Facile SNP detection using bifunctional, cross-linking oligonucleotide probes.
A facile, sensitive method for detecting specific sequences of oligonucleotides was developed. Detection of DNA sequences with single nucleotide discrimination is achieved by combining the selectivity of hybridization with an efficient cross-linking reaction. Readily synthesized bifunctional oligonucleotide probes containing a modified pyrimidine that is capable of forming interstrand cross-links under mild oxidative conditions internally, and biotin at their 5'-termini were used to discriminate between 16-nt long sites in plasmid DNA that differ by a single nucleotide. The target sequence was detected via fluorescence spectroscopy by utilizing conjugates of avidin and horseradish peroxidase in a microtiter plate assay. The method is able to detect as little as 250 fmol of target without using PCR and exhibits single nucleotide discrimination that approaches 200:1. In principle, this method is capable of probing any target sequence containing a 2'-deoxyadenosine.
Peng
Xiaohua
X
Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Greenberg
Marc M
MM
D004591:Electrophoresis, Polyacrylamide Gel; D015345:Oligonucleotide Probes / Q000737:chemistry*; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013936:Thymidine / Q000737:chemistry
D015345:Oligonucleotide Probes; D013936:Thymidine