Sequencing & Genotyping
Automated DNA sequencing is currently being accomplished with two ABI Model 3130XL Genetic Analyzers that provide investigators with four-color fluorescence -based sequencing based on the Sanger method. Single or double stranded genomic, plasmid, or PCR produced DNAs are suitable substrates for the sequencing protocol.
Samples are submitted as template/primer mixes, with the ratio of DNA/primer dependent on the type of template employed. Following the incorporation of the labeled ddNTPs in the extension products, the reactions are purified by gel filtration, dried down, re-suspended in formamide, and run on the analyzer. Depending on the quality of the sample, a run can extend over 700bp. The process usually takes 2 days to complete from the time the MRC receives the sample(s). Present capacity is about 200 samples/15 hrs.
DNA sequencing reactions purified with the BigDye® XTerminator™ Purification Kit result in high signal strength when analyzed on a DNA sequencer. When you prepare sequencing samples for purification with the BigDye XTerminator reagents, you may need to decrease the amount of DNA template in the sequencing reactions to keep the fluorescence signals on scale during analysis. See DNA quantity guidelines for additional information.
Data is e-mailed as either ab.1 files that can be opened with ABI software, or programs such as FinchTv, Sequencher, or Chromas, or text files can be returned to the investigator along with electropherograms from the run. Electropherograms are graphic printouts of the sequencing data and can be picked up at the MRC following the run. You will need to establish a dnaLIMS account, see Setting up / Accessing Your Core LIMS Account, for instructions on what is required to submit your sequencing samples.
GeneScan analysis is done on the
ABI Prism 3130XL Genetic Analyzer. The 3130XL is
an automated capillary electrophoresis system that can separate, detect and
analyze up to 16 samples of fluorescently labeled DNA fragments in one run. It
separates a mixture of DNA fragments according to their lengths, provides a
profile of the separation, determines the length of each fragment (in base
pairs), and estimates the relative concentration of each fragment in the sample.
See 3130XL GS Notes for more detailed information on this process.
For additional information please contact Dr. Tom Cunningham.
Don Thomason, Ph.D.
Molecular Resource Center
360 Cancer Research Building
Memphis, TN 38163
Phone: (901) 448-2656