file name dnaseq1.html
Sydney University Macromolecular Analysis Centre,
University of Sydney, NSW 2006,
ph. +612 351-3288 Fax: +612 351-4726
"Always remember this, my son; there are but three types of people in this world: those who can count, and those who can't."
I am considering adding DNA sequencing to our protein sequence facility. It has been at least 6 years since I was directly involved with DNA sequencing so I would be interesting in the answers/observations related to the following topics:
1. What are the problems with running a service facility for DNA sequencing?
2. What sort of suggestions would you have on requirements for submission of samples?
3. How well do the automated sequencers work? Are they reliable?
4. What are the strengths and weaknesses of the various DNA sequencers?
5. Any information you think is relevant about this topic.
Parke-Davis Pharmaceutical Research
Phone: 313-996-3512 FAX: 313-998-2716
We are planing to add the DNA sequencing facility at our core facility. I guess there are three main automated DNA sequencers in the market; Applied Biosys. 373. Li-Cor, 4000 and Pharmacia's ALF. All of these3 use fluorescent dyes. Does anyone has any experience regarding the Li-Cor and ALF DNA sequencers? Are they comparable to ABI's 373? Any suggestion will be useful.
1) Problems: the biggest one is not unique to DNA sequencing but I'll bring it up anyway: The DNA template. If template purification is done by the customer you have to have a form of negative feedback to keep the templates consistent. ( Our negative feedback is we make them pay for blank runs). Because we use ABI 373 automated sequencers it is crucial to also quantitate the template. The laser has only one shot to excite the dyes. 2) Requirements for sample submission: Each run consumes 1.2ug template and 20 ng primer. We like to get from 3-10 ug template from first time users per primer and 20ng/ul primers.
3) Do automated sequencers work. Yes.
4) Strengths of the various sequencers. In a service facility environment the ABI 373 has one advantage that makes it the obvious choice. Dye terminators. One sample, one rxn tube, one lane. That means 36 lanes each night on 14 hour gel runs. With the "stretch" upgrade we are getting 400 to 720 bases off double strand template using dye terminator cycle sequencing. The competition will blow smoke about their sequencers getting 2 runs a day. If you compare number of samples completed, manpower involved and length of runs the dye terminators win.
5) Relevant info: Buy the ABI 373, HP 1200C/ps color printer, 230mb MO "optical" drive from APS for archiving, an additional Mac for processing (fastest available) and the Sequencher program from Gene Codes to build contigs etc. Also get an ethernetted Internet connection to talk to the Blast servers and Nentrez down at NCBI.
What not to buy: Any printer that ABI recommends. The Hew/Pack. 1200C/ps is cheaper to buy, cheaper to run and faster. Also don't buy any software from ABI. There are much better alternatives on the market.
-Hope this helps, Paul
Paul Morrison Dana1030
Molecular Biology Core Facility
Dana Farber Cancer Institute
44 Binney Street
Boston, MA 02115
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW, UK
Tel: +44 (223) 333639 (including answerphone)
FAX: +44 (223) 333345