Firma:              Perkin Elmer

Modell:            ABI 310

Kommentar:   Dokumente engl.

About the ABI PRISM 310 Genetic Analyzer

The ABI PRISM 310 Genetic Analyzer (Figure 1-1) is an automated instrument System capable of determining base sequence or size and quantity of DNA fragments. The System consists of an electrophoresis instrument, a Macintosh® Computer, including software for data collection and analysis, and a printer (optional). Accessories for Standard runs, such as capillaries, polymer, sample tubes, and buffer vials are included.

The ABI PRISM 310 data collection software has a similar look and feel to the ABI PRISM 377 software from an operational point of view. Two software modules are available for analyzing the raw data produced by the instrument. With the Sequencing Analysis software you can analyze DNA sequence data. With the GeneScanTM Analysis software you can analyze DNA fragments for size and quantification. One or both of these programs is shipped with the instrument, depending an your order. If you ordered only one of the programs, you can order the second program later.

Figure 1-2 shows the instrument with labeled parts. Please note that some 310 models have the Tray button located where the LED Status indicators are shown below.

Sequence Analysis on the ABI PRISM 310 Genetic Analyzer

For sequence analysis on the ABI PRISM 310 Genetic Analyzer, you perform sequencing reactions with fluorescent labels instead of the usual radioactive labels. Four different dyes identify the A, C, G, and T extension reactions. You can incorporate the dye labels using either 5'-dye labeled primers or 3'-dye labeled dideoxynucleotide terminators. Use commonly employed polymerases, such as AmpliTaq® and T7 DNA polymerase, for primer extension (both are provided in kits with the dye terminator and the dye primer chemistries from Perkin Eimer). The labeling method you use depends on the sequencing strategy and on your personal preference. See the ABI PRrsMTM DNA Sequencing Chemistry Guide for more information.

GeneScan Analysis on the ABI PRISM 310 Genetic Analyzer

For GeneScanTM analysis on the ABI PRISM 310 Genetic Analyzer, which provides DNA size and quantitation data, you can perform labeling reactions using one fluorescent dye for each sample or one dye for severai sample. A standard experiment is three samples and one internal size standard in a single capillary. Label each of the samples in a set with a different color, and you can run a total of nine samples in one capillary. Running a size standard in the Same capillary (with a fourth dye color) promotes accurate results.

How the ABI PRISM 310 Genetic Analyzer Works

After your DNA fragments are labeled, the ABI PRISM 310 Genetic Analyzer automatically loads and detects them, providing data for analysis by the Sequencing Analysis or GeneScan Analysis software programs.

Electrophoresis and Detection

The ABI PRIsM 310 Genetic Analyzer is a laser-induced fluorescence capillary electrophoresis system. You combine and load DNA samples labeled with up to four different fluorescent dyes onto the autosampler. You can load up to 96 samples in the autosampler. The ABI PRISM 310 automatically introduces the samples into a polymer-filled capillary for electrophoresis.

The dye-labeled DNA fragments electrophorese through the polymer, and DNA fragments separate according to size. As the labeled samples travel through the capillary and into the window, they are illuminated. The fluorescent dyes attached to the fragments are excited by the laser and emit light at a specific wavelength for each dye. The light is collected and separated by a spectrograph according to wavelength. It is collected onto a charge-coupled device (CCD) camera, so all four types offluorescent emissions can be detected simultaneously. The data collection software collects the light intensities using software selectable filters (spectral or wavelength) and stores them as electrical Signals for eventual processing.

Observing Real Time Status and Data

The hard disk of the Macintosh computer stores the digitized output Signal. As data is collected for one sample, another sample can be analyzed. The computer screen displays the newest information received from the ABI PRISM 310 as it is generated.

You can view instrument status and unprocessed data in several windows on the computer screen by simply choosing the window name from a menu.

•    The Status window displays current instrument conditions.

•    The Electrophoresis History window displays actual values for the electrophoresis power supply and heat plate temperature.

•    The Raw Data window displays the raw data; a different colored line representing each dye color.

Data Analysis

At the end of a run, die computer can automatically analyze the collected data and print electropherograms on a color printer. Correct spectral (color) interpretation of the collected data is dependent on the use of matrix files, which are created for each specific separation and dye chemistry. Depending on your application, this is performed by either die Sequencing Analysis or the GeneScan Analysis software. Both programs are briefly described in Software forData Analysis on page 1-11, and more completely described in the separate analysis software user manuals. You can display die raw and analyzed data on the computer screen, or edit and print it at any time.