Promega Corporation

Edward E Pahuski

Leopoldo G Mendoza

Randall L Dimond

James Robert Hartnett

John E Monahan

James W Schumm

Richard A Martinelli

Mark A Maffitt

Steven J Ekenberg

Katharine M Miller

Geoffrey R Hudson

Christopher L Jones

John H Priest

Kathleen K Stebnitz

Lisa Zandt

The present invention entails methods and kits for carrying them out based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (&ldquo;DDRP&rdquo;) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2&prime;-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase.

Nucleic acid amplification with DNA-dependent RNA polymerase activity of RNA replicases

The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (&ldquo;DDRP&rdquo;) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2&prime;-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. The replicase catalyzes synthesis, from the DNA segment, of the RNA, which the replicase then autocatalytically replicates. The invention entails use of the amplification methods in detecting nucleic acid analytes, as in nucleic acid probe hybridization assays. Such assays of the invention include those wherein a nucleic acid analyte is hybridized with one or more nucleic acid probes, which include or are processed to generate a DNA segment which is amplifiable through production from the segment, catalyzed by the DDRP activity of an RNA replicase, of an autocatalytically replicatable RNA, which is autocatalytically replicated to provide an abundance of readily detectable reporter molecules. The invention permits replacement of an RNA, that is autocatalytically replicatable with an RNA replicase and employed as a reporter or label in prior art assays, such as nucleic acid probe hybridization assays or immunoassays, with a nucleic acid comprising a DNA segment with the same base sequence as the RNA. The invention also includes the methods of the invention with Mn , Co , or Zn in the solutions in which the DDRP activity occurs.

The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Q.beta. replicase, has DNA-dependent RNA polymerase ('DDRP') activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2'-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. Assays of the invention include those wherein a nucleic acid analyte is hybridized with one or more nucleic acid probes, which include or are processed to generate a DNA segment which is amplifiable through production from the segment, catalyzed by the DDRP activity of an RNA replicase, of an autocatalytically replicatable RNA, which is autocatalytically replicated to provide an abundance of readily detectable reporter molecules.

Nucleic acid amplification with DNA-dependent RNA polymerase activity of

Processes are provided for removal of cells as cell pellets from liquid milk samples, or from cultures or extracts of other food materials or other materials of biological origin. The concentrated cells in the pellet can be analyzed by various techniques to determine the relative cell count, such as by lysing of the cells followed by measurement of ATP. Nucleic amplification, as by the polymerase chain reaction method, can be carried out using the cellular pellet directly, without need for isolation of nucleic acid from the cells. After the amplification, an assay can be carried out for amplified nucleic acid segment indicative of the presence of cells of interest in the sample. The invention thus provides methods for obtaining cellular components from samples of milk, and cultures or extracts of other materials, including food materials, and for determining relative contamination of milk and such other materials by microorganisms. The invention also provides kits for carrying out its various methods.

Methods and kits for separation, concentration and analysis of cells

Methods and kits for detection of cells in food materials

Growing community of inventors

Marshall, WI, United States of America

Edward E Pahuski