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Anti-freeze protein enhanced nucleic acid amplification

a nucleic acid amplification and anti-freeze technology, applied in the field of anti-freeze protein can solve the problems of particularly problematic non-specific amplification, achieve enhanced nucleic acid amplification, improve enzyme stability, and improve the performance of nucleic acid amplification reactions.

Inactive Publication Date: 2010-02-11
EPPENDORF AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides compositions and methods for improving the performance of nucleic acid amplification reactions. These compositions provide the beneficial effect of improving enzyme stability over the course of numerous freeze / thaw events and significantly enhance the performance of the amplification reaction and improve signal intensity and signal-to-noise ratios in associated detection and quantification methods. The present invention provides amplification reaction mixture compositions comprising an anti-freeze protein, a carrier protein, and optionally a polyol. The methods and compositions improve the stability and performance of nucleic acid amplification reactions and related detection and quantification methods.

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.

Method used

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  • Anti-freeze protein enhanced nucleic acid amplification

Examples

Experimental program
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Effect test

example 1

Anti-Freeze Proteins (AFPs) Enhance Stability of Real Time-PCR Master Mix During Extended Periods of Storage at −20°

[0087]The effects of AFP1 on the freeze / thaw stability of real-time PCR buffers were investigated. Initially, AFP1 was titrated into real time-PCR reactions to determine what concentration of AFP1 did not detrimentally affect amplicon yield. Real-time PCR was performed as described in Eppendorf RealMasterMix Probe + / − ROX (catalog #00320900.525), and products visualized on ethidium bromide stained 1% agarose gels. As shown in FIG. 1, product yields were not detrimentally affected until approximately 100 μg / ml AFP1 was included in the reaction mix. Conversely, the addition of 50 μg / ml AFP1 to the real time-PCR had little or no effect on the reaction yields (compare the lane having 0 AFP1 to the lane having 50 μg / ml AFP1).

[0088]Prior to determining the freeze / thaw capacity of Real-Time PCR buffers in the presence and absence of AFP1, the ability of AFP1 to enhance fresh ...

example 2

pH Modification and Buffer Type have Significant Effect on Optimizing Sorbitol, AFP1 Containing Real-Time PCR Buffer

[0093]Several real time-PCR buffer combinations were tested for signal amplification and for threshold cycle in the presence of a standard amount of both sorbitol and AFP1 / BSA. Both the buffering component and salt were modified to provide different ionic concentrations and pHs. In particular, 25 mM HEPES-KOH with 15 mM KCL pH 8.0; 25 mM TAPS-Tris with 15 mM K GLUTpH 8.0; 25 mM HEPES-Tris with 50 mM KCL pH 8.0; 25 mM Bicine-Tris with 15 mM K GLUT pH 8.7; 25 mM HEPES-KOH with 15 mM K GLUTpH 8.0; 25 mM Bicine-Tris with 50 mM K GLUT pH 8.0; 25 mM TAPS-KOH with 15 mM KCL pH 8.0; 25 mM Bicine-Tris with 15 mM KCL pH 8.0; 25 mM TAPS-Tris with 50 mM KCL pH 8.0; and 25 mM Bicine-Tris with 50 mM KCl pH 8.4 were compared for ability to support highly accurate real time-PCR (each buffer also included 50 μg / ml AFP1, 200 μg / ml BSA and 100 mM sorbitol).

[0094]As shown graphically in F...

example 3

AFP Containing PCR Master Mix Functionally Outperforms Competitor PCR Mix

[0097]An embodiment of a PCR Master Mix prepared in accordance with the present invention was functionally compared to Invitrogen Platinum qPCR Supermix-UDG for its ability to support long term stability within real time-PCR. Long term stability test experimental parameters are shown in Table 5. Cycling parameters included: 95° C. for one minute and forty cycles of 95° C. for twenty seconds, 56° C. for ten seconds and 68° C. for thirty seconds.

TABLE 5Long Term Stability TestComponentStockFinalμl / 50 μl rxn3.5Aliquot 70 μl of each of the RealMaster, 2.5X master mix into a biopure tube2.5X Real Master2.512070Mix + / − ROX(x)Primer-Probe Template Mix For 2.5X MMB2M fwd primer100.2122(μM)B2M rev primer100.2122(μM)B2M FAM probe7.50.15122(μM)gDNA male501122(50 ng / μl)MBGW26572Total30660Add 105 μl (3.5 rxn) of primer-probe-template mix to each aliquot of 2.5X master mix.Aliquot 87.5 μl of each of the competitors 2X master...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N9/96C12P19/34C07H21/04
CPCC12Q1/6848C12Q1/6853C12Q2527/125C12Q2525/121C12Q2525/101
Inventor WESTBERRY, RYAN SMITHPETERS, LARS-ERIKGREENLEE, JESSICA JACLYN
Owner EPPENDORF AG
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