Two-step cycle sequencing improves base ambiguities and signal dropouts in DNA sequencing reactions using energy-transfer-based fluorescent dye terminators.
The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods plays an important role in the actual effort to improve the efficiency of large-scale DNA analysis. While 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. Premature terminations are usually evidenced by base ambiguities and are often accompanied by diminished signal intensity from that point on in the sequence. I studied a two-step protocol for Taq cycle sequencing using the ABI BigDye terminator for reducing premature terminations in DNA sequences. I demonstrate that combining the annealing step with the extension step at one temperature (60 degrees C) reduces premature terminations in DNA sequences that regularly contain premature terminations when the three temperature steps are used. This modification significantly increases the number of accurately read bases in DNA sequences.
Wen
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Microchemical Core Facility, Biology Department, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4614, USA. lwen@sciences.sdsu.edu
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D005456:Fluorescent Dyes; D020648:Peptide Elongation Factor 1; D004247:DNA