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Related Experiment Videos

Cycle sequencing using degenerate primers containing inosines

Z Shen1, J Liu, R L Wells

  • 1Department of Radiological Health Sciences, Colorado State University, Fort Collins 80523.

Biotechniques
|July 1, 1993
PubMed
Summary

This study presents a novel cycle sequencing protocol for DNA amplified by polymerase chain reaction (PCR). The method efficiently sequences DNA amplified from degenerate primers, even when flanking sequences are unknown.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA sequencing is crucial for genetic analysis.
  • Polymerase chain reaction (PCR) amplifies DNA for sequencing.
  • Sequencing DNA amplified from degenerate primers presents challenges, especially when flanking sequences are unknown.

Purpose of the Study:

  • To describe a modified, efficient protocol for cycle sequencing of PCR-amplified DNA.
  • To enable sequencing of DNA amplified using degenerate primers without prior knowledge of flanking sequences.

Main Methods:

  • The protocol involves two sequential linear PCR amplifications.
  • It utilizes a small amount of double-stranded DNA template and stringent annealing temperatures.
  • Key steps include alpha-35S-dATP labeling of specific primers and dideoxy-ribonucleotide termination.

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Main Results:

  • The described method does not require end labeling of DNA.
  • It is effective for sequencing PCR-amplified DNA fragments derived from highly degenerate primers.
  • The protocol is particularly useful when flanking sequences are unknown.

Conclusions:

  • This modified cycle sequencing protocol offers a robust method for analyzing DNA amplified by PCR.
  • It overcomes limitations associated with sequencing from degenerate primers and unknown flanking regions.
  • The technique enhances the utility of PCR-based DNA sequencing in various genetic research applications.