External-loop free energy affects dye-labeled terminators premature terminations in DNA cycle-sequencing reactions.
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.
Wen
Long
L
Microchemical Core Facility, Biology Department, San Diego State University, 5500 Capanile Drive, San Diego, CA 98=2182-1030, USA. lwen@sciences.sdsu.edu
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004735:Energy Transfer; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010957:Plasmids / Q000235:genetics; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D016324:Sequence Tagged Sites; D013696:Temperature; D013816:Thermodynamics
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA