Gen-probe, Inc.

Anne-Laure Shapiro

David Opalsky

Steven T Brentano

Dmitry Lyakhov

Matthew C Friedenberg

Norman C Nelson

Jijumon Chelliserry

Jason F Rhubottom

James T Tuggle

Sangeetha Vijaysri Nair

Jennifer L Tidd

Ankur H Shah

Alberto A Lio

Timothy J Scheer

Matthew Friedenberg

The invention provides compositions and methods for making closed nucleic acid structures in which one or both strands are continuous. The closed nucleic acid structures can be used as sequencing templates among other applications.

Closed nucleic acid structures

The invention provides methods of forming a circular template for sequencing a target nucleic acid. The circular template is generated by amplification of a segment of the target nucleic acid with chimeric primers with complementary 5' ends. The circular template has a single nick or gap providing a site for initiation of template-directed extension for sequence analysis. Sequencing of a single template generates reads of alternating segments of the same strand of the target nucleic spaced by primer segments. The different reads of the same strand of the target nucleic acid can be compiled to generate a consensus sequence. Because only one strand of the target nucleic acid is sequenced per reaction, the present method avoids errors introduced by unwittingly combining sequences of both strands of a heteroduplex PCR product. Because only one strand of the target nucleic acid is sequenced per reaction, the present method avoids errors introduced by unwittingly combining sequences of both strands of a heteroduplex PCR product.

Circularized templates for sequencing

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La Jolla, CA, United States of America

Anne-Laure Shapiro