The Molecular Genetics Core Facility (Corelab) at Morehouse School of Medicine (MSM) has been set up to assist investigators who need to do DNA sequence analysis. DNA templates and primers should be submitted to Ms. Qi Yang, in MEB 217 (Tel. X1866).
Sequencing reactions are performed using Applied Biosystems, Inc. (ABI) protocols for thermocycler-based sequencing with Taq DNA polymerase and BigDye TM fluorescent dideoxy terminators. Reaction products are resolved on the capillary-based ABI 3100 Genetic Analyzer, which automatically scans each capillary to detect labeled DNA. Data from fluorescent DNA peaks are collected automatically, analyzed and formatted at the end of each gel run. Sequencing results are sent by email to investigators. Printed copies can also be furnished if desired. Software for downstream analysis of DNA sequences is available on Corelab computers for use by MSM investigators. The Corelab is ready to consult with MSM investigators regarding DNA sequence analysis software options.
Sequencing reaction requirements are:
Template : 200-400 ng plasmid DNA or 20-100 ng PCR product DNA (this is based on size of plasmid or PCR amplicon; larger templates require more DNA)
Primer : 3-6 pmol oligonucleotide (either a commercial vector-specific primer or your own gene-specific 18-20mer). Some primers may be less efficient, and may require as much as 20-30 pmol/reaction.
Procedural Notes -
- Complete the DNA Sequence Order Form. Submit a completed copy to the Corelab with your primers and templates. This will help us (and you) keep a record of work performed.
- The template and primer must be sufficiently concentrated to accommodate a volume of 6 µ l . (Reactions consist of 6 µ l primer-template, plus 4 µ l reaction mix = 10 µ l total volume).
- Templates and primers should be dissolved in water, NOT in TrisCl-EDTA buffer, since sequencing reactions are inhibited by EDTA.
- The Corelab can supply small amounts of the M13(-21) forward primer and some other vector-specific primers for pilot sequencing projects. Please inquire.
- Make sure your DNA is pure! Estimate template concentration by running agarose gels, as optical density readings can be misleading.
- Corelab personnel will perform sequencing reactions as well as clean-up (removal of unincorporated fluorescent terminators) and loading of the 3100 DNA sequencer.
- Our standard policy is that sequencing reactions will be performed within 1 working day of template-primer submission. This means that when you submit reactions they will be electrophoresed within 24-36 hr and you should receive the data 48 hr after submission. Unless there is an urgent need to do otherwise (deadline for meeting, etc.), sequencing reactions will be performed in the order submitted. First come, first served!
Pricing and Payment
An account is kept of the number of reactions performed for each investigator. Payment can be made by one of two methods: (1) (preferred method) You pre-pay for sequencing by submitting a purchase order with your templates. Funds can deposited in the Molecular Genetics Core for future use. (2) After sequencing, we will send you an invoice, for which you write a requisition payable to the Molecular Genetics Core (please attach a copy of the invoice to your purchase requisition). The cost to Morehouse School of Medicine investigators for sequencing reaction and electrophoresis is $8.00 per primer-template combination. The cost to outside investigators is $12.00 per primer-template combination
You should receive readable sequence data in the range 600-650 nucleotides, depending on the quantity and purity of DNA templates. (We recommend the use of kits from Qiagen for DNA purification.) Data quality may vary due to binding properties of each primer-template pair or because of unusual template DNA sequence. We will work with investigators in order to obtain the best data possible. Sequence data are saved as electropherograms (*.ab1 files) which show reaction products graphically as a series of peaks; and as nucleotide sequence text files (*.seq files). These files are sent to investigators via e-mail. Investigators may also request printed copies of electropherograms (check box on Order Form).
Computer Analysis of DNA Sequence Data
Sequence data files (*.ab1) may be further analyzed using Sequencher and ClustalX in the Corelab; or with DSGene, available in the Corelab or on investigator's PC after installation of client software. First time users will consult with Dr. Roth (MEB 212, Tel. X1947) before using the Corelab computers
The Corelab no longer offers nucleotide synthesis; as it is readily and inexpensively available from commercial vendors. There are many on-line resources for oligonucleotide design. A good one is Primer 3.0 at MIT's human genome site:
First time users will consult with Dr. William Roth (MEB 212, Tel. (404)752-1947) before using the Corelab PC or Macintosh computers.
NOTE: Please advise us of any publications that include data generated by the Molecular Genetics Core Facility. Your publications will be cited in reports or advertisements concerning the Core Lab.
SNP analysis and DNA fragment analysis to be added in Autumn 2004!
The Corelab is upgrading the software for the ABI 3100 sequencer . As a result of this upgrade, we will offer the added features of SNP analysis and DNA fragment analysis. In addition, we will have improved accuracy and flexibility for DNA sequence analysis.