Allele-specific primer extension

Pastinen T, Raitio M, Lindroos K, Tainola P, Peltonen L, Syvanen AC. A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays. Genome Res 2000 10 1031-1042. 

O Meara D, et al. SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays. Nucleic Acids Res 2002 30(15) e75. 

DASH, dynamic allele-specific hybridization AS-PCR, allele-specific polymerase chain reaction AS-PE, allele-specific primer extension APEX, arrayed primer extension FP-TDI, fluorescence polarization template directed dye terminator incorporation MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry OLA, oligonucleotide ligation assay RCA, ... 

SNP genotyping by MALDI-TOF MS takes advantage of mass differences between allele-specific primer extension products. At present, three related assays are used, the PROBE— primer oligo base extension assay, which was further developed to the MassExtend assay by SEQUENOM— the PinPoint, and the GOOD assay (8). A representative scheme is depicted in Fig. 1. 

Figure 3 The MassEXTEND reaction. Following PCR amplification of the locus of interest, a primer-extension reaction is performed using an extend primer (ME) that is designed to anneal next to the SNP. The key feature of this scheme is the use of a terminator mixture that yields extension products that differ in length in an allele-specific manner, thus creating mass separations between alleles equal to the mass of a nucleotide. In this example, a normal dG base is used along with ddA, ddC and ddT. For the A allele (A), a ddA is incorporated, extending the primer to 20 mer. For the G allele (G), the SNP calls for incorporation of the normal dG residue prior to incorporation of a ddT, extending the 19-mer primer to 21-mer.

Many other applications have been carried out using semi-quantitative primer extension based MALDI-TOF MS analysis with unknown analyte concentrations. One example involves the quantification of differential allele-specific expression. Medelian inheritance of allele-specific expression has been shown in humans (40). Allele-specific expression is of great importance for the identification of genetic components, which lead to population... 

Allele-specific differences between regulatory polymorphisms associated with the ability of RNA polymerase II to bind and assemble its transcription complex at the start site of transcription for several eukaryotic promoters has also been measured using MALDI-TOF MS coupled with primer extension (42). This technique provides a powerful tool for identifying important regulatory SNPs and haplotypes in vivo. 

Essentially all genotyping approaches used to date fall into one of four categories allele-specific hybridization, primer extension, oligonucleotide ligation, or invasive cleavage. Figure 5.7-7 illustrates these different approaches. We will first... 

Allele-specific quantification can be achieved using a number of different approaches of which primer extension with detection by mass spectrometry will be described here (see Note 13). A number of steps are involved which potentially introduce variance into the experiment for this reason duplication should be included at each step (see Note 14 [1132]). 

Although a number of quantitative assays are available for allele frequency estimation, most are difficult and expensive to develop or implement. In this chapter, we describe the most versatile and least expensive assays for allele frequency estimation, namely, the template-directed dye-terminator incorporation assay with fluorescence quenching detection (the FQ-TDI assay [3]). The FQ-TDI assay is a real-time homogeneous primer extension assay based on two principles, namely, that deoxyribonucleic acid (DNA) polymerase catalyzes the allele-specific incorporation of a dye-terminator at the polymorphic site and that the fluorescence intensities of a fluorescent dye decreases significantly when it is incorporated into primers. 

Allele-specific expression levels can be assessed in two ways. Expression of mRNA and cDNA with different alleles at heterozygote SNPs is assessed using oligonucleotides and TaqMan/real-time PCR 5 nuclease assays (ABI, Foster City, CA) or the primer extension method (SnapShot, ABI, Foster City, CA and MALDI TOF, Sequenome). 

A primer extension reaction is used to generate allele-specific products. A primer is chosen upstream of the SNP that is to be genotyped. Primers can be placed on either strand of the PCR product. They are synthesised with functionalities that will result in the final products being H-1 or -1 in net charge. Primers with chemical modifications are shown in figure 3. 

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