Gen-probe, Inc.

Other

Dmitry Lyakhov

Steven T Brentano

Norman C Nelson

James D Carlson

Michael McClellan Becker

Lyle J Arnold, Jr

Lyle J Arnold

Matthew C Friedenberg

Anne-Laure Shapiro

Jijumon Chelliserry

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

Compositions, reaction mixtures, and methods for performing an amplification reaction, including multiplex amplification reaction, wherein the method comprises using one or more amplification oligomer complexes comprising linked first and second amplification oligomer members. In one aspect, the amplification oligomer complex is hybridized to a target nucleic acid, the target nucleic acid with hybridized amplification oligomer complex is then captured, and other components are washed away. Target sequences of the target nucleic acids are pre-amplified to generate a first amplification product. The first amplification product is amplified in one or more secondary amplification reactions to generate second amplification products.

Methods and compositions for nucleic acid amplification

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

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San Diego, CA, United States of America

Dmitry Lyakhov