Anti-freeze protein enhanced nucleic acid amplification

Abstract

Methods and compositions are provided for enhanced signal intensity and storage stability of standard nucleic acid amplification buffers, real-time PCR buffers or both. Buffers in accordance with the present invention include anti-freeze protein(s) (AFPs), optionally with a carrier protein, such as BSA.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Patent Application No. 60 / 541,999 titled “METHODS AND COMPOSITIONS TO ENHANCE AMPLIFICATION EFFICIENCY AND SIGNAL,” filed Feb. 4, 2004, which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION[0002]The invention generally relates to methods and compositions for increasing amplification yields in nucleic acid amplification reactions, for enhancing signal intensity and improving the signal to noise ratio in nucleic acid detection and quantification methods, such as real time polymerase chain reactions (real time PCR), and for increasing the stability of amplification enzyme solutions for use in such reactions over repeated freeze / thaw cycles.BACKGROUND OF THE INVENTION[0003]The ability to prepare large amounts of nucleic acid molecules is requisite to a number of protocols in molecular biology, as well as a basic requirement in numerous downstream uses in biotechno...

AI Technical Summary

Benefits of technology

[0010]The present invention provides compositions and methods for improving the performance of nucleic acid amplification reactions, where the reaction typically comprises at least one cycle of a denaturation step, an annealing step, and an extension step. Compositions of the present invention provide the beneficial effect of improving enzyme stability over the course of numerous freeze / thaw events and, more surprisingly, also significantly enhance the performance of the amplification reaction and dramatically increased signal intensity and improve signal to noise ratios in associated detection and quantification methods. The present invention provides amplification reaction mixture compositions comprising an anti-freeze protein (AFP), an AFP combined with a carrier protein such as, e.g., BSA and the like, an AFP combined with a polyol, and an AFP combined with a carrier protein and a polyol.

[0011]In one aspect of the present invention, methods and compositions are provided for improving the stability of an enzyme in an amplification enzyme solution, where the enzyme solution is subjected to at least one freeze / thaw cycle before use in a subsequent amplification reaction. In one embodiment, the enzyme solution comprises AFP, and optionally further comprises a carrier protein and / or a polyol. In a preferred embodiment, the AFP is added to the enzyme solution prior to, or contemporaneous with, the freeze / thaw cycle.

[0012]In another aspect of the present invention, methods and compositions are provided for improving the performance of nucleic acid amplification reactions in general, and related detection and quantification methods in particular, comprising including in the amplification reaction mixture at least one anti-freeze protein optionally in combination with a carrier protein in a zwitterionic buffer. As demonstrated herein, these novel amplification reaction mixtures improve amplicon yield and signal intensity, increase the signal-to-noise ratio, and enhance the overall sensitivity of the reactions.

[0013]In a particular embodiment, improved nucleic acid detection and quantification methods are provided comprising a nucleic acid amplification reaction in which the reaction mixture comprises at least one anti-freeze protein to improve the signal-to-noise ratio of the reaction. Preferred anti-freeze protein(s) have one or more alanine-rich motifs for enhancement of signal and sensitivity. In particularly preferred embodiments, a carrier protein such as, e.g., BSA or other like protein, is included with the anti-freeze protein in the reaction mixture to maximize the signal-to-noise ratio. Surprisingly, the combination of AFP and carrier protein provides a synergistic improvement in the signal-to-noise ratio over AFP alone or carrier protein alone.

[0014]In a preferred embodiment, a polyol is included in amplification reaction mixtures comprising a template nucleic acid having a degree of secondary structure. In a particularly preferred embodiment, the polyol is selected from the group consisting of sorbitol, maltitol, adonitol, arabitol and mannitol. Ancillary materials can also be included with these reaction mixtures, including, but not limited to single-stranded binding protein, n-propyl sulfoxide, and the like, to further facilitate the amplification reaction.

[0015]Additional embodiments of the present invention include compositions for maximizing signal amplification during real time PCR where release of fluorescent signal upon 5′-3′ nucleolytic degradation is anticipated. Preferred compositions and methods of the present invention employ reaction mixtures comprising at least one anti-freeze protein, preferably having one or more alanine rich motifs (for example exhibited by AFP type I), alone or in combination with a carrier protein. In particularly preferred embodiments, the reaction mixtures are prepared with a zwitterionic buffer having a pH of between about 7.9 and about 8.1. In another preferred embodiment, the nucleic acid quantification method is real time PCR and the addition of the anti-freeze protein and carrier protein provides for synergistic signal amplification.

Problems solved by technology

Each real time assay relies upon the release of a detectable signal upon production of a PCR product, and nonspecific amplification is particularly problematic when the starting material is small.

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