Colleen M Davis

US Patent:

7229765, Jun 12, 2007

Filed:

Nov 28, 2001

Appl. No.:

10/432176

Inventors:

Michael Ziman - San Francisco CA, US
Colleen P. Davis - Seattle WA, US

Assignee:

Rosetta Inpharmatics LLC - Seattle WA

International Classification:

C12Q 1/68

US Classification:

435 6

Abstract:

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.

US Patent:

20070255053, Nov 1, 2007

Filed:

May 7, 2007

Appl. No.:

11/745386

Inventors:

Michael Ziman - San Francisco CA, US
Colleen Davis - Seattle WA, US

Assignee:

ROSETTA INPHARMATICS LLC - Seattle WA

International Classification:

C07H 21/04

US Classification:

536025300

Abstract:

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.

US Patent:

20100297728, Nov 25, 2010

Filed:

May 28, 2010

Appl. No.:

12/790234

Inventors:

MICHAEL ZIMAN - SAN FRANCISCO CA, US
COLLEEN P. DAVIS - SEATTLE WA, US

Assignee:

LIFE TECHNOLOGIES CORPORATION - CARLSBAD CA

International Classification:

C12N 9/12

US Classification:

435194

Abstract:

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.

US Patent:

20120208199, Aug 16, 2012

Filed:

Feb 13, 2012

Appl. No.:

13/372320

Inventors:

Michael ZIMAN - San Francisco CA, US
Colleen P. Davis - Seattle WA, US

Assignee:

LIFE TECHNOLOGIES CORPORATION - Carlsbad CA

International Classification:

C12Q 1/68

US Classification:

435 612

Abstract:

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.

US Patent:

20130344491, Dec 26, 2013

Filed:

May 17, 2013

Appl. No.:

13/896872

Inventors:

COLLEEN DAVIS - SEATTLE WA, US

Assignee:

LIFE TECHNOLOGIES CORPORATION - Carlsbad CA

International Classification:

C12N 15/10

US Classification:

435 612, 435 9121

Abstract:

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.