diversity. DNA does not actually make the organism but makes proteins. The DNA is then transcribed into mRNA and then it is translated into the protein that forms the organism. By changing the DNA sequence, the protein formed will also change. This results in either a different protein or an inactive protein. We now know what DNA is. Recombinant DNA combines two different strands of DNA. So, the name is recombinant! which is sometimes also called “chimera”. By combining different strands of DNA, a scientist can create a different combination of DNA. How is recombinant DNA made? There are three different ways to produce recombinant DNA: 1. Transformation;2. Introduction to phages; and3. Non-bacterial transformation. Obtaining the DNA: Step 1: The DNA fragment containing the sequence of the gene to be cloned (alsocalled "insert") is isolated.Step 2: Cut the DNAStep 3: Join the DNAStep 2: Insert these DNA fragments into a host cell using a "vector" (carrying DNA molecule )Step 3: The rDNA molecules are generated when the vector self-replicates in the host cell. Step 4: Transfer of the rDNA molecules into a suitable host cell. Step 5: Selection of the host cells carrying the molecule of rDNA using a markerStep 6: Replication of cells carrying rDNA molecules to obtain a population or clone of genetically identical cells. The first step in making recombinant DNA is to isolate the donor and vector DNA. The procedure used to obtain vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified from bacterial genomic DNA. Ultracentrifugation: A protocol for extracting plasmid DNA can be carried out by ultracentrifugation. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by poking a hole in the plastic centrifuge tube. Alkaline Lysis: Another protocol is based on the observation that at a specific alkaline pH, bacterial genomic DNA denatures, but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but the plasmids remain in solution. Phages such as? can also be used as vectors to clone DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from phage lysate. Cutting DNA The restriction enzyme EcoRI cuts a circular DNA molecule carrying a target sequence, resulting in a linear molecule with single-stranded sticky ends. Joining DNA: Insertion A vector is any DNA molecule capable of multiplying inside the host into which our gene of interest is integrated for cloning. In this process, restriction enzymes work like scissors to cut DNA molecules. The ligase enzyme is the linker enzyme that connects the vector DNA to the gene of interest. this will produce the recombinant DNA.Definition:It is a process of inserting foreign DNA into bacteria, which could be used to reliably introduce DNA into bacteria.Methods1. Calcium chloride transformation: In calcium chloride transformation, cells are prepared by cooling the cells in the presence of Ca2+ (in a CaCl2 solution), which makes the cell permeable to plasmid DNA. The cells are incubated on ice with the DNA and then briefly heat shocked (e.g. at 42°C for 30 to 120 seconds). This method works very well for circular plasmid DNA. Non-commercial preparations should normally yield 106 to 107 transformants per microgram of plasmid; a poor preparation will be ~104/µg or less, but a good preparation of competent cells can yield up to ~108 colonies per microgram of plasmid. There are, however, protocols for making super competent cells that can produce transformation efficiencies greater than 109. The chemical method, however, generally does not work well for linear DNA, such as chromosomal DNA fragments, probably because exonuclease enzymes native to the cell. rapidly degrade linear DNA. In contrast, naturally competent cells are generally transformed more efficiently with linear DNA than with plasmid DNA.(4)2. Electroporation: Electroporation, or electropermeabilization, is a microbiology technique in which an electric field is applied to cells toto increase the permeability of the cell membrane, thus allowing the introduction of chemicals, drugs or DNA into the cell.[1] In microbiology, the process of electroporation is often used to transform bacteria, yeast or plant protoplasts by introducing new encoding DNA. If the bacteria and plasmids are mixed, the plasmids can be transferred into the bacteria after electroporation, although, depending on what is transferred, cell-penetrating peptides or CellSqueeze can also be used. Electroporation works by passing thousands of volts over a distance of one to two millimeters of cells suspended in an electroporation cuvette (1.0 to 1.5 kV, 250 to 750 V/cm). Next, the cells must be carefully handled until they have had a chance to divide, producing new cells containing replicated plasmids. This process is approximately ten times more efficient than chemical transformation. Selectable markers may relate to antibiotic resistance, color changes, or any other characteristics to distinguish transformed hosts from untransformed hosts. Different vectors have different properties that make them suitable for different applications. Some properties may include symmetrical cloning sites, size, and high copy number. Antibiotic Selection This is a technique in which transformed bacterial cells are plated on agar plates with different antibiotics, to identify recombinant bacteria and untransformed cells. Procedure The transformed bacteria are spread on agar plates containing an antibiotic-ampicillin or any other. Untransformed bacteria cannot grow in the presence of ampicillin because they lack ampicillin plasmids containing an ampicillin resistance gene (ampR). Disadvantage Antibiotic selection alone does not distinguish bacteria transformed with a non-recombinant plasmid that has recircularized from recombinant plasmids. technique used to distinguish recombinant bacteria from non-recombinant bacteria (containing a plasmid without foreign DNA). Procedure In this case, the agar plates also contain a chromogenic (color-producing) substrate for B-gal called X-gal (5-bromo-4-chloro-3-indolyl-BD-galactopyranoside). X-gal is similar to lactose in structure and turns blue when cleaved by B-gal. As a result, nonrecombinant bacteria—those that contain a plasmid that has ligated to itself without inserting DNA—contain a functional LacZ gene, produce B-gal, and turn blue. Conversely, recombinant bacteria are identified in the form of white colonies. Since these cells contain the plasmid with foreign DNA inserted into the lacZ gene, B-gal is not produced and these cells cannot metabolize X-gal. Therefore, through blue-white selection, untransformed and non-recombinant bacteria are selected and white colonies are identified or selected as desired colonies containing recombinant plasmids. Non-bacterial transformation This process is very similar to transformation. The only difference is that the non-bacterial system does not use bacteria such as E.Coli for the host. In microinjection, DNA is directly injected into the nucleus of the cell undergoing transformation. In biolistics, host cells are bombarded with high-velocity microprojectiles, such as DNA-coated gold or tungsten particles. Phage introduction Phage introduction is the process of transfection, which is equivalent to transformation, except that a phage is used tothe place of bacteria. In vitro packaging of a vector is used. This uses lambda or MI3 phages to produce phage plaques containing recombinants. Created recombinants can be identified by differences between recombinants and non-recombinants using various selection methods. APPLICATIONS OF RECOMBINANT DNA TECHNOLOGYThe three important applications are (1) applications in crop improvement; (2) applications in medicines; and (3) Industrial applications.I. Applications in Crop Improvement: Genetic engineering has several potential applications in crop improvement, such as those given below: 1. Remote hybridization: With advances in genetic engineering, it is now possible to transfer genes between distant species. The barriers to gene transfer between species, and even between genera, have been overcome. Desirable genes can even be transferred from lower to higher organisms through recombinant DNA technology.2. Development of transgenic plants: Genetically transformed plants that contain foreign genes are called transgenic plants. Resistance to diseases, insects and pests, herbicides, drought; tolerance to metal toxicity; induction of male sterility for plant breeding purposes; and quality improvement can be achieved through this recombinant DNA technology. BT cotton, resistant to bollworms, is a striking example.3. Development of root nodules in cereal crops: Legumes have root nodules that contain the nitrogen-fixing bacteria Rhizobium. This bacterium converts free atmospheric nitrogen to nitrates in root nodules. The bacterial genes responsible for thisNitrogen fixation can now be transferred to grain crops like wheat, rice, corn, barley, etc. thanks to genetic engineering techniques, thus making these crops also capable of fixing atmospheric nitrogen.4. C4 Plant Development: Yield improvement can be achieved by improving the photosynthetic efficiency of crop plants. The rate of photosynthesis can be increased by converting C3 plants into C4 plants, which can be achieved either by protoplasm fusion or by recombinant DNA technology. C4 plants have a higher potential rate of biomass production than C3 plants. Most C4 plants (sorghum, sugarcane, corn, certain grasses) are grown in tropical and subtropical zones. Applications in medicine: Biotechnology, especially genetic engineering, plays an important role in the production of antibiotics, hormones, vaccines and interferon in the field of medicines. .1. Antibiotic production: Penicillium and Streptomyces fungi are used for the mass production of the famous antibiotics penicillin and streptomycin. Genetically effective strains of these fungi have been developed to significantly increase the yield of these antibiotics.2. Insulin hormone production: Insulin, a hormone used by diabetics, is usually extracted from the pancreases of cows and pigs. This insulin has a slightly different structure than human insulin. As a result, this leads to allergic reactions in approximately 5% of patients. The human gene for insulin production has been incorporated into bacterial DNA and these genetically modified bacteria are used for large-scale insulin production. This insulin does not cause allergies.3. Vaccine production: Vaccines are now produced by transferring genes encoding antigens to pathogenic bacteria. Such antibodies.