Published online before print October 4, 2007

This Article Full Text (Rapid PDF) All Versions of this Article: jmoldx.2007.070022v1 9/5/598    most recent Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lin, M.-T. Articles by Eshleman, J. R. Search for Related Content PubMed PubMed Citation Articles by Lin, M.-T. Articles by Eshleman, J. R.

Copyright © 2007 American Society for Investigative Pathology
Journal of Molecular Diagnostics, doi:10.2353/jmoldx.2007.070022

Accepted for publication June 11, 2007.

Article

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 D. Gocke^*, and James R. Eshleman^*@

From the Departments of Pathology,* and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Clinical Engineering Services, Johns Hopkins Hospital, Baltimore, Maryland; and the Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan

^@ To whom correspondence should be addressed. E-mail: jeshlema{at}jhmi.edu.

	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.