Johnson & Johnson Clinical Diagnostics, Inc.

John W Backus

Richard C Sutton

John Bruce Findlay

John W Sutherland

JoAnne H Kerschner

William Harold Donish

Tobias E Ekeze

Ignazio S Ponticello

David J Sharkey

Marlene M King

Jerome C Swartz

John Bruce Findley

John William H Sutherland

Marcia L Kramer

Joseph Falvo

Nucleic acids can be amplified and detected using a very rapid polymerase chain reaction procedure. This procedure includes a series of steps which have critically defined temperature and time parameters. Each polymerase chain reaction cycle requires generally less than about two minutes, and in most cases less than 90 seconds. At least 5 units/100 &mgr;l of solution of thermostable DNA polymerase are used, and other preferred levels of primer concentrations facilitate the quick cycling in the amplification. In preferred embodiments, only two temperatures are used in the amplification.

Nucleic acid amplification and detection methods using rapid polymerase chain reaction cycle

The present invention relates to a method for amplifying and detecting a target nucleic acid. The method comprising contacting a sample suspected of containing the target nucleic acid with a thermostable DNA polymerase and two primers that are substantially complementary to the target nucleic acid, under conditions such that the target nucleic acid is amplified. The amplified target nucleic acids are then denatured to form single stranded nucleic acids. Following amplification, the sample is subject to a pre-detection incubation step. The sample is incubated for between 1 second and 30 minutes at between 95&deg; C. and 120&deg; C. to inactivate said polymerization agent. Finally, the presence or absence of the amplified target nucleic acids is determined. Preferably, amplification, incubation and detection are carried out in a closed reaction vessel.

Amplifying and detecting target nucleic acids using a post amplification incubation step

Multiple target nucleic acids are amplified using polymerase chain reaction in the presence of a nonionic, polymeric volume exclusion agent. The amplification efficiency of low copy target nucleic acids is increased in the presence of the volume exclusion agent even though reduced primer levels are used. In this manner, amplification efficiency of a given target nucleic acid can be more readily manipulated.

Coamplification of target nucleic acids using volume exclusion agent in

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

Nucleic acids can be made available for amplification or other treatment after lysis by contacting the lysate with polyethyleneimine to form a precipitate with the nucleic acids. The nucleic acids are then released from the precipitate by contact with a strong base, and the released nucleic acids are kept in solution with an anionic phosphate ester surfactant. This method for preparing specimen samples is simple and quite rapid.

Methods for capture and selective release of nucleic acids using

Nucleic acids can be made available for amplification or other treatment after lysis by contacting the lysate with specific weakly basic polymers to form a precipitate with the nucleic acids at acidic pH. After removing non-precipitated materials, the pH is then made basic, thereby releasing the nucleic acids from the polymer. This method for preparing specimen samples is simple and quite rapid, and the released nucleic acids can be further treated in hybridization assays or amplification procedures. The weakly basic polymers are water-soluble and cationic at acidic pH, but neutral in charge at basic pH.

Methods for capture and selective release of nucleic acids using weakly

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.

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Williamson, NY, United States of America

John W Backus