Covalent modification of primers improves PCR amplification specificity and yield.

Amar P Gupta,Karen K Y Young,Nancy J Schoenbrunner,Stephen G Will

doi:10.1093/biomethods/bpx011

Amar P Gupta, Karen K Y Young + Show 2 more

Open Access

https://doi.org/10.1093/biomethods/bpx011

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Abstract

We report a method for covalent modification of primers that enhances the specificity of PCR and increases the yield of specific amplification products at the end of PCR. The introduction of thermally stable covalent modifications, such as alkyl groups to the exocyclic amines of deoxyadenosine or cytosine residues at the 3′-ends of primers results in enhanced specificity of reactions. This higher specificity can result in greater sensitivity of detection by reducing competition with non-productive reactions. The reduction in the amplification of unintended byproducts is most apparent when both primers are modified at their respective 3′-ends. The TMs of such modified primers are only slightly affected by the inclusion of these modifiers. The principal mode of action is believed to be driven by the poor enzyme extension of substrates with closely juxtaposed bulky alkyl groups, such as would result from the replication of primer dimer artifact.

Highlights

The broad application of the PCR process in many areas of molecular biology and to in vitro diagnostic testing is a powerful testament to the specificity and sensitivity of the method
We report a method for covalent modification of primers that enhances the specificity of PCR and increases the yield of specific amplification products at the end of PCR
We have developed an alternative method for specificity improvement using chemically stable primer modifications as a convenient and broadly applicable solution to this problem

Summary

Introduction

The broad application of the PCR process in many areas of molecular biology and to in vitro diagnostic testing is a powerful testament to the specificity and sensitivity of the method. There are circumstances where the specificity of the PCR process is compromised through the generation of nonspecific artifacts, including primer dimer and other forms of non-specific amplification products, which may limit the ability to sensitively amplify and detect specific sequences These circumstances include high concentration of total nucleic acids, high concentrations of oligonucleotides, low thermal stringency and partial homology of the primers to non-target sequences. The initiation of non-specific amplification is believed to occur during low temperature PCR setup, where there is low but sufficient polymerase activity to extend a primer which is bound weakly or transiently to an unintended, partially complementary templating sequence This templating sequence could be from the biological sample, e.g. human DNA or from the PCR oligonucleotides themselves.

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