BASIC SCIENCE: Techniques and Technologies.
(Updated: April 15, 2004)
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There are a vast number of techniques used in laboratories around the world, especially for those in the biological sciences. Which ever method is used really depends on the types of questions a researcher is trying to answer. Obviously, I can't mention and describe all techniques that are used, but the ones below are common. For more detailed protocols, visit my research.
Gel Electrophoresis is a method used to separate or purify samples of DNA, RNA, or protein molecules. A gel is made by dissolving agarose in some buffer solution, which is then allowed to set in a gel tray. The gel tray has combs attached to create wells in the gel (i.e. the wells are holes in the gel where the DNA, RNA or protein sample is added). For DNA, the samples are prepared and added to the well, and then an electric current is run through the gel apparatus. The DNA fragments are separated by charge (e.g. large fragments move more slowly than small fragments) and the relative sizes of the fragments are determined by comparing to a standard DNA ladder (which is run alongside the fragments in a well of the same gel). The SDS-PAGE (SDS Polyacrylamide Gel Electrophoresis) is used to separate proteins by size rather than by charge. Gel electrophoresis is used anytime DNA, RNA or protein needs to be visualised or purified. The Polymerase Chain Reaction (PCR): The Polymerase Chain Reaction (PCR) is a method used to amplify a specific sequence of DNA. The reaction mixture is prepared in which a DNA template (e.g. plasmid or chromosomal DNA containing the specific sequence of interest) is added to a mixture of nucleotides (i.e. the four bases: Adenine, Guanine, Cytosine, and Uracil), buffer solutions, Magnesium, primers, and DNA-dependent DNA polymerase (i.e. a heat stable polymerase, such as Taq polymerase). DNA polymerase is an enzyme that simply copies your DNA segment (i.e. it polymerizes nucleotides by reading off of the template DNA). The reaction mixture is put under several cycles of heating and cooling, which is electronically controlled, in a thermocycler. At a particular temperature , the primers anneal to the template DNA and are used as starting points for the Polymerase enzyme to carry out amplification. The temperature is increased to denature (or separate) the template DNA strand from the newly synthesized strand, and then the reaction is cooled again to start DNA replication again. This technique is very sensitive -- DNA segments are amplified exponentially in a matter of hours -- and only very small amounts of template DNA is required. PCR has only been around since 1983 (invented by Kary Mullis who won the Nobel Prize in 1993), and yet it has many applications across the biological sciences. Southern Blotting: Southern blotting is technique used to identify DNA sequences. Gel electrophoresis is used to separate DNA fragments. A blotting "aparatus" is set up by placing a nitrocellulose or nylon membrane (or filter) on top of the gel, and then a stack of blotting paper is placed on top of the filter. Buffer solution is added to reservoirs of the apparatus, and the system is left overnight (or the weekend). The DNA fragments are transferred to the filter in the same relative positions that they were in the gel. After the transfer, the radioactive labels (i.e. radioactively labelled DNA or RNA sequence that is complementary to some part on the sample DNA) are added to the filter in a hybridization step. The labels become attached to the complementary regions in the sample DNA that is embedded in the filter. In this way, qualitative analysis of DNA fragments can be carried out. Simmilarly, Northern blotting is used for RNA, and Western blotting is done for proteins. Simple Cloning:
There are a variety of methods of cloning, but I will describe a simple example. After having isolated a DNA sequence and ligated it into a plasmid vector (a plasmid is an extra-chromosomal circular DNA molecule that carries non-essential genes in many species of Bacteria; the plasmid is also referred to as a 'vector'; thus, a vector is a plasmid, engineered or naturally derived, that is used to carry a DNA segment in a bacterial cell). The plasmid that contains the DNA segment of interest can be transformed into a bacterium, such as Escherichia coli, which can then carry the plasmid and replicate it during growth. This is cloning -- the cells that took up your plasmid DNA, will have replicated it and passed it on to their daughter cells. Now all that needs to be done is to screen for cells that carry the plasmid DNA. Screening can be done by selecting for strains that grow with the presence of a particular antibiotic (e.g. plamids usually carry some antibiotic resistance gene that enable the cell to survive and grow on media containing the antibiotic), and checked using PCR.
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