![]() (B) Whereas treatment with BamHI produces fragments with “cohesive” or “sticky” ends. (A) Treating the DNA with SmaI results in fragments with blunt ends. Example of how restriction enzymes cut DNA. Although there are several different types of restriction enzymes, those most useful for rDNA technology recognize specific short sequences in DNA and cleave the DNA at that site to produce cohesive (sticky) or blunt-ended fragments (Figure 2). Restriction endonucleases are a group of enzymes derived (primarily) from bacteria. Digestion of DNA with restriction endonucleases Other compounds, for example, proteins can also be easily separated from DNA by altering the concentration of salt in the extraction buffer. Therefore, the final step in many DNA purifications protocols involves precipitation using alcohol. However, the polar sugar phosphate groups of the DNA are repelled by non-polar solutions. For example, DNA is negatively charged, making it soluble in aqueous solution. To achieve this, scientists take advantage of the chemical and physical properties of different molecules inside the cell. These compounds must be separated from the DNA during isolation. Also, tissues and organs from the same plant, or different plants often contain different composition of metabolites, for example, proteins, lipids, and carbohydrates. The challenge is that plant and animal cells produce numerous other compounds that often act as contaminants and may inhibit cloning or sequencing of the DNA. Isolation of DNA, restriction digestions and its separation on an agarose gel: Preparation of DNAįor recombinant DNA procedures to work, a pure DNA sample must be obtained. The first GMOs were Escherichia coli cells that were transformed with genes from human to produce various proteins for pharmaceutical purposes. The GMOs contain new traits from another organism. Transformation of cells with rDNA produces organisms called bioengineered or genetically modified organisms (GMOs). Table 1.Events related to the development and application of recombinant DNA technology. Image by Walter Suza.Īdvances in molecular biology in the early 1970s, including the success in creating, and transferring DNA molecules into cells, revolutionized both science and industry. The first genetically modified organisms were bacteria that made simple proteins of pharmaceutical interest, for example, insulin. As the technologies improved, other organisms including plants became amenable for improvement by rDNA technology. Table 1 provides important milestones in the development and application of rDNA technology. Recombinant DNA is made from combining DNA from different sources. Recombinant rDNA technology involves procedures for analyzing or combining DNA fragments from one or several organisms (Figure 1) including the introduction of the rDNA molecule into a cell for its replication, or integration into the genome of the target cell. To arrive at this several tools of molecular biology are used including, DNA isolation and analysis, molecular cloning, quantification of gene expression, determination of gene copy number, transformation of the appropriate host for replication or transfer into crop plants and analyses of transgenic plants. The power of rDNA technology comes from our ability to study and modify gene function by manipulating genes and transform them into cells of plant and animals. ![]() Recombinant DNA (rDNA) technology has resulted in breakthroughs in crop and animal biotechnology. Understand polymerase chain termination reaction (PCR).Understand vectors and their application in gene cloning and expression.Understand restriction and ligase enzymes and their application in gene cloning.Learn isolation of DNA and its separation on an agarose gel.Understand the importance of recombinant DNA technology. ![]()
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