The Perkin-elmer Corporation

Life Technologies Corporation

Applied Biosystems, LLC

Applera Corporation

Pe Corporation (ny)

Perkin-elmer Corp., Applied Biosystems Division

Paul E Mayrand

John S Shigeura

George Stefan Golda

John A Bridgham

Norman M Whiteley

Louis B Hoff

Lincoln J McBride

William J Mordan

Douglas H Keith

Thomas Brennan-Marquez

Melvyn N Kronick

Chen-Hanson Ting

Michael L Metzker

Eric Lachenmeier

Marianne Hane

Harry A Guiremand

G Richard Cathcart

An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5'&#x2192;3&#x2032; exonuclease activity of a polymerase and the 5&#x2032;&#x2192;3&#x2032; extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such methods not requiring probes having their 5&#x2032; ends protected, wherein (i) the polymerase lacks 5&#x2032;&#x2192;3&#x2032; exonuclease activity, (ii) a 5&#x2032;&#x2192;3&#x2032; exonuclease inhibitor is included, and (iii) an exonuclease deactivation step is performed.

Methods and reagents for combined PCR amplification

Methods and reagents for combined PCR amplification and hybridization probing

An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5&prime;&rarr;3&prime; exonuclease activity of a polymerase and the 3&prime;&rarr;5&prime; extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such methods not requiring probes having their 5&prime; ends protected, wherein (i) the polymerase lacks 5&prime;&rarr;3&prime; exonuclease activity, (ii) a 5&prime;&rarr;3&prime; exonuclease inhibitor is included, and (iii) an exonuclease deactivation step is performed.

An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5&prime;&rarr;3&prime; exonuclease activity of a polymerase and the 5&prime;&rarr;3&prime; extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such methods not requiring probes having their 5&prime; ends protected, wherein (i) the polymerase lacks 5&prime;&rarr;3&prime; exonuclease activity, (ii) a 5&prime;&rarr;3&prime; exonuclease inhibitor is included, and (iii) an exonuclease deactivation step is performed.

Method and reagents for combined PCR amplification and hybridization probing

An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5'.fwdarw.3' exonuclease activity of a polymerase and the 3'.fwdarw.5' extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such methods not requiring probes having their 5' ends protected, wherein (i) the polymerase lacks 5'.fwdarw.3' exonuclease activity, (ii) a 5'.fwdarw.3' exonuclease inhibitor is included, and (iii) an exonuclease reactivation step is performed.

Methods for combined PCR amplification and hybridization probing using

A liquid-handling instrument has a worksurface with registration for modular stations to support containers of liquid, pipette apparatus with a pipette tip coupled to a sensing circuit, a robotic translation system for moving the pipette tip, and a control system with an iconic user interface for programming and editing. A gauge block registered on the worksurface provides for calibration using the sensing tip, and register cavities on the worksurface provide for modular stations. There is a wash station fop the pipette tip on the worksurface. An automated laboratory based on the liquid-handling system has heating and cooling and a sealable incubation station as well as a magnetic separation station. Methods are disclosed using the apparatus to convey droplets of liquid, to aspirate with minimum tip contamination, to mix liquids in containers, and to validate the worksurface. A duck-billed closure is disclosed for minimizing evaporation and cross-contamination during processing, and is a part of a container disclosed for storing and transporting liquids.

Automated molecular biology laboratory

Growing community of inventors

Pacifica, CA, United States of America

Paul E Mayrand