Agilent Technologies, Inc.

Diane D Ilsley

Douglas J Dellinger

Joel Myerson

Mark Andrew Troll

Douglas A Amorese

Robert A Ach

SueAnn C Dahm

Geraldine Fulcrand

Methods and compositions for producing labeled probe nucleic acids from genomic nucleic acid template are provided. In the subject methods, a conserved coding consensus region primer is employed to enzymatically generate a select set of labeled probe nucleic acids corresponding to coding regions of genes from a genomic template via a primer extension protocol. The subject methods find use in a variety of different applications, and are particularly suited for use in the preparation of labeled probe nucleic acids for use in array based comparative genomic hybridization applications. Also provided are kits for use in practicing the subject methods.

Methods and compositions for producing labeled probe nucleic acids for use in array based comparative genomic hybridization applications

Methods are disclosed for conjugating one moiety to another moiety. In the method the moieties are reacted with one another in a protic solvent. Reaction between the moieties and the protic solvent during the conjugating is negligible or reversible. A stable bond is formed between the moieties to produce a product that is not subject to &bgr;-elimination at elevated pH. Usually, one of the moieties comprises an unsaturation between two carbon atoms. One of the carbon atoms is or becomes an electrophile during the conjugating. The other of the moieties comprises a functionality reactive with the electrophile carbon atom to form a product that comprises the unsaturation. Compounds comprising both of the moieties as well as precursor molecules are also disclosed. Methods are also disclosed for determining an analyte in a sample employing compounds as described above.

Methods for preparing conjugates

A signal amplification method for detecting a target nucleic acid analyte having a homopolymeric region and a target sequence includes steps of: contacting an analyte under hybridizing conditions with a multiplicity of reporter probes, each reporter probe including a signal region and an oligonucleotide sequence which is complementary to and capable of forming a stable hybrid with the analyte homopolymeric region to form an analyte:reporter probe hybrid; and forming an analyte:capture probe hybrid by contacting the analyte target sequence with a capture probe under hybridizing conditions. The analyte:reporter probe hybrid may formed prior to contacting the analyte target sequence with the capture probe, so the result of contacting the analyte target sequence with the capture probe results in formation of an analyte:reporter probe:capture probe complex. The analyte:capture probe hybrid may be immobilized on a solid generally planar surface in an array format. Multiple reporter probes may form triple helix structures for further signal enhancement. Multiply-labeled hairpin reporter probes may be employed according to the invention. Also, a kit for carrying out the invention includes one or more capture probes immobilized on a surface, reporter probes each having a signal region and a sequence for binding analyte homopolymer regions, and, optionally, reagents for addition of homopolymer regions to nucleic acid analytes and for carrying out the hybridization reaction.

Hybridization assay signal enhancement

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San Jose, CA, United States of America

Diane D Ilsley