Nucleic acid amplification in the presence of modified randomers

Abstract

The present invention is directed to a composition comprising a DNA Polymerase which is preferably thermostable, Deoxynucleotides, at least one primer oligonucleotide or a pair of amplification primers, and randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety Such a composition is specifically useful for performing hot start PCR.

Description

RELATED APPLICATIONS[0001]This application claims priority to EP 08005100.6 filed Mar. 19, 2008.FIELD OF THE INVENTION[0002]The present invention relates to the field of template dependent polymerase catalyzed primer extension reactions. In particular, the invention provides a new method for nucleic acid amplification by means of performing a polymerase chain reaction (PCR). More precisely, the present invention provides a new method for performing a hot start PCR characterized in that unspecific primer dimer amplification is avoided.BACKGROUND[0003]A major problem with nucleic acid amplification and more especially with PCR is the generation of unspecific amplification products. In many cases, this is due to an unspecific oligonucleotide priming and subsequent primer extension event prior to the actual thermocycling procedure itself, since thermostable DNA polymerases are also moderately active at ambient temperature. For example, amplification products due to eventually by chance ...

AI Technical Summary

Benefits of technology

[0091]One major advantage of the present invention is the ease of use and the short activation time to eliminate the inhibition of the polymerase at low temperatures. Simply, a randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety needs to be added to a PCR reaction set up. During PCR thermocycling the denaturation time prior to the first cycle which is usually required to separate double stranded DNA templates into single strands is sufficient to eliminate the interaction between the conjugated randomers and the PCR primers.

[0092]Furthermore, random hexamers can be synthesized according to standard phosphoramidate chemistry methods which are well established in the art. Moreover, also 5′ modifications can be introduced into the oligonucleotide using Phosphoramidate chemistry with a respectively modified terminal Phosphoramidate according to standard methods very easily. Thus the production costs for the inventive PCR additive are fairly low as compared to other hot start solutions.

[0093]In addition, the inventive methods, compositions and kits can be generically used for any kind of primer extension, reverse transcription or PCR amplification, irrespective of what specific target nucleic acid sequence shall be prepared, amplified, detected, or analyzed.BRIEF DESCRIPTION OF THE FIGURES

[0094]FIG. 1 Amplification of genomic DNA in the presence of Pyrene-capped hexamers according to

Problems solved by technology

A major problem with nucleic acid amplification and more especially with PCR is the generation of unspecific amplification products.

In many cases, this is due to an unspecific oligonucleotide priming and subsequent primer extension event prior to the actual thermocycling procedure itself, since thermostable DNA polymerases are also moderately active at ambient temperature.

Both methods, however are time consuming and inconvenient to perform.

Therefore, the reproducibility and quality of chemically modified enzyme preparations may vary and can hardly be controlled.

However, the N-terminal truncated cold sensitive mutant form requires low salt buffer conditions, has a lower processivity as compared to the wild type enzyme and thus can only be used for the amplification of short target nucleic acids.

Moreover, since the truncated form lacks 5′-3′ exonuclease activity, it can not be used for real time PCR experiments based on the TaqMan detection format.

However, it is not known, to which extent the excess of competitor DNA influences the yield of the nucleic acid amplification reaction.

Due to the selection process, however, all so far available Aptamers can only be used in combination with one particular species of DNA polymerase.

This leads to a substantial time consuming prolongation of the amplification as a whole, especially if protocols for rapid thermocycling are applied (WO 97 / 46706).

Yet, digestion of the unspecific primers dependent on the duration of the preincubation time may lead to a substantial and uncontrolled decrease in primer concentration, which in turn may affect the amplification reaction itself.

However, it is major drawback of the disclosed alternatives that for each PCR reaction, modified primers are required, which lead to increased requirements regarding increase the cost for each individual assay.

This association is disrupted when heating during PCR.

Yet, the inhibitor tends to reduce the obtainable product yield, when primers with lower annealing temperatures are used.

On the other hand, such a solution does not allow the preparation of mastermixes containing all reagents except primer and target nucleic acid which are necessary to perform a nucleic acid amplification reaction.

As a consequence, inter-assay data reproducibility and data comparisons are complicated.

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