Becton Dickinson and Company Limited

David J Wright

James G Nadeau

J Bruce Pitner

Douglas Byron Sherman

Colleen M Nycz

Terry J Amiss

Michael C Little

James L Schram

George T Walker

G Terrance Walker

Helen V Hsieh

Melinda S Fraiser

Catherine A Spargo

Mark Van Cleve

Cheryl H Dean

Deborah R Howard

Margaret S Dey

Maria A Milla

The invention is directed to compositions of mutated binding proteins containing reporter groups, analyte biosensor devices derived therefrom, and their use as analyte biosensors both in vitro and in vivo.

Binding proteins as biosensors

The present invention provides methods for detecting and identifying sequence variations in a nucleic acid sequence of interest using a detector primer. It has been found that the reduced efficiency of primer extension by DNA polymerases when the 3' end of a primer does not hybridize perfectly with the target can be adapted for use as a means for distinguishing or identifying the nucleotide in the target which is at the site where the diagnostic mismatch between the detector primer and the target occurs. The detector primer hybridizes to the sequence of interest and is extended with polymerase. The efficiency of detector primer extension is detected as an indication of the presence and/or identity of the sequence variation in the target. The inventive methods make use of nucleotide mismatches at or near the 3&#x2032; end of the detector primer to discriminate between the nucleotide sequence of interest and a second nucleotide sequence which may occur at that same site in the target. The methods are particularly well suited for detecting and identifying single nucleotide differences between a target sequence of interest (e.g., a mutant allele of a gene) and a second nucleic acid sequence (e.g., a wild type allele for the same gene).

Methods for detecting single nucleotide polymorphisms

The invention is directed to compositions of mutated binding proteins containing reporter groups, analyte biosensor devices derived there from, and their use as analyte biosensor both in vitro and in vivo.

Binding protein as biosensors

Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of the Mycobacterium tuberculosis (M.tb) complex and a 16S rDNA target common to essentially all mycobacteria are described. In certain embodiments, the primers are optimized for efficient multiplex amplification in thermophilic SDA. The multiplex Strand Displacement Amplification methods of the invention are capable, in a single amplification reaction, of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of mycobacteria. Also disclosed are internal control sequences designed for coamplification with the two targets, allowing assessment of amplification efficiency and/or quantitation of the targets.

Detection of mycobacteria by multiplex nucleic acid amplification

Strand Displacement Amplification methods (thermophilic SDA) which can be performed over a broad temperature range (37.degree. C. to 70.degree. C.). The preferred temperature range for thermophilic SDA is 50.degree. C. to 70.degree. C. It has been found that certain thermophilic restriction endonucleases are capable of nicking the hemimodified restriction endonuclease recognition/cleavage site as required by SDA and dissociating from the site. It has further been found that certain thermophilic polymerases are capable of extending from the nick while displacing the downstream strand. Thermophilic SDA, because of reaction temperatures higher than previously possible with conventional SDA enzyme systems, has improved specificity and efficiency, reduced nonspecific background amplification, and potentially improved yields of amplification products. In addition, the need to add the enzymes in a separate step after the initial heat denaturation of double stranded targets is eliminated when enzymes capable of tolerating the denaturation temperature are used.

Strand displacement amplification using thermophilic enzymes

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Chapel Hill, NC, United States of America

David J Wright