The ABI 3700, which is a 96-capillary array system, offers even higher potential throughput and automation than the ABI 377. Sample injection of 96 samples is performed in parallel. In a 24-hour period of unattended operation, more than 750 samples can be injected. Thus, approximately 190 000 sample injections can be theoretically processed per year. The ABI 3730 is widely used in human genome centers for DNA sequencing and offers even higher throughput compared to the ABI 3700 even through both systems utilize arrays with 96-capillaries.
The ABI 3700 is used in several U.S. forensic laboratories including the Florida Department of Law Enforcement (Tallahassee, FL) and the New York State Police (Albany, NY) for performing STR typing of convicted offender samples going into DNA databanks.
HITACHI FMBIO II AND FMBIO III FLUORESCENCE IMAGING SYSTEMS
An alternative solution to capillary systems for processing STR samples is to run them on a polyacrylamide gel and then perform a post-electrophoresis fluorescent scan of the gel. The Promega Corporation's PowerPlex® 1.1, 2.1, and 16 BIO STR kits have been made compatible with the Hitachi FMBIO II and FMBIO III Fluorescence Imaging Systems. The STR amplicons are labeled with two different dyes, fluorescein and tetramethylrhodamine (usually referred to as TMR), in the case of PowerPlex® 1.1 and 2.1 or three different dyes for the PowerPlex® 16 BIO kit (fluorescein, JOE, and Rhodamine Red-X). An additional dye, carboxy-X-rhodamine or CXR, is attached to an internal lane standard for DNA fragment sizing purposes. The PowerPlex® 16 BIO kit uses an ILS600 size standard labeled with the dye Texas Red-X.
Unfortunately, in the fall of 2003 MiriaBio/Hitachi Genetic Systems decided to stop making the FMBIO instruments. Forensic laboratories that have an FMBIO II or FMBIO III instrument will be supported for several years but many of these laboratories are understandably looking to other instrument platforms
Figure 14.6 Schematic of FMBIO II Fluorescence Imaging System. Following electrophoretic separation of STR alleles, a gel is placed in the FMBIO and scanned to reveal the presence of fluorescently labeled PCR products. The FMBIO utilizes a solid-state, green laser (1) to excite fluorophores at 532 nm using a polygon scanning mirror (2). The resulting fluorescent light signals emitted from the excited fluorophores attached to DNA fragments are then collected by two optical fiber arrays (3). Specific fluorescent dye signals are isolated using separate interference filters
(4) and are converted to electrical signals with two photomultiplier tubes
(5). Figure used with permission from Hitachi Genetic Systems web page.
for STR typing such as those described earlier in this chapter or technologies under development that are described in Chapter 17. Recognizing that this portion of the book may soon become obsolete for many working forensic laboratories that are currently using the FMBIO systems, we hope that this section will still provide a valuable historical perspective in the post-FMBIO world of the future.
The FMBIO II Fluorescence Imaging System from Hitachi Genetic Systems (Alameda, CA) consists of a scanning unit (Figure 14.6) that is controlled by a Macintosh or Windows computer and three software programs. The hardware for the system features a 20 mW solid-state Nd:YAG (neodinium yttrium aluminum garnet) laser that emits light at an excitation wavelength of 532 nm. The instrument can scan an area of 20 cm X 43 cm and has a reported linear dynamic range of four orders of magnitude. Newer instrument platforms include the FMBIO IIe, III, and III+ with multiple lasers to expand the possible excitation wavelengths to 635 nm and 488 nm and a larger scan area that is capable of examining two gels at once. Two photomultiplier tubes are used for simultaneous detection of emitted light from two or more different fluorescent dyes. Band pass interference filters are used to achieve multicolor imaging.
Up to four filters can be stored in the instrument at any one time and accessed through the data collection software. The FMBIO II takes approximately 15-20 minutes to scan two dyes at once. In a typical scan of PowerPlex samples, the gel image is produced after electrophoresis using a 505 nm bandpass filter to detect amplification products containing a fluorescein label and a 585 nm band-pass filter to detect amplicons labeled with TMR. A 650 nm band-pass filter is used to observe the CXR-labeled internal size standard DNA fragments. These images can then be overlaid into a three-color image or viewed separately by color for closer inspection of the data.
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