Draw a diagram of the structure of plasmid pBR322

Draw a diagram of an experiment in genetic engineering (design recDNA) and give a description of the main stages

Molecular cloning is the laboratory process used to create recombinant DNA. It is one of two widely used methods (along withpolymerase chain reaction, abbr. PCR) used to direct the replication of any specific DNA sequence chosen by the experimentalist. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free of living cells.

Formation of recombinant DNA requires a cloning vector, a DNA molecule that will replicate within a living cell. Vectors are generally derived from plasmids or viruses, and represent relatively small segments of DNA that contain necessary genetic signals for replication, as well as additional elements for convenience in inserting foreign DNA, identifying cells that contain recombinant DNA, and, where appropriate, expressing the foreign DNA. The choice of vector for molecular cloning depends on the choice of host organism, the size of the DNA to be cloned, and whether and how the foreign DNA is to be expressed.^[5] The DNA segments can be combined by using a variety of methods, such as restriction enzyme/ligase cloning or Gibson assembly.

In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into the host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening for clones with desired DNA inserts and biological properties.

Construction of recombinant DNA, in which a foreign DNA fragment is inserted into a plasmid vector. In this example, the gene indicated by the white color is inactivated upon insertion of the foreign DNA fragment.

 

 

)Describe the method of microinjection of DNA in the germ cells of animals

Microinjection is an effective method for creating transgenic animals, for RNAi of selected genes, and for introducing various types of molecules directly to cells. For DNA transformation, the easiest approach is to inject DNAs into the distal arm of the gonad. The distal germ line of C. elegans contains a central core of cytoplasm that is shared by many germ cell nuclei.Therefore, DNAs injected here can be delivered to many progeny. This approach usually leads to the formation of large extrachromosomal DNA arrays. Microinjection directly into oocyte nuclei can induce chromosomal integration of transgenes, but this technique is relatively difficult to do. For RNAi experiments, most progeny of injected animals can be affected by simply injecting dsRNA into a single gonad or intestinal cell because of a very efficient RNA transport system. While RNAi by feeding is best for high throughput experiments, RNAi by microinjection is more effective for at least some genes.

Microinjection
Microinjection is a technique of delivering foreign DNA into a living cell (a cell, egg, oocyte, embryos of animals) through a glass micropipette. One end of a glass micropipette is heated until the glass becomes somewhat liquified. It is quickly stretched which forms a very fine tip at the heated end. The tip of the pipette attains to about 0.5 mm diameter which resembles an injection needle. The process of delivering foreign DNA is done under a powerful microscope. Cells to be microinjected are placed in a container (Fig. 4.15). A holding pipette is placed in the field of view of the microscope. The holding pipette holds a target cell at the tip when gently sucked. The tip of the micropipette is injected through the membrane of the cell. Contents of the needle are delivered into the cytoplasm and the empty needle is taken out.

 

	Fig. 4.15. A method of microinjection of DNA preparation in egg.

 

Xenopus oocytes have been widely used for the study of transcription by microinjection because oocytes contain between 6,000 and 100,000 or more RNA polymerase molecules than somatic cells. Microinjection is technically easy because of large size of oocytes. Some of the endogenous pattern of gene regulation during development has been characterized (Wickens and Laskey, 1981). The injected DNA integrates randomly with nuclear DNA and its expression could be possible only when the foreign DNA is attached to a suitable promoter sequence.

73 Basic properties of cryoprotectors and their components A cryoprotectant is a substance that is used to protect biological tissue from freezing damage (i.e. that due to ice formation). Without protection, cells will rupture when they freeze as a result of expanding water, causing severe injury or death to living organisms, and ruining tissue samples or frozen food products. The compound can work in a number of different ways. A common approach is to lower the freezing point, keeping the tissue flexible at temperatures that would normally result in freezing. Others bond to specific molecules to help tissue retain its structure under the intense pressures of cold temperatures. Cryoprotectants must (1) easily penetrate cells, and (2) not be toxic to the cell. Conventional cryoprotectants are glycols (alcohols containing at least two hydroxyl groups), such as ethylene glycol[citation needed], propylene glycol, and glycerol. Ethylene glycol is commonly used as automobile antifreeze, and propylene glycol has been used to reduce ice formation in ice cream. Dimethyl sulfoxide (DMSO) is also regarded as a conventional cryoprotectant. Glycerol and DMSO have been used for decades by cryobiologists to reduce ice formation in sperm and embryos that are cold-preserved in liquid nitrogen. Mixtures of cryoprotectants have less toxicity and are more effective than single-agent cryoprotectants. A mixture of formamide with DMSO (dimethyl sulfoxide), propylene glycol, and a colloid was for many years, the most effective of all artificially created cryoprotectants. Cryoprotectant mixtures have been used for vitrification (i.e. solidification without crystal ice formation). Vitrification has important applications in preserving embryos, biological tissues, and organs for transplant. Vitrification is also used in cryonics in an effort to eliminate freezing damage. Some cryoprotectants function by lowering the glass transition temperature of a solution or of a material. In this way, the cryoprotectant prevents actual freezing, and the solution maintains some flexibility in a glassy phase. Many cryoprotectants also function by forming hydrogen bonds with biological molecules as water molecules are displaced. Hydrogen bonding in aqueous solutions is important for proper protein and DNA function. Thus, as the cryoprotectant replaces the water molecules, the biological material retains its native physiological structure and function, although they are no longer immersed in an aqueous environment. This preservation strategy is most often utilized in anhydrobiosis.