Sequence-enabled reassembly of β-lactamase (SEER-LAC): A sensitive method for the detection of double-stranded DNA

Aik T. Ooi, Cliff I. Stains, Indraneel Ghosh, David J. Segal

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), was demonstrated in vitro to produce a DNA-concentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM-1 β-lacatamase. This system could distinguish target DNA from nontarget DNA in less than 5 min, representing a more than 1000-fold improvement over our previous SEER design. A single base-pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. Signal intensity was not affected by genomic DNA when present in equal mass to the target DNA. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences.

Original languageEnglish (US)
Pages (from-to)3620-3625
Number of pages6
JournalBiochemistry
Volume45
Issue number11
DOIs
StatePublished - Mar 21 2006

ASJC Scopus subject areas

  • Biochemistry

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