Welcome to UCMP’s Molecular Labs (cont.)

The PCR Set-up Lab
Once DNA is extracted, the samples are off to the PCR Set-up Lab where researchers, including Scott Nichols, Carole Hickman’s graduate student, feverishly set up PCR experiments (Polymerase Chain Reaction). PCR, a technique used in DNA sequencing, duplicates specified regions of DNA, producing numerous copies that can then be manipulated and studied. Scott is attempting to determine the amount of variation present in the sequences of the gene Cytochrome Oxidase I (COI) in two marine sponge groups that are found around the world. From these data he hopes to identify the physical factors acting as barriers to gene flow for sessile marine invertebrates that have short larval stages and limited dispersal.
Scott makes a gel
Grad student Scott Nichols makes a gel in which he’ll place amplified DNA for sequencing. (photo by David Smith)
  Molecular Phylogenetics Laboratory
After PCR set-up, the next stop is the Molecular Phylogenetics Laboratory (MPL), a joint research facility supported by both the UCMP and the University/Jepson Herbaria. The establishment of the MPL has enabled UCMP users to gain an understanding of evolutionary relationships through the analysis of DNA sequences. The MPL is well equipped with three thermocyclers, machines in which PCR (and thus, the duplication of genes) takes place. These machines run 24 hours a day with the capability of generating 288 copies every four hours (that’s 1,728 copies per day!). Because chemical reaction conditions for each PCR varies between organisms and from gene to gene, getting it to work for different organisms can sometimes be tricky. To determine whether a particular set of temperatures and reaction conditions have produced the desired PCR result, our researchers carry out an electrophoretic analysis of the PCR products.
Gel electrophoresis is the process of placing the DNA in a gelatin-like slab and applying an electrical current to the gel. Because DNA has a negative electrical charge it is attracted by and moves towards the positive side of the gel. How far it moves will be determined by its size. The DNA is stained with a special dye to make it visible and then compared against a DNA standard. The Laboratory is equipped with many gel boxes to carry out multiple runs of DNA electrophoresis and a video documentation system to permanently record the results. While a typical PCR amplifies (copies) about 1000-2000 bases (or “letters”) of the DNA code, Kirsten Lindstrom, a graduate student in Roy Caldwell’s laboratory, has been performing long PCRs. This has enabled her to isolate the entire mitochondrion of three stomatopod crustaceans (that’s 15,000 bases of DNA per mitochondria!).

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