Recombinant DNA A recombinant DNA molecule is produced by joining together two or more DNA segments usually from different organisms. More specifically, a recombinant DNA molecule is a vector (e.g. , phage or virus) into which the desired DNA fragment has been inserted to enable its cloning in an appropriate host. Genetically engineered DNA prepared by splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated. The idea for recombinant DNA was first proposed by Peter Lobban, a graduate student of Prof. Dale Kaiser in the Biochemistry Department at Stanford University Medical School. The first publications describing the successful production and intracellular replication of recombinant DNA appeared in 1972 and 1973. Recombinant DNA molecules are produced with one of the following three objectives: 1) To obtain a large number of copies of specific DNA fragments. 2) To recover large quantities of the protein produced by concerned gene. 3) To integrate the gene in question into the chromosome of a target organism where it express itself. Recombinant DNA technology: A series of procedures that are used to join together (recombine) DNA segments. 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 on its own or after it has been integrated into a chromosome. Vector In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA.

Fig. The pGEX-3x plasmid 1

The four major types of vectors are - i. : Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin of replication. Modern plasmids generally have many more features, notably including a "multiple cloning site" which includes nucleotide overhangs for insertion of an insert, and multiple consensus sites to either side of the insert. Plasmids may be conjugative/transmissible and non- conjugative.  Conjugative: mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population; e.g., F plasmid, many R and some col plasmids.  Nonconjugative: do not mediate DNA through conjugation, e.g., many R and col plasmids. ii. Viral vectors: Viral vectors are generally genetically-engineered viruses carrying modified viral DNA or RNA. Viral vectors frequently are lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Viral vectors are often designed for permanent incorporation of the insert into the host genome, and thus leave distinct genetic markers in the host genome after incorporating the transgene. For example, retroviruses leave a characteristic retroviral integration pattern after insertion that is detectable and indicates that the viral vector has incorporated into the host genome. iii. Cosmid: A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as a cloning vector) that contains a Lambda phage cos sequence. Cosmids' (cos sites + plasmid = cosmid) DNA sequences are originally from the lambda phage. Cosmids can be used to build genomic libraries. Characteristics of cosmid: a) Cosmids are able to contain 37 to 52 kb of DNA, while normal plasmids are able to carry only 1–20 kb. b) They can replicate as plasmids if they have a suitable origin of replication, for example SV40 ori in mammalian cells. c) They frequently also contain a gene for selection such as antibiotic resistance. These cells which did not take up the cosmid would be unable to grow.

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iv. Bacterial artificial chromosome: It is a DNA constructing, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150-350 kbp. A similar cloning vector called a PAC has also been produced from the bacterial P1-plasmid.

Properties of a good vector: i. Origin of replication: The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. ii. Promoter: In , a promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the genes they transcribe, on the same strand and upstream on the DNA. Promoters can be about 100–1000 base pairs long. iii. Cloning site: A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~ 20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during procedures involving molecular cloning or subcloning. Extremely useful in , bioengineering, and , MCSs let a microbiologist insert a piece of DNA or several pieces of DNA into the region of the MCS. This can be used to create transgenic organisms, also known as genetically modified organisms (GMOs). iv. Genetic markers: A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites. v. Antibiotic resistance: Antibiotic resistance is a form of drug resistance whereby some (or, less commonly, all) sub-populations of a microorganism, usually a bacterial species, are able to survive after exposure to one or more antibiotics; pathogens resistant to multiple antibiotics are considered multidrug resistant (MDR) or, more colloquially, superbugs. Microbes, rather than people, develop resistance to antibiotics. 3

vi. Epitope: An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized are also epitopes. vii. Reporter genes: In , a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. viii. Targeting sequence: Protein targeting or protein sorting is the biological mechanism by which proteins are transported to the appropriate destinations in the cell or outside of it. ix. Protein purification tags: Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis. Tags are attached to proteins for various purposes.

Applications of recombinant DNA technology Recombinant DNA is widely used in biotechnology, medicine and research. Many additional practical applications of recombinant DNA are found in industry, food production, human and veterinary medicine, in agriculture, and in bioengineering. Some specific examples are identified below: 1. Recombinant chymosin: Found in rennet, is an enzyme required to manufacture cheese. It was the first genetically engineered food additive to be used commercially. Traditionally, processors obtained chymosin from rennet, a preparation derived from the fourth stomach of milk-fed calves. 2. Recombinant human insulin: Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. Recombinant insulin is synthesized by inserting the human insulin gene into E. coli, which then produces insulin for human use. 3. Recombinant human growth hormone (HGH, somatotropin): Administered to patients whose pituitary glands generate insufficient quantities to support normal growth and development. 4. Recombinant blood clotting factor VIII: A blood-clotting protein that is administered to patients with forms of the bleeding disorder hemophilia, who are unable to produce factor VIII in quantities sufficient to support normal blood coagulation.

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5. Recombinant hepatitis B vaccine: Prevention of hepatitis B infection is controlled through the use of a recombinant hepatitis B vaccine. It contains a form of the hepatitis B virus surface antigen that is produced in yeast cells. 6. Diagnosis of infection with HIV: Each of the three widely-used methods for diagnosing HIV infection has been developed using recombinant DNA. The antibody test (ELISA or western blot) uses a recombinant HIV protein to test for the presence of antibodies that the body has produced in response to an HIV infection. 7. Golden rice: A recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis. This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population. Golden rice is not currently in use, pending the resolution of intellectual property, environmental and nutritional issues. 8. Herbicide-resistant crops: Commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed which incorporate a recombinant gene that results in resistance to the herbicide glyphosate (trade name Roundup), and simplifies weed control by glyphosate application. These crops are in common commercial use in several countries. 9. Insect-resistant crops: Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties. The bacterium has been applied to crops as an insect-control strategy for many years, and this practice has been widely adopted in agriculture and gardening.

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