Other

Yale University

Joseph C Kaufman

Paul M Lizardi

Darin R Latimer

Matthew E Roth

Li Feng

Shane Crawford Weber

Cesar E Guerra

The described method provides, methods, and kits to produce, identify, catalog and classify a comprehensive collection of nucleic acid targets produced from a nucleic acid sample. The method, referred to as Cataloging and Classification of Sequence Tags, involves generating a set of target nucleic acid fragments; coupling the target nucleic acid fragments to a nucleic acid bridge comprising, for example, two or more primer binding sites and two recognition sites for cleavage at a site offset from the recognition site to the fragment's end; and cleaving the fragments to generate chimeric nucleic acids of known length. The nucleic acid bridge is thus disposed between the two nucleic acid fragments in the chimeric nucleic acid. The resulting duplex nucleic acids comprise a set of sequence tags (i.e., by amplification using universal primers), comprising an addressable portion, a target nucleic portion and a portion of the nucleic acid bridge. Single-stranded or partial duplex sequence tags may be captured by coupling to a complementary capture probe. Capture probe-sequence tag hybrids, may be detected employing a labeled detector probe. The method allows a complex sample of nucleic acids to be cataloged in a reproducible and sequence-specific manner. The method further provides methods for analysis of the above sample to classify the sequence tags; determine the presence and relative amounts of sequences of interest; derive expressed genes signatures and differential gene expression signatures; and identify putative expressed sequence tags (EST).

Producing, cataloging and classifying sequence tags

Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method, referred to as Binary Encoded Sequence Tags (BEST), involves generation of a set of nucleic acid fragments; adding an adaptor to the ends containing recognition site for cleavage at a site offset from the recognition site; cleaving the fragment to generate fragments having a plurality sticky ends; indexing of the fragments into sets based on the sequence of sticky ends. The fragments are indexed by adding a offset adaptor to newly generated ends. A different adaptor will be coupled to each different sticky end. The resulting fragments&mdash;which will have defined ends, be of equal lengths (in preferred embodiment), and a central sequence derived from the source nucleic acid molecule&mdash;are binary sequence tags. The binary sequence tags can be used and further analyzed in numerous ways. For example, the binary sequence tags can be captured by hybridization and coupling, preferably by ligation, to a probe. The probe is preferably immobilized in an array or on sortable beads. One form of the BEST method, referred to as modification assisted analysis of binary sequence tags (MAABST), assesses modification of sequences in nucleic acid molecules by detecting differential cleavage based on the presence or absence of modification in the molecules.

Binary encoded sequence tags

Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method, referred to as Fixed Address Analysis of Sequence Tags (FAAST), involves generation of a set of nucleic acid fragments having a variety of sticky end sequences; indexing of the fragments into sets based on the sequence of sticky ends; associating a detector sequence with the fragments; sequence-based capture of the indexed fragments on a detector array; and detection of the fragment labels. Generation of the multiple sticky end sequences is accomplished by incubating the nucleic acid sample with one or more nucleic acid cleaving reagents. The indexed fragments are captured by hybridization and coupling, preferably by ligation, to a probe. The method allows a complex sample of nucleic acid to be quickly and easily cataloged in a reproducible and sequence-specific manner. One form of the method allows determination of associations, in a nucleic acid molecule, of different combinations of known or potential sequences. Another form of the method assesses modification of sequences in nucleic acid molecules by basing cleavage of the molecules on the presence or absence of modification.

Fixed address analysis of sequence tags

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Hamden, CT, United States of America

Joseph C Kaufman