Rapid SLT gene detection on polyethylene-coacrylic acid film without molecular labels or surface-fouling agents.
The commercially available copolymer of 10 mol% acrylic acid and polyethylene is easily formed into a nonfluorescing, non-polynucleotide-adsorbing film. The film has surface carboxylate functions whose concentration can be increased by heating to 80 degrees C in 30% NaOH. The carboxylate groups will react at pH approximately 7 with commercially available, oligo-DNA, 2-8 ng/microl, that has been synthesized with a C(12)-alkylamino tail on the 5'-end. The reaction is mediated with water-soluble carbodiimide reagent and is assumed to result in a primary amide bond between the polymer film and the modified oligo-DNA. The tethered oligo-DNA retains its hybridization activity, and its surface concentration is sufficient to permit qualitative, labelless detection of hybridized target by fluorescence after brief staining with ethidium bromide. The film is used to detect Shiga-like toxin gene II (SLT-II) from Escherichia coli O157:H7 after asymmetric, capillary, PCR amplification, and a 4-h hybridization. Captured target may be removed from the film using distilled water, after which the film can be used again without noticeable loss of activity. The method provides relatively rapid detection of PCR amplimers without having to use molecular labels, or surface-fouling agents.
Zhang
P
P
Department of Chemistry and Biochemistry, Southern Station, University of Southern Mississippi, Hattiesburg, Mississippi 39406, USA.
Fawcett
N C
NC
Evans
J A
JA
Hurt
T
T
Harvey
K G
KG
Craven
R C
RC
D000179:Acrylates / Q000737:chemistry*; D001427:Bacterial Toxins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D004996:Ethidium; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry*; D005453:Fluorescence; D005798:Genes, Bacterial; D009693:Nucleic Acid Hybridization / Q000379:methods; D020959:Polyethylene / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011108:Polymers / Q000737:chemistry; D022641:Shiga Toxin 2; D012972:Sodium Hydroxide; D013698:Templates, Genetic
D000179:Acrylates; D001427:Bacterial Toxins; D011108:Polymers; D022641:Shiga Toxin 2; D012972:Sodium Hydroxide; D020959:Polyethylene; D004996:Ethidium; C036658:acrylic acid; D005022:Ethyldimethylaminopropyl Carbodiimide