JP-2026076257-A - Enzyme preparations and reaction mixtures for nucleic acid amplification

Abstract

[Problem] To provide a polymerase preparation and reaction mixture that mitigate enzyme reaction inhibition caused by residual detergents. [Solution] A formulation and reaction mixture comprising at least one polymerase, α-cyclodextrin, and polysorbate 20 is disclosed. Related methods for preparing the disclosed formulation and reaction mixture, as well as related kits and methods for nucleic acid amplification, are also disclosed. The formulation and reaction mixture are useful, for example, to mitigate the inhibitory effect of sample extraction washing agents on nucleic acid amplification reactions. The formulation may be a lyophilized polymerase formulation. This formulation may further contain a cryoprotectant. [Selection Diagram] None

Inventors

• ビンセント ジェイ． シャボー
• マーク イー． フィリポウスキー

Assignees

• ジェン－プローブ・インコーポレーテッド

Dates

Publication Date

20260511

Application Date

20260122

Priority Date

20210701

Claims (1)

1. The invention described herein.

Description

Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63/217,560, filed on 1 July 2021, which is incorporated herein by reference in its entirety. Sequence Listing Reference This application includes a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on 8 June 2022, is named "GPR_7910 PC_20220608_Seq_Listing_ST 25" and has a size of 4,974 bytes. Background: Nucleic acid amplification methods are widely used in many bioscience applications, including in vitro detection assays. Methods for amplifying target nucleic acids from biological samples typically involve a sample extraction step (of which there may be one or more) that leaves one or more residual detergents in the extracted sample, which are often inhibitory to polymerase enzymes. The inhibitory effect of such residual detergents presents challenges in the formulation of polymerase enzymes and amplification reaction mixtures. Abstract In one embodiment, the present invention provides an aqueous polymerase formulation. In some embodiments, the formulation generally comprises at least one polymerase, α-cyclodextrin at a concentration of about 10 mg/mL to about 40 mg/mL, polysorbate 20 at a concentration of about 0.002% to about 0.05% (v/v), and at least one buffer. In certain modifications, the formulation further comprises a cryoprotectant. A particularly suitable cryoprotectant is trehalose, which may be present at a concentration of, for example, about 0.2 M to about 0.4 M. In another aspect, the present invention provides a method for preparing a lyophilized polymerase formulation. This method generally comprises (a) providing the aqueous formulation described above, and (b) lyophilizing the aqueous formulation to form a lyophilized polymerase formulation. In another aspect, the present invention provides a lyophilized polymerase formulation prepared by the method described above. In another aspect, the present invention provides a lyophilized polymerase formulation that enables reconstitution into the above-mentioned aqueous formulation. In another aspect, the present invention provides a method for preparing an aqueous polymerase formulation. This method generally includes (a) providing the above-mentioned lyophilized polymerase formulation, and (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation. In another embodiment, the present invention provides a kit having a first sealed container containing the above-described lyophilized polymerase formulation. In some embodiments, the kit further includes a second sealed container containing a diluent. In another embodiment, the present invention provides an amplification reaction mixture. The reaction mixture generally comprises at least one polymerase, α-cyclodextrin at a concentration of about 5 mg/mL to about 20 mg/mL, polysorbate 20 at a concentration of about 0.001% to about 0.025% (v/v), and at least one buffer. In some variations, the reaction mixture further comprises nucleotide triphosphates suitable for nucleic acid amplification. In other non-exclusive embodiments, the reaction mixture comprises at least one amplification oligomer configured to amplify a target region of a target nucleic acid, and/or at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region. In yet another non-exclusive embodiment, the reaction mixture further comprises the target nucleic acid to be amplified and/or at least one cleaning agent, such as an anionic cleaning agent. Particularly preferred anionic cleaning agents include sodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS). In another embodiment, the present invention provides a method for preparing an amplification reaction mixture. In some embodiments, the method generally comprises (a) providing the aqueous polymerase formulation described above, and (b) mixing the aqueous formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid. In some such variations, the method further comprises mixing the mixture produced in step (b) with a sample containing or suspected to contain the target nucleic acid. In other embodiments, the method generally comprises (a) providing the lyophilized polymerase formulation described above, (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation, and (c) mixing the reconstituted formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid. In some such variations, the method further comprises mixing the mixture produced in step (c) with a sample containing or suspected to contain the target nucleic acid. In some of the above embodimen