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Related Experiment Videos

Mismatch cleavage by single-strand specific nucleases.

Bradley J Till1, Chris Burtner, Luca Comai

  • 1Basic Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.

Nucleic Acids Research
|May 14, 2004
PubMed
Summary
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Single-strand specific nucleases effectively cleave DNA mismatches for mutation discovery. Diverse nucleases and crude plant extracts show promise for high-throughput analysis of genetic variations.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Mutation detection and single-nucleotide polymorphism (SNP) analysis are crucial in genetic research.
  • Heteroduplex DNA analysis relies on identifying mismatches for variant discovery.

Purpose of the Study:

  • To investigate the efficacy of single-strand specific (sss) nucleases in cleaving single base pair mismatches.
  • To evaluate the utility of various nucleases and crude extracts for mutation and polymorphism discovery.

Main Methods:

  • Utilized the Targeting Induced Local Lesions IN Genomes (TILLING) protocol with LI-COR gel detection.
  • Assayed cleavage of amplified heteroduplexes containing induced mutations and natural polymorphisms.
  • Tested purified nucleases from celery (CEL I), mung bean, and Aspergillus (S1), as well as crude plant extracts.

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Main Results:

  • Purified CEL I, S1, and mung bean nucleases efficiently cleaved nearly all tested single base pair mismatches.
  • Optimal cleavage conditions involved higher pH, temperature, and divalent cations.
  • Crude plant extracts demonstrated comparable efficacy to purified enzymes.

Conclusions:

  • Diverse S1 family sss nucleases effectively cleave DNA heteroduplexes at mismatches.
  • Single-base mismatches are cleaved due to the smallest single-stranded regions available for enzyme binding.
  • A variety of sss nucleases and extracts are suitable for high-throughput mutation and polymorphism discovery.