Johnson & Johnson Clinical Diagnostics, Inc.

Eastman-kodak Company

Clinical Diagnostic Systems, Inc.

Cetus Corporation

John W Sutherland

John Bruce Findlay

Thomas J Cummins

Lynn Bergmeyer

JoAnne H Kerschner

Susan Melissa Atwood

Jon N Eikenberry

John W Backus

David Robert Patterson

Steven Evans

Patrick J Sheridan

Richard L Detwiler

Karl J Sanford

Louis M Mezei

William Harold Donish

Marlene M King

Arthur R Mack, Ii

Marsha D Bale Oenick

The present invention provides admixtures and methods for PCR amplification of a target nucleic acid in which amplification efficiency is increased by including an antibody specific for a polymerization agent and at least one of an exonuclease and a glycosylase in the PCR reaction mix. Kits for amplification of a target nucleic acid are also provided.

Use of exonuclease and/or glycosylase as supplements to anti-polymerase

An aqueous composition containing primers for opposing strands of two or more target nucleic acids can be used in polymerase chain reaction to provide simultaneously rapid and efficient amplification and detection of those nucleic acids. The primers for each target DNA differ in length by no more than 5 nucleotides and have a T.sub.m within the range of from about 65.degree. to about 74.degree. C., while the T.sub.m 's are within about 5.degree. C. of each other. Such compositions are useful in diagnostic test kits and methods for amplification and detection of multiple nucleic acids, or in 'multiplexing', using multiple capture probes. All of the capture probes have T.sub.m 's which are greater than 50.degree. C. and are within 15.degree. C. of each other.

Diagnostic compositions, elements, methods and test kits for

Nucleic acids can be amplified and detected using a very rapid polymerase chain reaction procedure in which two different nucleic acid sequences are present. This method allows one to preferentially modulate (for example, suppress) the degree of amplification of one or more nucleic acid sequences relative to other nucleic acid sequences. This modulation is achieved by exploiting differences in the relative primer melt temperatures, or by using certain ratios of primers. Each PCR cycle is very fast, that is less than about 90 seconds. This method is particularly useful for amplification and detection of DNA associated with infectious agents that may be present in a specimen in very small quantities compared to other nontargeted nucleic acids.

Rapid method for preferential coamplification of two different nucleic

Diagnostic primers and probes

A highly sensitive homogeneous assay allows for quantitative detection of amplified nucleic acids. This detection is achieved during or after amplification with a high affinity fluorescent dye which is from the class of unsymmetrical cyanine dyes having at least two positive charges and a binding constant (K.sub.b) within the range of from about 1.times.10.sup.4 to about 5.times.10.sup.5 (molar.sup.-1). The reagents used for the assay can be contained in a kit designed for amplification such as by polymerase chain reaction.

Homogeneous method for assay of double-stranded nucleic acids using

A method for amplification and detection of a low copy target nucleic acid includes coamplification of a high copy target nucleic acid. After a number of conventional amplification cycles which include a denaturation step, several cycles are carried out during which the denatured products are renatured for a brief period of time. This intermediate step in later cycles of the amplification process reduces the effective concentration of the high copy target nucleic acid available for amplification in later cycles, thereby making more DNA polymerase available for amplification of the low copy target nucleic acid.

Method of amplification using intermediate renaturation step

A dry multilayer analytical element for assaying transaminases wherein the analytical element comprises:

Dry multilayer analytical elements for assaying transaminases

A convenient method for the quantitative determination of a DNA polymerase includes contacting an aqueous test specimen suspected of containing the enzyme with the following: a single-stranded DNA template present in a concentration of at least about 10.sup.-8 molar bases, a DNA primer complementary to the template, a source of a metal polymerase cofactor, sufficient deoxyribonucleoside triphosphates to synthesize double-stranded DNA in the presence of the polymerase, and a colorimetric or fluorescent dye which is capable of providing a detectable signal when a primed single-stranded nucleic acid is converted to double-stranded DNA by the polymerase. The rate of signal generation is then measured and can be correlated with the level of DNA polymerase in the specimen using graphical or mathematical means. The results of this method are precise, having a covariance of less than about 10%. A test kit includes the reagents needed for carrying out this method.

Quantitative determination of a DNA polymerase and a test kit useful in

A dye development composition comprising 1,10-phenanthroline and 1,7-dihydroxynaphthalene is disclosed for use in assaying high levels of hydrogen peroxide.

Compositions for the detection of high levels of hydrogen peroxide

An analytical method for the determination of hydrogen peroxide utilizes a hydrazide or a hydrazine substituted with one or more electron withdrawing groups. The hydrazides or hydrazines react with phenolic color couplers in the presence of hydrogen peroxide and a peroxidative compound to form a dye detectable at a wavelength of 600 nm or greater. These materials can also be used to assay analytes, such as glucose and cholesterol, which can react to produce hydrogen peroxide.

Analytical composition, element and method for the determination of

Enzymes which are activated by pyridoxal phosphate are assayed in a dry analytical element which includes a high coverage of pyridoxal phosphate. The coverage of pyridoxal phosphate is such that the multiplication product of the coverage and the spreading coefficient of the element results in a reagent mixture concentration of at least about 0.9 mmole per liter when a liquid sample is contacted with the element thereby eliminating the need for a preincubation of the sample with a solution containing pyridoxal phosphate. The reactions for the determination of the enzymes are initiated by contact of the dry elements with a liquid sample.

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Rochester, NY, United States of America

John W Sutherland