A molecular fraction collecting tool for the ABI 310 automated sequencer.
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.
Lin
Ming-Tseh
MT
The Sol Goldman Pancreatic Cancer Research Center, Departments of Pathology and Oncology, Johns Hopkins School of Medicine, 1550 Orleans Street, Baltimore, MD 21231, USA.
Rich
Roy G
RG
Shipley
Royce F
RF
Hafez
Michael J
MJ
Tseng
Li-Hui
LH
Murphy
Kathleen M
KM
Gocke
Christopher D
CD
Eshleman
James R
JR
D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification; D004252:DNA Mutational Analysis; D019075:Electrophoresis, Capillary; D020440:Gene Duplication; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
D011948:Receptors, Antigen, T-Cell; D004247:DNA