of Molecular Markers
Detecting DNA PolymorphismsBecause any DNA molecule greater than 10 base pairs contains essentially the same mass-to-charge ratio, any procedure that separates the molecules based on mass alone will be useful to uncover DNA polymorphisms. Currently, gel electrophoresis is the most often used procedure to detect these polymorphisms. But as we move into the post genomic era, techniques that rapidly screen large numbers of samples are emerging. The most recently widely applied procedure is capillary array electrophoresis. In the near future, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) may replace many of the current procedures.
Gel ElectrophoresisGel electrophoresis is most widely adapted technique for detecting polymorphism. Samples are loaded into a gel and allowed to migrate in an electric field. Since DNA is negatively charged, the samples are loaded near the negative pole, and they migrate toward the positive pole. Separation of the molecules is strictly based on size: the smallest fragments move farther in the gel because they can navigate through the small pores in the gel better than large molecules.
The two gel matrices used to separate the molecules are agarose and polyacrylamide. The separation capabilites of each molecule is function of the concentration of the polymer in the gel. The table below show the resolution that can be obtained with different polymer concentrations.
You choose the type of polymer to use based on the range of fragment that need to be distinguished. For RFLP and RAPD procedures, agarose is the polymer of choose. Because microsatellites and AFLPs procedures generate smaller fragments for comparison, polyacrylamide is typically used.
Following gel electrophoresis to separate molecules based on mass, the polymorphism is revealed using a detection agent. The dye ethidium bromide is typically used to reveal RAPD polymorphisms in agarose genes. Silver nitrate is procedure used to detect polymorphisms in polyacrylamide gels. This has been used for both microsatellites and AFLPs. With these marker systems, it also an option to include a radio-labelled nucleotide during the PCR step. If this option is chosen, the gel is used to expose autoradiographic film. The film is then analyzed to uncover the polymorphism.
Laser technology has also been applied to the microsatellite marker system. When using this approach, the primer is labelled with a fluorescent dye. Samples are then separated in a polyacrylamide gel. As the samples flow through the bottom of the gel, fragments are detected by a laser that detects the presence of the fluor. Computer programs output data in a form that can be analyzed.
Detecting RFLPs requires the use of a southern hybridization procedure. Briefly, molecules are separated in agarose gel and transferred to a nylon membrane. The membrane will then contain a faithful representation of the fragment distribution found in the gel. A probe to a sequence of interest is then hybridized to the membrane, and unbound probe is removed by a series of stringent washes. The membrane is then used to expose an autoradiographic film. Polymorphisms are determined by studying the film.
Capillary Array ElectrophoresisSmall DNA fragments can be separated rapidly in narrow capillaries. Because heat is lost rapidly from these thin capillaries, molecules can be separated rapidly using high voltage. This greatly speeds the processing of samples. Recently, instruments that contain capillaries arrays have reached the market. These arrays consist of eight (Beckman-Coulter) or 96 (Applied Biosystems) cappillaries. These machines simultaneously introduce samples into each of the arrays. Laser technology is then used to detect the samples as they exit the capillary.
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)MALDI-TOF MS is the newest procedure in use to detect microsatellite polymorphisms. In general, mass spectrometry involves the ionization of a molecule, accelerating the ions in an electric field, and passing the ions through a magnetic field. For DNA genotyping purposes, the larger molecules will travel through the magnetic field faster. MALDI-TOF is a recent improvement of mass spectrometry. The molecule to be analyzed is mixed with a matrix that does not ionize, but aids in the ionization of large molecules (up to 100,000 daltons) such as DNA. Time-of-flight allows for high throughput (and thus high sensitivity) of ions. Samples can be analyzed in a couple of seconds using this procedure.
A basic procedure follows. PCR amplification is performed with microsatellite specific primers. It is best if the primers are located as close to the repeat as possible. This ensures that smaller molecules are amplified. The primer contains an attached biotin molecule. The product is recovered from the PCR reaction with streptavidin magnetic beads. The product is mixed with the matrix an analyzed. Both alleles of heterozygous individuals have been easily scored. In some cases the alleles, differ by a single copy of a tetranucleotide repeat.
The following image shows the separation of alleles at four human loci. Each of the loci is a tetrnucleotide repeat. The mobility difference among the loci is a reflection of the variation in the size of the amplified product.
Copyright © 2000. Phillip McClean