by John Cannon
Isolation
The first thing to do when recombining DNA is to isolate a plasmid from the cells or bacteria that are being engineered. Plasmid DNA may be removed from bacteria or yeast cells, while genomic DNA is isolated from eurkaryotic or prokaryotic cells. By isolating the DNA from the cell membrane of the bacteria or other cell, the DNA can be genetically engineered. The cell wall is usually broken down by using a detergent, such as SDS (sodium dodecyl sulfate), or by using lysozyme for bacteria. Isopropyl or ethyl alcohol is then used to precipitate the DNA out of solution. Plasmid DNA can be pelleted out using a centrifuge, while genomic DNA may be spooled out using a glass rod.
Restriction
By restricting, or cutting, the DNA at predetermined locations in the plasmid, the piece of DNA containing the new gene can be added to form recombinant, genetically-engineered DNA. The plasmid is cut using restriction enzymes (also called endonucleases) that cleave the DNA at certain base pairs. The restriction site of each enzyme is known – this tells the scientist which enzyme to use at the base pair that he wants to cut in a sequence. If the correct enzymes are used, they will cut each strand of the DNA at different points so that the double helix has staggered ends or ‘sticky ends.’ The sticky ends allow the complementary sequences of the new DNA to anneal to the plasmid in the intended section. Click here for Action of the Restriction Enzyme
Ligation
The process of ligation is nothing more than inserting the DNA piece with the new gene into the plasmid or genomic DNA. This result is accomplished by mixing the fragments and the plasmids together and allowing the mixture to incubate. Many of the pieces of DNA with the new gene should anneal to the plasmid at the complementary sticky ends. Because the annealing process is random, many of the pieces will not anneal to the plasmid.
Transformation
This process involves transforming the ligated DNA into living cells that can reproduce the DNA into mass numbers. E. coli bacteria cells have proven to be an ideal medium to generate large quantities of recombinant DNA. The transformation of the bacteria is often accomplished by treating the bacteria with a CaCl solution and ‘heat shocking’ the cells into receiving the foreign DNA. By suddenly exposing the bacteria to heat, the cell membranes are disrupted just enough to allow the plasmid DNA to pass through the membrane. Click here for illustration of Plasmid Insertion
Electrophoresis
Electrophoresis is a method frequently used during the genetic engineering process to confirm that each of the steps worked appropriately. Electrophoresis utilizes an agarose gel and applies a current through it. The DNA samples, along with a dye, are placed into wells in the gel. As the current flows through the gel, the DNA migrates further on the gel relative to the weight of the DNA (heavier molecules will not travel as far as the smaller DNA molecules). After running the current, the size of the molecules can be compared to a standard that is also run on the gel. By knowing the size of the molecules after performing each step, the scientist can determine if the step was successful.
Electrophoresis can also be used after the transformation process to purify the recombinant DNA. This purification can be achieved by cutting out the bands in the gel that contain the desired recombinant DNA. This DNA and gel is then purified by using a series of procedures to separate the DNA from the gel. Once the DNA has been purified, RNA polymerase can be added to eventually produce the protein.
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