A molecular fraction collecting tool for the ABI 310 automated sequencer

Ming-Tseh Lin, Roy G. Rich, Royce F. Shipley, Michael J. Hafez, Li Hui Tseng, Kathleen M. Murphy, Christopher Gocke, James Eshleman

Research output: Contribution to journalArticle

Abstract

Several methods exist to retrieve and purify DNA fragments after agarose or polyacrylamide gel electrophoresis for subsequent analyses. However, molecules present in low concentration and molecules similar in size to their neighbors are difficult to purify. Capillary electrophoresis has become popular in molecular diagnostic laboratories because of its automation, excellent resolution, and high sensitivity. In the current study, the ABI Prism 310 Genetic Analyzer was reconfigured into a fraction collector by adapting the standard gel block to accommodate a collection tube at the distal end of capillary. The time to collect the desired peaks was estimated by extrapolating from standard capillary electrophoresis using the original gel block. Fraction collection from a mixture of DNA fragments amplified from wild type and several internal tandem duplication mutations of the FMS-like tyrosine kinase 3 (Flt3) gene yielded highly purified DNA fragments containing internal tandem duplication mutations and predictable electrokinetics using the reconstructed gel block. The reconfigured instrument could successfully isolate DNA amplicons from extremely low-amplitude peaks (110 relative fluorescent units), which were undetectable using polyacrylamide gel electrophoresis. In addition, we successfully isolated bands that were only three bases apart that comigrated on polyacrylamide gel electrophoresis. DNA sequencing 'was used to confirm that the correct peaks were recovered at sufficient purity.

Original languageEnglish (US)
Pages (from-to)598-603
Number of pages6
JournalJournal of Molecular Diagnostics
Volume9
Issue number5
DOIs
StatePublished - Nov 2007

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Polyacrylamide Gel Electrophoresis
Gels
DNA
Capillary Electrophoresis
Mutation
Molecular Pathology
Agar Gel Electrophoresis
Automation
DNA Sequence Analysis
Protein-Tyrosine Kinases
Genes

ASJC Scopus subject areas

  • Molecular Biology

Cite this

A molecular fraction collecting tool for the ABI 310 automated sequencer. / Lin, Ming-Tseh; Rich, Roy G.; Shipley, Royce F.; Hafez, Michael J.; Tseng, Li Hui; Murphy, Kathleen M.; Gocke, Christopher; Eshleman, James.

In: Journal of Molecular Diagnostics, Vol. 9, No. 5, 11.2007, p. 598-603.

Research output: Contribution to journalArticle

Lin, Ming-Tseh ; Rich, Roy G. ; Shipley, Royce F. ; Hafez, Michael J. ; Tseng, Li Hui ; Murphy, Kathleen M. ; Gocke, Christopher ; Eshleman, James. / A molecular fraction collecting tool for the ABI 310 automated sequencer. In: Journal of Molecular Diagnostics. 2007 ; Vol. 9, No. 5. pp. 598-603.
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