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Related Experiment Videos

Fluorescence-based cycle sequencing with primers selected from a nonamer library

J H Bock1, J L Slightom

  • 1Upjohn Company, Kalamazoo, MI 49007, USA.

Biotechniques
|July 1, 1995
PubMed
Summary

Short DNA primers called nonamers can successfully prime sequencing reactions on the Applied Biosystems 373A fluorescence-based sequencer. This method, using modified Taq DNA polymerase, achieved an average read length of 257 bp.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Nonamers have previously been shown to prime manual T7 polymerase sequencing.
  • The utility of nonamers for automated fluorescence-based sequencing remained to be explored.

Purpose of the Study:

  • To determine if nonamers can prime sequencing reactions for the Applied Biosystems Model 373A sequencer.
  • To optimize conditions for nonamer-primed cycle sequencing.

Main Methods:

  • Tested Applied Biosystems T7 and Taq DNA Polymerase DyeDeoxy Terminator methodologies.
  • Developed a modified Taq DNA polymerase cycle sequencing procedure.
  • Optimized denaturation, annealing, and extension temperatures and times.
  • Utilized linear, alkaline-denatured double-stranded DNA with a doubled reaction mixture and 2% dimethyl sulfoxide.

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Main Results:

  • Successful nonamer-primed sequencing was achieved using a modified Taq DNA polymerase method.
  • Optimized conditions: 96°C (30s) denaturation, 20°C (5min) annealing, 60°C (4min) extension, repeated 50 times.
  • Approximately 50% of 121 tested nonamers yielded successful reactions.
  • Achieved an average reading length of 257 bp.

Conclusions:

  • Nonamers are effective primers for fluorescence-based DNA sequencing on the Applied Biosystems 373A.
  • Modified Taq polymerase cycle sequencing with optimized conditions and specific DNA preparation significantly improves nonamer-primed reactions.
  • This approach offers a viable method for DNA sequencing using short primers.