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Methods of sequencing fluorophore-quencher fret-aptamers

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Methods of sequencing fluorophore-quencher fret-aptamers

The present invention describes methods for the production and selecting of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase high performance liquid chromatography (HPLC) or other types of chromatography. Finally, FRET-aptamer structures and the specific locations of F and Q within FRET-aptamer structures are determined by digestion with exonucleases and mass spectral nucleotide sequencing analysis. Alternatively, single DNA or RNA intrachain FRET-aptamers can be sequenced and the locations of F and Q within the structure can be determined by nanopore sequencing and the locations of F and Q within the structure can be verified by nucleic acid “combing” coupled to high-powered fluorescence microscopy.

Browse recent Pronucleotein Biotechnologies, LLC patents - ,
Inventors: John G. Bruno, Joseph Chanpong
USPTO Applicaton #: #20120270221 - Class: 435 612 (USPTO) - 10/25/12 - Class 435 

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The Patent Description & Claims data below is from USPTO Patent Application 20120270221, Methods of sequencing fluorophore-quencher fret-aptamers.

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