Sequence-enabled reassembly of beta-lactamase (SEER-LAC): a sensitive method for the detection of double-stranded DNA.
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 beta-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.
Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721, USA.
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Cliff I
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Indraneel
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D020137:Base Pair Mismatch; D001483:Base Sequence; D002511:Cephalosporins / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D005057:Eukaryotic Cells / Q000201:enzymology / Q000378:metabolism; D005240:Feasibility Studies; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011387:Prokaryotic Cells / Q000201:enzymology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D016335:Zinc Fingers; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002511:Cephalosporins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D004247:DNA; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; C021720:nitrocefin