OVERVIEW OF DNA MANIPULATION
1993 Woodrow Wilson Biology Institute
Working with DNA in the high school biology laboratory can be made relatively simple, and the labs needn't be costly. The flow chart shows the steps for isolation of DNA, restriction (cutting), ligation (linking), transformation (changing), and selection (choosing) of transformants. This series of steps actually allows one to transfer a gene from one organism to another and then witness its expression. I will explain each of these steps in more detail in the paragraphs that follow.
The Flow Chart
The removal of plasmid or genomic DNA from cells is termed isolation. Plasmid DNA may be removed only from certain bacteria or yeast cells, whereas genomic DNA may be isolated from many cells, both prokaryotic and eukaryotic.
Isolation usually involves the breaking of the cell's membrane (and possibly nuclear membrane) and possibly also a cell wall (plant cells). Mixing the cells with detergents like SDS (sodium dodecyl sulfate) or a common liquid detergent (such as Palmolive Green or Woolite) will break down the cell walls (if present) and cell membranes. Lysozyme may be added to bacterial cells as an aid for lysis. Mixing proteases such as papain or meat tenderizer and chloroform (plant cells) with this homogenate will help to remove protein molecules (such as histones) from the DNA. The addition of a salt solution will help to keep the DNA and proteins separated.
Finally, in order to collect the DNA, it first must be precipitated out of solution using isopropyl and ethyl alcohols. At this point, plasmid DNA may be pelleted by centrifugation and genomic DNA may be "spooled" by attaching it to a clean glass rod that is slowly twirled in the ethanol. In terms of cost and materials, isolation of genomic DNA is definitely a possibility for most high school biology teachers. It may be isolated from calf thymus, onion, spinach, or wheat germ. Time and expense may be saved by purchasing pre-isolated plasmids which contain the gene for antibiotic resistance (usually ampicillin).
Restriction (cutting) of DNA at predetermined locations along the molecule is accomplished using endonucleases, naturally occurring protective enzymes that have been isolated from the nuclei of certain cells. The idea here is to cut the plasmid at a certain location so that a piece of DNA containing a particular gene might be inserted.
Endonucleases (also known as restriction enzymes) predictably cleave DNA at certain base sequences that they recognize along the chain, and around 70 different restriction enzymes and their recognition sequences are known. When deciding which endonuclease to use, it is necessary to know which recognition sequences the plasmid and genomic DNA have in common. You would also want to avoid cutting the plasmid at the gene sequence that confers antibiotic resistance. Thus, an enzyme used to cut a specific plasmid or genomic DNA would be chosen because a particular base sequence is known to exist in both of these DNA's. DNA's cut with the same enzymes will have "sticky ends" with complementary sequences and thus be able to anneal (stick together). (See Obbink, Jane; Teaching Biotechnology by Analogies and Models).
In the laboratory, tiny amounts of the chosen endonuclease are added to separate microtubes; one tube contains the plasmids and endonuclease, and the other tube contains the isolated genomic DNA and the same endonuclease. These mixtures are incubated at a certain temperature for restriction to occur and then deactivated, usually by heating the mixture enough to denature the enzyme.
Ligation involves the linking together of a fragment of genomic DNA and a plasmid that has been cut open (linearized) at a particular location by the chosen endonuclease. This can be accomplished by mixing the linearized plasmids and the genomic fragments together in the same tube, and then allowing them to "incubate" at room temperature for 2-3 hours. Through random associations, the sticky ends of the cut DNA molecules will anneal, and with the addition of DNA ligase, the weak bonding of the base pairs will be supplemented by bonds between the phosphate groups. Of course, not all ligations will result in fragments of genomic DNA being inserted into plasmids with antibiotic resistance (this is the desired result); some plasmids will simply recircularize (these would also have antibiotic resistance), and some fragments may recombine to form longer fragments.
The transformation step involves getting the ligated DNA into living cells that can then reproduce large numbers of cells with the new genetic information in them. Introducing this DNA into E. coli cells usually involves treating the bacteria with calcium chloride solution and then "heat shocking" them by subjecting them to 42degC for 90 seconds. This step disrupts the cell membrane enough for the DNA to pass through it and into the cell. Plasmids with antibiotic resistance should be introduced into cells that don't have this gene; this will allow us to recognize transformed cells later. Introducing the ligated DNA into yeast cells involves suspending the yeast cells in an LTE solution, adding the ligated DNA, adding PEG and heat pulsing them (this will get the DNA past the yeast cell membrane), and then allowing the cells to incubate. In any event, in order to determine what cells (if any) have picked up the ligated DNA, the next step, called selection, must be performed.
Selection involves taking the cells that you hope have been transformed and exposing them to a growth medium which will allow only the transformed cells to grow. For example, an easy way to select transformed bacteria cells is to add the genomic DNA fragment to a plasmid which contains a gene that codes for antibiotic resistance (such as ampicillin). If a sample of these cells is then plated onto agar containing ampicillin, only those cells that have the ampicillin resistance gene may grow into a colony. Cells that did not pick up the plasmid do not have ampicillin resistance, and will not survive on agar with ampicillin. This indicates whether or not the plasmid entered the cell. In order to check if the ligation was successful, an additional selection should be performed. If the plasmid also has a gene that codes for a blue substance, the colonies with these plasmids will appear blue. Insertion of the genomic fragment into this gene's sequence will cause cells to grow into white colonies when plated. An additional control should be set up by plating a sample of cells on agar without ampicillin.
Electrophoresis is a procedure that may be used as an analytical tool after the restriction, ligation, or selection steps. Mixtures of DNA fragments may be separated by applying the mixture to a gel of agarose or polyacrylamide and then running a direct current through the gel. The fragments of DNA will migrate in an electric field relative to their molecular weight; lighter (and shorter) fragments will migrate farther on the gel than heavier (and longer) fragments. After restriction, the process would be useful to check to see that fragments of the desired size are in the mixture. After ligation, it would be used to confirm that fragments of genomic DNA and plasmids did indeed join. And after selection, if some DNA is isolated from the transformants, electrophoresis can be used to provide further evidence that the fragment was incorporated into the plasmid.
7. BLOT TRANSFER
A blot transfer (called a Southern blot) may be used after electrophoresis if a particular DNA sequence is to be studied. The double stranded DNA in the gel is denatured with sodium hydroxide and then transfered to a nitrocellulose filter using capillary action. This filter is then bathed in a solution with probes (fragments of DNA with a specific base sequence) that are labeled with radioactivity or some other means for visualization. After hybridization, the filter is then exposed to an X-ray film; DNA sequences that complement the probes will produce a line on the film at a position that may be compared to their position on the gel.