Competitive enzymatic reaction to control allele-specific extensions

Abstract

Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian,A., Gharizadeh,B., O'Meara,D., Odeberg,J. and Lundeberg,J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 5' end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.

Figure 1

Schematic drawing of the PrASE procedures. A nested multiplex PCR amplification is performed with biotinylated inner PCR primers, generating short PCR products. The biotinylated PCR products are immobilized to streptavidin-coated magnetic beads, and ssDNA is generated by alkali elution of the non-biotinylated strand followed by annealing of ASE primers to the immobilized DNA strands. A washing step, where unannealed extension primers are removed, is then followed by PrASE with Cy5-labeled dCTPs and dUTPs. The last steps involve removal of unreacted nucleotides and PrASE enzymes, release of PrASE products by alkali elution and hybridization to tag microarrays. All procedures except the last step of hybridization to tag arrays are automated.

Figure 2

The effect of a protease on extension length. Four synthetic templates have been analyzed by conventional ASE and PrASE with three different amounts of Proteinase K (20, 40 and 80 μg). The templates only contain one G-nucleotide, at different distances downstream of the extension primer, and differ from each other only by the G position. The primers are then extended with Cy5 labeled dCTPs together with native dGTPs, dATPs and dUTPs, generating a signal only if the incorporated nucleotides cover the G-nucleotide position. The fluorescent signal obtained by ASE (0 μg Proteinase K), where the extension is not hindered, has been used to normalize the fluorescence signals acquired by the PrASE reactions. Note that the fluorescent signals from the extended primers are normalized to the signals from the ASE reactions (black bars). The standard deviations are based on analysis of nine data points.

Figure 3

The effect of removal of unannealed extension primers on signal intensities on the arrays. Six individuals have been analyzed by PrASE on tag arrays both with and without the washing away of excess primers. The different individuals in this analysis are indicated on the x-axis. The y-axis shows an average of total signal intensities (for 13 SNPs) obtained for each individual.

Figure 4

The effect of multiplexing on the sensitivity of PrASE. The logarithmic value of the total signal intensity for each SNP (y-axis) is plotted toward the number of SNPs, i.e. the degree of multiplexing in the PrASE reaction. A total of 12 different PrASE reactions were performed, from a simplex to a 12-plex, where SNP 1 has been analyzed in all 12 reactions down to SNP 12, which only has been analyzed in the 12-plex. To achieve the different degrees of multiplexing, simplex PCR products for each of the 12 SNPs were pooled.

Figure 5

Array image for one individual analyzed by both PrASE (left) and ASE (right). The 13 SNPs are in triplicates, where in the first replicate the SNP positions are marked with white squares in the order of SNP 1 to 13 from upper left to lower right corner. In the second replicate, the SNPs with similar results in both PrASE and ASE are marked (white ovals), while the third replicate indicates conflicting results between PrASE and ASE (marked with white ovals). Note that all spotted tags (48) were not used in this assay (indicated with white slashed boxes).

Figure 6

Genotyping results for PrASE (blue upper panels) and ASE (red lower panels). The 36 analyzed samples are visualized in 13 cluster diagrams for each SNP. The x-axes represent allelic fractions that are calculated by the equation spot1/(spot1 + spot2), where spot1 and spot2 correspond to fluorescent signal intensity from primer extension of the first and the second allele, respectively. The y-axes represent logarithmic value of the total fluorescent signal intensity. Circles indicate the different genotypes, where samples scored as heterozygous are situated in the middle circle with an optimal allelic fraction close to 0.5. Homozygous samples for the first allele and the second allele are located in the circles with allelic fractions close to 1 and 0, respectively. Note that, for SNP 10, the scoring with ASE is impossible, while PrASE generates distinct clusters.