Gen-probe, Inc.

Biomerieux, Inc.

Matthew C Friedenberg

Steven T Brentano

Dmitry Lyakhov

Anne-Laure Shapiro

Norman C Nelson

Michael McClellan Becker

Ali Laayoun

Jijumon Chelliserry

Kenneth A Browne

Lionel Menou

Christelle Tora

Aloke R Banerjee

Fred F Hajjar

Aloke Raj Banerjee

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

A process of fragmenting and labeling a synthetic or natural nucleic acid, comprising the steps of providing a mixture containing a nucleic acid, a labeling agent containing a detectable label, and at least one multivalent metal cation in a substantially aqueous solution; chemically fragmenting the nucleic acid in the mixture to produce a multiplicity of nucleic acid fragments; and attaching at least one label to at least one of the nucleic acid fragments to produce a detectably labeled nucleic acid fragment.

Process for labeling a nucleic acid

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San Diego, CA, United States of America

Matthew C Friedenberg