DNA isolation and analysis

  • Introduction

  • DNA isolation
  • Restriction enzyme digestion
  • Size fractionation by electrophoresis
  • PCR analysis

    Fatty acids provide the fuel needed for sustained muscle activity. Thus, muscles that are engaged in endurance activity (such as the heart or flight muscles in migratory birds or insects) depend on sufficent amounts of free fatty acids. Fatty acids are delivered to the tissue as glycerides, which in turn are transported by lipoproteins. Following their hydrolysis of the muscle cell membrane (sarcolemma), free fatty acids enter the muscle cell. Alternatively, free fatty acids may be transported directly to the muscle; in this case, the fatty acid is also protein-bound (serum albumin). After entering the muscle cell, fatty acids need to reach the mitochondria, where they are metabolized by beta-oxidation, i.e., the process that leads to ATP production.

    Since fatty acid are relatively hydrophobic, they are only poorly soluble in aqueous media, and thus an intracelluar transport mechanism is required as well. Within the muscle cell, fatty acids are transported by a small fatty acid binding protein (FABP). This protein is needed in higher concentrations if more fatty acids are utilized. While skeletal muscles which utilize only limited amounts of fatty acids possess relatively small quantities of FABP, tissues that use fatty acid preferentially have much more of this protein. In mammalian heart, approx. 5 % of the soluble cytosolic proteins are FABP, and in locust flight muscle, which can sustain extreme levels of muscle activity, up to 20 % of all cytosolic proteins may be FABP (Haunerland, 1994).

    Under physiological conditions the carboxy group is dissociated, and therefore a fatty acid molecule has a negative charge and thus a polar head group, as well as a hydrophobic tail. Thus, it can act like a detergent and disrupt membranes and other cellular components. The binding of fatty acids to FABP prevents the accumulation of unbound free fatty acids, and therefore FABP not only assures effient transport to the mitochondria, but also prevents cell damage caused by an excess of free fatty acids. In fact, it has been shown that increased fatty acid utilization in a muscle stimulates the expression of the FABP gene, probably because more FABP is required to bind all the fatty acids.

    If one wants to understand the regulation of protein expression, knowledge of the structure of its gene is necessary. In this experiment, we will identify the gene that codes for this fatty acid binding protein. We will isolate genomic DNA and produce restriction fragments, which are separated by agarose electrophoresis. Using a cDNA probe for FABP, one could detect the size of the restriction fragment containing parts of the gene in a Southern Blot. Alternatively, one could elute the restriction fragments from the gel and detect the presence of the gene by PCR. Such fragments would be useful for producing a partial genomic library (subgenomic library), from which the gene could be cloned.

    The FABP genes from rat and locust muscle have been cloned in our laboratory (Zhang et al., 1999; Wu et al., 2000). Although FABP itself is a very small protein (133 amino acid residues, which means that the cDNA is approx. 400 nucleotides long) its gene is much bigger, because it contains several introns. The overall organization of the three genes is shown below.

    While the intron size varies widely, the location of the introns within the coding sequence is conserved, except that in the locust the second intron is missing. Therefore, we can use PCR primers that recognize sequences within the coding region to determine the size of the introns.

    In this experiment, we will prepare genomic DNA from rat heart. We will also isolate the insert from a genomic and a cDNA clone of rat heart FABP. In week 2, we will check our DNA samples with PCR for the presence of the FABP gene.


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