Abstract
Allele-specific extension reactions (ASERs) use 3′ terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3′ terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3′ terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.
Highlights
Allele-specific extension reactions (ASERs) are used for a broad range of applications, especially in the detection of single nucleotide polymorphisms (SNPs) for medical usage, disease association studies, and routine molecular diagnostics [1,2,3]
We demonstrated the deep impact of optimal probe design and probe length on specific primer extension reactions
Our results clearly demonstrate that length differences of only few base pairs can determine haplotype-specific extraction (HSE) efficiency
Summary
Allele-specific extension reactions (ASERs) are used for a broad range of applications, especially in the detection of single nucleotide polymorphisms (SNPs) for medical usage, disease association studies, and routine molecular diagnostics [1,2,3]. Thermostable Taq DNA polymerase is used in HSE for the extension of an allele-specific probe, followed by 20 minutes of elongation with the incorporation of biotinylated nucleotides and an extraction step with streptadivin-coated magnetic beads (Figure 1). HSE allows the separation of coherent chromosomal fragments or haplotypes, as well as numerous applications for different scientific approaches [8,9,10]. For all applications of ASER, the reliability and specificity of the approach is dependent on the ability of the DNA polymerase to discriminate between the extension of mispaired and canonically paired primers. Because polymerase binds with similar affinities to matched and mismatched primer templates, the non-specific extension of a 39 nucleotide mismatch is often a significant fraction of the overall extension product, which can be controlled with rational probe design [11]
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