05 - Biotechnology Basics: My First Electrophoresis
negative charge migrate towards the positive electrode (anode) while those with a net positive charge migrate towards the negative electrode (cathode).
4. Molecular shape: Molecules with a more com- pact shape, like a sphere, move through the gel more quickly than those with a looser confor- mation. Because molecules with different properties travel at different speeds, they become separated and form discrete “bands” within the gel (Figure 1). THE POLYMERASE CHAIN REACTION (PCR) In 1984, Dr. Kary Mullis revolutionized the field of molecular biology when he devised a simple and elegant method to copy specific pieces of DNA. Recognizing that an initial step in DNA replication in a cell’s nucleus is the binding of RNA primers, Mullis discovered that he could replicate DNA in vitro using short, synthetic DNA primers and DNA polymerase I. Furthermore, because researchers
Figure 1: Separation of dyes by agarose gel electrophoresis.
can specify a primer’s sequence to target a specific gene, this method allowed for the rapid amplification of a selected DNA sequence. For the development of this tech - nique, known today as the Polymerase Chain Reaction (or PCR), Mullis was awarded the Nobel Prize in Chemistry in 1993. In order to amplify DNA, purified double-stranded DNA is mixed with the short DNA primers, a thermostable DNA polymerase (Taq) and nucleotides. The mixture is heat- ed to 94°C to “denature” (i.e., unzip into single strands) the DNA duplex. Next, the sample is cooled to 45-60°C, allowing the primers to base pair with their target DNA sequences (a step known as “annealing”). Lastly, the temperature is raised again, to 72°C, the optimal temperature at which Taq polymerase will extend the primer to synthesize a new strand of DNA. Each cycle (denaturation, annealing, extension) doubles the amount of target DNA. Today, a specialized machine, called a “thermal cycler” or “PCR machine”, is used to rapidly heat and cool the samples. As a result, a PCR cycle can be completed in less than 5 minutes; 20-40 cycles produce sufficient DNA for analysis. Because of its ease of use and its ability to rapidly amplify DNA, PCR has become indispensable in medical and lifesciences labs, replacing the time-intensive Southern blot as the method of choice. For example, today’s research laboratories can quickly create copies of a specific region of DNA for cloning applications. Medical diagnostics use PCR to identify genetic mutations and infectious agents. In addition, because am- plification by PCR requires very little starting material, it is ideal for forensic analysis of biological samples or determination of paternity.
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