Three types of DNA targets were generated to address various issues surrounding the adaptation of the SNaPshot approach for the quantitative evaluation of C/T (G/A) ratios:

A fragment of the promoter region of the gene encoding human catecholamine O-methyltransferase (COMT) was amplified as follows. 10× PCR Buffer, 2 mM MgCl2, 2.5 mM dNTP, 1 M Betaine, 0.4 mM primers and 1 U of Taq polymerase (New England Biolabs), primers: comtF1 5′-agaccacaggtgcagtcagcacag-3′ and comtR1 5′-caccctatcccagtgttccacccta-3′ at 95°C for 5 min, 30 cycles (94°C for 1 min, 61°C for 1.5 min and 72°C for 1 min), and 72°C for 5 min. CCGG and GCGC sites of the amplicon were subsequently methylated using M-HpaII and M-HhaI, respectively, in two separate fractions. The third fraction of the amplicon was left unmethylated. Both methylated and unmethylated DNA samples were then subjected to bisulfite modification (21 (link)). Briefly, DNA was boiled for 5 min, cooled on ice and denatured for 15 min at 50°C after adding 4 μl of fresh 2 M NaOH in a total reaction volume of 25 μl. Two volumes of 2% LMP agarose in distilled water was added and 10 μl aliquots of this solution was pipetted into cold mineral oil and placed immediately back into dry ice to create beads. The mineral oil was removed and a solution of 1.9 g sodium metabisulfite in 2.5 ml H2O, 720 μl of 2 M NaOH and 500 μl of 1 mM hydroquinone was added. Samples were incubated on ice for 30 min followed by incubation at 50°C for 3.5 h. The agarose beads were washed four times for 15 min with 1 ml TE, two times for 15 min with 0.2 M NaOH, three times for 10 min with 1 ml TE and two times for 15 min with H2O. This was followed by semi-nested PCR using identical reaction and cycling conditions as above with semi-nested primers: BisF1, 5′-gaagggggttatttgtggttagaa-3′, BisF2 5′-gatttttgagtaagattagattaag-3′ and BisR1 5′-aacaaccctaactaccccaa-3′. C (metC in the amplicon) containing templates were mixed with the T (unmethylated C in the amplicon) containing fraction to create a standard curve from 100 to 0% of C signal in increments of 5%: (100% C: 0% T), (95% C: 5% T), (90% C: 10% T), …, (0% C: 100% T). This was done for those templates containing C at M-HpaII sites and M-HhaI sites separately. The M-HpaII sites were interrogated with three forward primers, while three primers for the M-HhaI sites were added as negative controls. In a similar way, M-HhaI sites were interrogated with all six forward primers (three of which were for the M-HpaII sites as negative controls) in one run and the three reverse primers in a second run (Figure 1). The interrogating primers were designed to have a Tm close to 50°C to allow for similar annealing dynamics in the multiplexed reaction. To vary the length of the primers, non-complementary tails were designed on the 5′ end of each primer by repeating the sequence GACT (shown in brackets): at least two sets of the GACT for oligos with the total length <40 nt and by one set of GACT for those >40 nt. The interrogating SNaPshot primers were

5′-agtaagattagattaagaggt-3′, 5′-[gact]1gatatttttatgaggatattt-3′ and 5′-[gact]6ttatggtttgtgtttgttat-3′ for the HpaII sites;

5′-[gact]4ggatattttggttattgttg-3′, 5′-[gact]6ttttgattttattttatttgttg-3′ and 5′-[gact]7agtgtttttttaatttttgtag-3′ for the HhaI sites (direct primers);

5′-ccacaataaatatccac-3′, 5′-[gact]2tataacaaacaaaatacaaaac-3′ and 5′-[gact]3acactacaaaaattaaaaaaac-3′ for the remaining three HhaI sites (reverse primers).

In order to investigate the effects of quantitative G/A and C/T polymorphisms in the SNaPshot primer binding region, sets of oligonucleotides containing variable C/T and G/A were synthesized. Five polymorphic positions were investigated: −2, −5, −10, −15 and −18 (A−2/G−2, A−5/G−5, T−10/C−10, A−15/G−15 and T−18/C−18) upstream of the nucleotide that was interrogated (‘target’ nucleotide, Ntarget) (Figure 2). SNaPshot primers in the experiment were named according to the polymorphic site, while the DNA template itself was named by the nucleotide in the polymorphic position and also the target nucleotide. Therefore, the primer T−2 is fully complementary to the templates A−2Atarget and A−2Gtarget, but not complementary at the −2 position to the templates G−2Atarget and G−2Gtarget. It is evident that the T−2 primer will preferentially bind to (and interrogate) the DNA sequence that contains A−2, in comparison to G−2 at the upstream position. The degree of such bias, however, is unknown as is the impact of the location of the mismatch proximal to the target nucleotide. To elucidate the degree of bias, DNA templates containing an upstream polymorphism, e.g. G−2Gtarget and A−2Atarget, were added in equal amounts which resulted in a polymorphic G/A site at the −2 position. This template mix was tested in two different primer scenarios: first with primer T−2, then with primer C−2. All other polymorphic sites at positions −5, −10, −15 and −18 were analyzed in the same way. For the mismatch bias correction, numerous DNA template combinations with varying percentages of polymorphic nucleotides in the primer binding site were tested using different primer combinations (see Results).

To verify that degenerative SNaPshot primers are able to interrogate numerous polymorphic C/T and A/G containing DNA sequence targets, two types of DNA templates were used:

Six oligonucleotide templates were synthesized with quantitative G/A polymorphisms in different positions of the SNaPshot primer annealing region (Figure 3). The target nucleotide A/G proportions were synthesized to be 50%:50% in each template while the upstream A/G ratios were synthesized according to Figure 3. SNaPshot primers contained a 50%:50% proportion of C/T at degenerative positions corresponding to polymorphic positions in the templates.

Two human genomic DNA samples from brain and placenta were bisulfite modified and subjected to both SNaPshot interrogation and to cloning plus sequencing-based measurement of metC density. The two selected CpG island regions were identified as exhibiting DNA methylation differences according to our microarray-based DNA methylation profiling (A. Schumacher, A. Petronis, et al., unpublished data). These regions are located between 28 and 276 bp upstream of known genes coding for LGALS1 (lectin, galactoside-binding, soluble, 1), otherwise referred to as galectin 1 and humanin, respectively. Three CpG positions were selected for each CpG island and will be referred to as gal1, gal2 and gal3 for galectin 1 and hum1, hum2 and hum3 for humanin (Figure 4).

Bisulfite modification reactions were performed as described above. Target sequences were amplified using fully nested PCR. PCR conditions were as follows: 10× PCR Buffer, 2 mM MgCl2, 2.5 mM dNTP, 0.4 mM primers and 1 U of Taq polymerase. The first PCR was performed using primers, for galectin 1: 22_f1 5′-gtagaatgttaattttgggtagaaataat-3′ plus 22_r1 5′-ctcaaccatcttctctaaacacc-3′; and for humanin: 52_f1 5′-agtttgtattaaggagatttataaggatag-3′ plus 52_r1 5′-aaccaacaaaacacacaaacc-3′. The second (nested) PCR used primers, for galectin 1: 22_f2: 5′-gttattgaggtttagaaaagagaaggtat-3′ plus 22_r2 5′-acttataaacctaactcatcatcaaactat-3′ and for humanin: 52_f2: 5′-aatttagattttgagtttttgaaag-3′ plus 52_r2 5′-aacacaacataacaacaaacaaaac-3′ site. Two successive rounds of touch down PCR were used with the following cycling conditions: 95°C for 3 min, 10 cycles [94°C for 1 min, 60°C for 30 s (minus 1°C/cycle), 72°C for 40 s], 30 cycles of (94°C for 1 min, 50°C for 30 s, 72°C for 40 s) and 72°C for 5 min. The sequences of the interrogating SNaPshot primers were gal1 5′-gttattgggggyggagtt-3′, gal2 5′-[gact]2gaggatgttttygggtagg-3′ and gal3 5′-[gact]4gatyggatygggtgagttt-3′. Primers for humanin were hum1 5′-acagttyggatttttygaaaggggg-3′, hum2 5′-aactcccaatatcrtacratac-3′ and hum3 5′-ygagggtgatagggaag-3′. Amplicons were cloned into the pDrive plasmid (Qiagen) that were used for transformation of DH5-α competent cells. Individual colonies were grown at 37°C for 15 h followed by plasmid purification using the Qiagen Spin Miniprep kit. Sequencing of 12–15 plasmid inserts per template was carried out with M13 reverse primer using ABI Big Dye Terminator kit 3.1. Six CpG positions were investigated using SNaPshot primers individually and five were multiplexed in two groups (gal1, gal2 and gal3, and hum1 and hum3). The differences in length of degenerative primers to be multiplexed were in accordance with the specifications recommended by the manufacturer (ABI). All SNaPshot experiments on bisulfite-modified DNA were repeated in quadruplicate.