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Updated: Aug 19, 2026

Studying DNA Looping by Single-Molecule FRET
Published on: June 28, 2014
External-loop free energy affects dye-labeled terminators premature terminations in DNA cycle-sequencing reactions
1Microchemical Core Facility, Biology Department, San Diego State University, 5500 Capanile Drive, San Diego, CA 98=2182-1030, USA. lwen@sciences.sdsu.edu
Abstract:
Although dideoxy terminators labeled with energy-transfer dyes (BigDyes) provide the most versatile method of automated DNA sequencing, premature terminations result in a substantially reduced reading length of the DNA sequence. I recently demonstrated that combining the annealing step with the extension step at a single temperature (60 degrees C) reduces premature terminations of DNA sequences that ordinarily contain premature terminations when three temperature steps are used in sequencing. I studied a novel class of DNA sequences of 100-bp length and located upstream from the point that causes premature terminations. I determined the thermodynamics of 49 DNA sequences with premature terminations at three temperature steps by using DNA mfold profiles. Sequencing results for 28 samples were improved with two-step cycle sequencing, whereas two-step cycle-sequencing reactions did not improve the results for 21 sequences. Nearest-neighbor thermodynamic parameters for all 49 sequences were compared at temperatures 50 degrees C and 60 degrees C. The parameters predicted that thermodynamic free-base (external-loop) energies (delta delta G degree) were significantly different for these two study groups of samples. The results indicate that changes in free energy in single-strand base (external-loop) sequences can have a significant effect in reducing premature terminations in DNA sequencing reactions run with energy-transfer-based fluorescent-dye terminators.
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