Triple-helical capture assay for quantification of polymerase chain reaction products.
This method for rapid, automated analysis of polymerase chain reaction (PCR) products makes use of PCR primers containing 5'-polypyrimidine sequences. Polypyrimidine-"headed" primers confer to the PCR product the ability to form triple helical complexes with a third polypyrimidine oligonucleotide. Third-strand oligonucleotides are modified to serve as either capture reagents or detection reagents for PCR products. Automated quantitative measurement of the PCR product is achieved by using latex bead-based fluorescence analysis. The use of triple-instead of double-helical interactions avoids the usual requirements of complex blocking reagents, time- and labor-intensive washing steps, and long times for color development. The method also provides rapid, sequence-specific capture and detection of PCR products without the need to denature the double-stranded PCR product. The assay is demonstrated with use of both PCR primer-derived and endogenous triple-helix-forming sequences resulting from PCR of several bacterial and viral target nucleic acids.
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Idexx Corp., Portland, ME 04101.
D001322:Autoanalysis / Q000379:methods*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis; D015497:HIV-1 / Q000235:genetics; D007202:Indicators and Reagents; D008863:Microspheres; D008969:Molecular Sequence Data; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011743:Pyrimidines
D004269:DNA, Bacterial; D004279:DNA, Viral; D007202:Indicators and Reagents; D009841:Oligonucleotides; D011743:Pyrimidines; C030986:pyrimidine