University of California

Hugh Edward Olsen

Mark Albert Akeson

Miten Jain

David W Deamer

Bakthan Singaram

Rebecca L Braslau

Wenonah Vercoutere

Frank Cappuccio

Derek Steiner

Logan Mulroney

Provided are methods of analyzing capped ribonucleic acids (RNAs). The methods include translocating an adapted RNA through a nanopore of a nanopore device. The adapted RNA includes an RNA region, a 5' cap, and an adapter polynucleotide attached to the 5′ cap. The methods include monitoring ionic current through the nanopore during the translocating, translocating the 5′ cap through the nanopore, and identifying one or more ionic current features characteristic of the 5′ cap (e.g., a triphosphate linkage between the 5′ cap and nucleotide N1 of the RNA region, a 5′ to 5′ orientation of the 5′ cap and nucleotide N1 of the RNA region, and/or the like), translocating through the nanopore. Also provided are computer-readable media, computer devices, and systems that find use, e.g., in practicing the methods of the present disclosure.

Methods of analyzing capped ribonucleic acids

Provided are methods of producing size-selected nucleic acid libraries. The methods include contacting a nucleic acid sample and a nucleic acid binding reagent including an affinity tag, under conditions in which nucleic acids of less than a desired length are substantially bound to the nucleic acid binding reagent and nucleic acids of the desired length are substantially not bound to the nucleic acid binding reagent. The conditions include the duration of the contacting, the concentration of the nucleic acid binding reagent, or both. The methods further include separating, using the affinity tag, the nucleic acids of less than the desired length bound to the nucleic acid binding reagent from the nucleic acids of the desired length not bound to the nucleic acid binding reagent, to produce a size-selected nucleic acid library. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure, are also provided.

Methods of producing size-selected nucleic acid libraries and compositions and kits for practicing same

Disclosed are methods for polynucleotide sequencing that detect the location of selected nucleobases with greater precision. The methods can be used to determine the location and nature of modified bases in a polynucleotide, that is, non-canonical bases, or to improve accuracy of sequencing of 'problem' regions of DNA sequencing such as homopolymers, GC rich areas, etc. The sequencing method exemplified is nanopore sequencing. Nanopore sequencing is used to generate a unique signal at a point in a polynucleotide sequence where an abasic site (AP site, or apurinic or apyrimidinic site) exists. As part of the method, an abasic site is specifically created enzymatically using a DNA glycosylase that recognizes a pre-determined nucleobase species and cleaves the N-glycosidic bond to release only that base, leaving an AP site in its place.

Methods for determining base locations in a polynucleotide

Targeted molecular bar codes and methods for using the same are provided. The subject targeted molecular bar codes include a molecular bar code and a member of a specific binding pair, where the specific binding pair member is generally bonded to the bar code through a linking group. The subject molecular bar code may be read during translocation through a single nano-meter scale pore. The subject targeted molecular bar codes find use in a variety of different applications involving analyte detection, such as screening and diagnostic applications.

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Santa Cruz, CA, United States of America

Hugh Edward Olsen