rick@GENEMAN.WUSTL.EDU (Rick Wilson) (06/20/91)
I wasn't going to say anything but after reading Donald Lehn's comments, I feel I must ... First of all, the "370A" isn't "...the current state..." of DNA sequencers; the 373A, with the Macintosh environment and different software is a much different instrument than the old 370A. If the people who are complaining about the 370's user unfriendliness, etc. (and there are many!) would simply buy a Mac and upgrade their instruments, they would see a world of difference. Secondly, and perhaps more critical to successes with the automatic DNA sequencers, the chemistry has greatly improved. T7 DNA polymerase chemistry and the Taq linear amplification sequencing method have made the 373A and Pharmacia's ALF extremely useful and easy to use. We routinely sequence from c. 400 ng of double-stranded plasmid DNA, prepared using a standard mini-prep procedure. The sequencer usually gives good reads (99%+ accuracy) of 470-500 base pairs, and we typically analyze 48 samples per instrument per day. I've run a lot of gels (and read a lot of films) and I've got to tell you that I've gone fluorescent and I'll never go back. As an example of how easy the 373A is to run these days, I have an undergrad in my lab who didn't know a dideoxynucleotide from a diode two weeks ago. Now, she is running one of our 373s everyday (24 templates per run), and will probably finish sequencing one or two cosmids before the end of the summer program. So much for the need for a "skilled technician". For those who are contemplating purchase of an automated DNA sequencer, you have some choices. The ABI 373A is easy to use, is compatible with T7 (Sequenase) or Taq sequencing methods, allows high-throughput sequencing with good accuracy, bu t is currently limited to dye-primer sequencing. Furthermore, since the 373A uses 4-color chemistry, synthesis and use of custom dye-primers is tricky. The ALF gets around this problem with its one-color dye-primer chemistry, and custom primers may be labeled with fluorescein amidite as part of primer synthesis. The ALF is lower throughput than the ABI (10 samples per run), but accuracy appears to be roughly the same over 500 base pairs. Software on the ALF is still a little rough, but it's a new machine, and I expect the software to improve significantly with the next release (I've seen a beta version). Cost of the two machines is about the same; reagent cost will vary depending on the chemistry you choose. As far as software for editing, assembling, analyzing and managing sequencing data, it is quite simple to send all of the raw data files (not sequence text files) over an network connection to another computer. A second Mac can be used with the ABI software to view, edit and print data; a Sun workstation (a SPARCstation IPC, cost about $7000) can be set up with Rodger Staden's new Xwindows programs for viewing, editing and assembling data from either the 373A or ALF sequencers. Alternatively, a Mac IIci with 8 MB of RAM running Multifinder seems to do just fine (thouhg a little slow) with other tasks while collecting data from the 373A. So, my advice to those considering fluorescent sequencers would be very positive. We have three of 'em, one ALF and two ABI, and good DNA sequence data is going into the computer faster than with any other method I've ever used. If you want some more info, give me a shout. Rick Wilson, St. Louis rick@geneman.wustl.edu Disclaimer: I don't work for ABI, Pharmacia, Sun Microsystems or Rodger Staden; I just sequence a lot of worm DNA.