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 language | English (US) |
|---|---|
| Pages (from-to) | 3620-3625 |
| Number of pages | 6 |
| Journal | Biochemistry |
| Volume | 45 |
| Issue number | 11 |
| DOIs | |
| State | Published - Mar 21 2006 |
| Externally published | Yes |
ASJC Scopus subject areas
- Biochemistry