US 7,465,562 B2Granted

Nucleic acid processing methods, kits and devices

AssigneeBrandeis University

InventorsLawrence J. Wangh, Cristina Hartshorn

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Abstract

Biological samples containing nucleic acids, RNA and DNA, are freed from bound proteins by incubation with a chaotropic agent such as a guanidium salt, and the mixture is readied for further processing by dilution of such agent to a concentration below 0.05 M without physical isolation of RNA and DNA from one another or from other components of the reaction mixture. Methods include such preparation and further processing, such as amplification and detection, which may be performed in a single container. Chaotropic agent may be supplied as a dried reagent adhered to a container. Kits may include such reagents, alone or with amplification reagents.

Claims28

Dependent claims

A single-container method for synthesizing cDNA from RNA in a biological sample using a reverse transcriptase and amplifying and detecting at least one selected sequence of said cDNA, comprising:

a) combining said biological sample with a disruption reagent comprising a chaotropic salt having a concentration of at least 2M that denatures or degrades all proteins to produce a disrupted sample containing RNA freed from bound proteins and inactivating nucleases with no more than twenty-five percent loss of RNA;
b) reducing the concentration of said chaotropic salt in said disrupted sample to less than 0.05M by diluting without washing the disrupted sample with at least one aqueous reagent before adding the reverse transcriptase;
c) incubating the diluted, disrupted sample with reverse transcriptase to transcribe said RNA to cDNA;
d) amplifying at least one selected sequence of said cDNA;
and
e) detecting the at least one amplified cDNA sequence
wherein steps b) through e) are performed without first separating the RNA from degraded proteins or from said chaotropic salt.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 10/557,280Filed 18 May 2004 · us provisional application
Priority: 19 May 2003
Published: US 2006/0275773 A17 Dec 2006
Granted: 16 Dec 2008
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12N 15/10
Recombinant DNA-technology: processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12N
Microorganisms or enzymes
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms

Family

Application60/471,895Us provisional application · filed 19 May 2003
US 2006/0275773 A1Pre-grant publication · published 7 Dec 2006
US 7,465,562 B2This patent

From the US filing history. Foreign family members aren’t listed.

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