Method of isolating nucleic acid from specimens in liquid-based cytology preservatives containing formaldehyde
AssigneeGen-Probe Incorporated
InventorsDeborah C. Jensen, Brett W. Kirkconnell, Timothy J. Wilson
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Abstract
Method, composition, kit and system for isolating amplifiable nucleic acid from specimens preserved in a liquid-based cytology preservative that contains formaldehyde. The technique relies on the use of 2-imidazolidone and a protease enzyme, such as proteinase K, at elevated temperatures. Advantageously, RNA can be isolated and used as a template in nucleic acid amplification reactions.
Claims39
A method of isolating a nucleic acid from processing a specimen that includes a clinical sample disposed in a liquid-based cytology preservative that comprises formaldehyde, the method comprising the steps of:
- (a) combining the specimen with a protease enzyme and a formaldehyde scavenger that is 2-imidazolidone to create a reaction mixture;
- (b) incubating the reaction mixture at an elevated temperature for a period of time sufficient to reverse chemical modifications of nucleic acid that may be contained in the specimen by formaldehyde in the liquid-based cytology preservative;
- and
- (c) isolating a nucleic acid from the reaction mixture after the incubating step.
A method of isolating RNA from a specimen that includes a clinical sample disposed in a liquid-based cytology preservative that comprises formaldehyde, the method comprising the steps of:
- (a) combining the specimen with a protease enzyme and a hydrazine- or hydrazide-containing formaldehyde scavenger;
- (b) incubating the reaction mixture at a temperature in the range of 80° C. to 100° C. for a period of time sufficient to reverse chemical modifications of RNA that may be contained in the specimen by formaldehyde in the liquid-based cytology preservative;
- and
- (c) isolating RNA from the reaction mixture after the incubating step.
A system for processing nucleic acid containing samples preserved in a liquid-based cytology preservative that comprises formaldehyde, the system comprising:
- a programmable controller;
- a pipetting device in communication with the programmable controller;
- a first holder for a reaction vial;
- a second holder for a reagent vial;
- and
- a heating element;
- wherein the programmable controller is configured by software instructions to cause the pipetting device to transfer an aliquot of liquid from the reagent vial to the reaction vial when the reagent vial contains a solution comprising a hydrazine- or hydrazide-containing formaldehyde scavenger;
- proteinase K, EDTA, and a pH buffer, and
- wherein the programmable controller is configured by software instructions to cause the heating element to heat the reaction vial to a temperature in the range of 80° C. to 100° C.
Dependencies are inferred from the claim text.