Oxford Nanopore Technologies Limited

Orchid Cellmark, Inc.

Brian McKeown

James White

James Anthony Clarke

Lakmal Nishantha Jayasinghe

John Hagan Pryce Bayley

Stephen Cheley

Roger Derbyshire

Paul Rowan

Robert Sung

The invention relates to constructs comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme. The pore subunit is covalently attached to the enzyme such that both the subunit and enzyme retain their activity. The constructs can be used to generate transmembrane protein pores having a nucleic acid handling enzyme attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect its component nucleotides by stochastic sensing.

Enzyme-pore constructs

The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.

Adaptors for nucleic acid constructs in transmembrane sequencing

The invention provides compositions and methods for performing primer extension reactions, including employment of amplification primers having 5' tags to incorporate into amplicons variant nucleotides of interest from target nucleic acids at known ratios, with or without sequences surrounding the variant nucleotides of interest. The invention provides identifying the variant nucleotides generated from the target nucleic acid and generated from the 5&#x2032; tags, comparing the results, evaluating the efficiency of the primer extension reactions, and monitoring the efficacy of such reactions. The invention accounts for DNA sequence and experimental variables that may affect efficiency of incorporation of nucleotides, and provides a reference point for the interpretation of polymorphisms. The invention also provides methods of breeding scrapie-resistant sheep populations.

Methods and compositions for genotyping

The invention comprises compositions and methods for performing primer extension reactions, including employment of amplification primers having 5' tags to incorporate into amplicons variant nucleotides of interest from target nucleic acids at known ratios, along with the sequences surrounding the variant nucleotides of interest. The invention comprises identifying the variant nucleotides generated from the target nucleic acid and generated from the 5&#x2032; tags, comparing the results, evaluating the efficiency of the primer extension reactions, and monitoring the efficacy of such reactions. The invention accounts for DNA sequence and experimental variables which may affect efficiency of incorporation of nucleotides, and provides a reference point for the interpretation of polymorphisms.

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Middle Barton Oxon, United Kingdom

Brian McKeown