DNA Sequencing Automated DNA sequencing is currently the gold standard test for detecting most DNA variations (e.g. point mutations). Many mutations are associated with hereditary diseases can be detected by automated DNA sequencing. We use the ABI PRISM 377 (ABI377) automated DNA sequencer, which has been the most commonly used automated sequencer throughout the human genome project. The ABI377 is an automated instrument designed for analyzing fluorescently-labeled DNA fragments by gel electrophoresis. Gel electrophoresis is the movement of charged molecules through a gel solution (such as polyacrylamide) in an electrical field. It is used to separate DNA fragments by size. Once the samples are loaded, voltage is applied, causing the fragments to electrophorese through the gel and separate according to size. The laser excites the fluorescent dyes attached to the fragments, and they emit light at a specific wavelength for each fluorescent dye. The light emitted is then separated according to wavelength by a spectrograph onto a cooled, charge coupled device (CCD) camera, so all four types of fluorescent emissions can be detected with one pass of the laser. The ABI377 is connected to a computer, and the data is collected by a "Date Collection" software. The collected data before it is analyzed is often referred to as "raw data". The file in which the raw data is stored is referred to as a "gel file". Additionally, complex matrix of information, including the raw data, exists in the gel file, organized in a number of channels. The gel file can be viewed as an image of the data for display and the raw scan data. Gel files range in size from 20 MB to 40MB. At the end of data collection, the analysis software (ABI PRISM DNA Sequencing Analysis) is used to manually or automatically process, analyze, and translate the collected complex data and matrix into easily readable base sequences.  The above electropherogram shows a homozygous mutation GNE-M712T (atg>acg), which is the most common mutation found in people affected with IBM2. It is the founder mutation in people of Iranian-Jewish heritage. The base change from "t" to "c" causes the amino acid number 712 to change from Methionine (encoded by "atg") to Threonine (encoded by "acg"). *Revenue generated from services are used to speed up development of a treatment for IBM2. |
 |