Single nucleotide polymorphisms (SNPs) are highly abundant in the genome and especially useful in the search for disease susceptibility genes via population-based association or linkage studies. Therefore, there is a strong need for high throughput and reliable methodologies to assess the SNP genotypes. Despite an unambiguous result of an SNP analysis, with the use of a commercial kit based on primer extension, subsequent sequencing analysis revealed that a proportion of the genotypes was not correctly assessed. The problem we have encountered may originate from specific structures in the genomic DNA sequence, rather than being a methodological problem.

Single nucleotide polymorphism (SNP) is the most common variation of the human genome with more than 9 million reported in public databases (1,2). Extensive studies are being made to characterize single SNPs, or haplotypes containing multiple SNPs, which can be used as prognostic and predictive disease markers or as tools to locate new disease genes.

A number of techniques to establish the genotype of a known SNP are available, as reviewed in Reference 3,. The choice of methodology is dependent on available technology platforms and whether the analysis demands a high or low throughput.

The SNaPshot Multiplex kit from Applied Biosystems (Foster City, CA, USA), based upon the primer extension principle, is widely used to assess multiple genotypes in population-based analyses. The output is in the range of 1640–41,280 genotypes per day and depends on the available type of electrophoresis instrument and type of polymer. Genetic analyzer instruments and analysis software are available to most laboratories, which is one reason why the SNaPshot method is straightforward and affordable to apply. The final results are fast and easy to interpret via the GenoTyper or GeneMapper software (Applied Biosystems), which further allows post-processing of the data with other software tools.

In the search for new cancer susceptibility genes and risk markers, we have established the SNP genotypes in different target genes. The SNaPshot Multiplex kit was used to assess the genotypes. When the results were confirmed by sequencing, we discovered that certain SNPs were not correctly interpreted, despite an unambiguous SNaPshot result.

The following procedure was used: Included in the study were 199 patients diagnosed with primary breast cancer and 512 healthy Danish medical students. The group of patients is described in Reference 4,. DNA was extracted from peripheral blood and purified by a modified salt precipitation procedure from each individual (5).

Sixteen SNPs were genotyped spanning the RNase L (RNASEL) and the regulator of G-protein signaling 16 (RGS16) genes, as well as seven SNPs within the 2′–5′ oligoadenylate synthetase 1 (OAS1) gene. The protocol given below is general, but the annealing temperatures are specific for the SNPs: rs3738579 (RNASEL), rs2660 (OAS1), rs1051042 (OAS1), and rs10774671 (OAS1).

The manufacturer's protocol was followed. In principle, a primer is designed to anneal to the target DNA just 5′ to the polymorphic nucleotide. The primers can complement either the forward or the reverse DNA strand. In a multiplex reaction a tail of tetramers (GACT) is linked to the 5′ end to ensure different lengths of the primers and separation during electrophoresis.

The criteria used for SNP primer design was in accordance with the SNaPshot protocol: no hairpin structures, no self-or cross-annealing, ∼50% GC content, T_m between 55° and 60°C, and 18–22 bp in length excluding the GACT tail. The location of the primer is restricted to the area flanking the SNP on either of the two DNA strands.

The test DNA fragments, containing one or multiple SNPs, are PCR-amplified using 20 ng of genomic DNA, 1 × NH4 buffer (supplied with the Taq polymerase), 5 pmol of each primer (10 pmol for amplification of the OAS1 fragment), 250 µmol dNTP, 0.5 U Taq polymerase (Ampliqon, Bie and Berntsen, Herlev, Denmark) and 1 M betaine (OAS1) in a final volume of 20 µl. Primers are described in (Table 1).

The PCR amplification of RNASEL exon 2 (rs3738579) consisted of an initial denaturing step of: 40 s at 96°C, followed by 34 cycles comprising 35 s at 93°C, 40 s at 56°C and 40 s at 72°C, and terminated by 6 min at 72°C. PCR amplification of fragments from OAS1 exons 3 and 7 consisted of an initial denaturing step for 1 min at 95°C followed by 32 cycles comprising 30 s at 95°C, 30 s at 60°C and 90 s at 72°C, and terminated by 7 min at 72°C.

The amplification products were treated enzymatically to remove unincorporated deoxynucleotides (dNTPs) and primers by adding 3.3 U shrimp alkaline phosphatase (SAP; Roche Diagnostics, Basel, Switzerland) and 1.34 U exonuclease 1 (Exo1; Medinova, Glostrup, Denmark) to a 10-µl PCR product, and incubating it for 75 min at 37°C followed by 15 min at 75°C to inactivate the enzymes.

For the primer extension, 2 pmol of the specific SNP primer and 3.0 µl SNaPshot mix were added to 6 µl of the enzymatically-treated PCR product in a final volume of 10 µl. Primer extension was performed in 25 cycles comprising 10 s at 96°C, 5 s at 50°C, and 30 s at 60°C. The product was left at 4°C for further processing by adding 1 U SAP and incubating at 37°C for 75 min followed by 15 min at 75°C.