Primers, probes sequences, genomic targets, and amplification conditions
Primers, molecular beacon probes, and thermal cycle conditions for symmetric and LATE-PCR amplification of the human HEXA normal and TSD Δ1278 alleles were as described . Purified genomic DNA homozygous and heterozygous for the TSD Δ1278 HEXA and the TSD G269 alleles were obtained from the Coriell Cell Repositories (Camden, NJ; nucleic acid samples NA11852 – homozygous normal for TSD G269, NA03441 – homozygous normal for TSD Δ1278 HEXA, and NA03575 – heterozygous for both TSD Δ1278 and TSD G269). Genomic DNA samples with various genotypes for the rs858521 and rs2270517 SNP site were a kind gift from Dr. Brian Reid (Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA). These samples are also available from the Coriell Cell Repository (nucleic acid samples NA10839 – homozygous CC-, NA10830 – heterozygous CG-, NA07348 – homozygous GG- for rs858521 SNP; nucleic acid samples NA18524 – homozygous CC-, NA18526 – heterozygous CT-, NA18562 – homozygous TT- for rs2270517 SNP).
LATE-PCR primers and ResonSense® probe sequences for TSD G269 locus were:
TSD G269 limiting primer: 5' CGAGGTCATTGAATACGCACGGCTCC 3'
TSD G269 excess primer: 5' TAACAAGCAGAGTCCCTCTGGT 3'
TSD G269 G allele mismatch-tolerant probe: 5' [Cy5]GGGACCAGGTAAGAA [Phos]
Primers and ResonSense® probe sequences for rs858521 SNP site (C/G alleles) located 41.5 Kbp upstream of the p53 gene in human chromosome 17 were:
rs858521 limiting primer: 5' TCCCCAGAGCCCAGCCGGTGTCATTTTC 3'
rs858521 excess primer: 5' CAATCCCTTGACCTGTTGTGGAGAGAA 3'
rs858521 C allele mismatch-tolerant probe: 5' [Cy5] CTTCAGCTCAAACAATA [Phos].
Primers and ResonSense® probe sequences for rs2270517 SNP site (C/T alleles) located downstream of the p53 gene in human chromosome 17 were:
rs2270517 excess primer: 5' GAGGCAGCCCGAGCAATG 3'
rs2270517 limiting primer: 5' GGTCAGCGCCGGGCTGCAAGTGTAGA 3'
rs2270517 C allele mismatch-tolerant probe: 5' [Cy5] AGCGGGTGGTAG [Phos].
Amplification reactions for the TSD G269 and rs858521 amplicons consisted of 1× Platinum Taq Buffer (Invitrogen, Carlsbad, CA), 3 mM MgCl2, 250 nM dNTP, 25 nM (for TSD G269) or 50 nM (for rs858521) limiting primer, 1000 nM corresponding excess primer, 0.24× Syber Gold I (Invitrogen, Carlsbad, CA), 1.25 units Platinum Taq (Invitrogen, Carlsbad, CA), 0.1× Primesafe (for TSD G269; Smiths Detection, Edgewood, MD) or 0.2× Primesafe (for rs858521; Smiths Detection, Edgewood, MD), 2.4 μM of the respective ResonSense® probe, and 100–1000 genome-equivalent of human DNA in a volume of 25 μl. Amplification reactions for the rs2270517 amplicon consisted of 1× Platinum Taq Buffer (Invitrogen, Carlsbad, CA), 3 mM MgCl2, 250 nM dNTP, 50 nM limiting primer, 1000 nM corresponding excess primer, 0.24× SyberGold I (Invitrogen, Carlsbad, CA), 1.25 units Platinum Taq (Invitrogen, Carlsbad, CA), 0.1× Primesafe (Smiths Detection, Edgewood, MD), 2.4 μM rs2270517 ResonSense® probe, and 100 genome-equivalents of human DNA in a volume of 25 μl. Amplification was carried out in an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The thermal cycling profile for TSD G269 amplification was 95°C for 3 min; followed by 15 cycles of 95°C for 10 sec, 65°C for 20 sec, and 72°C for 20 sec; then 30 cycles of 95°C for 10 sec, 65°C for 20 sec., 72°C for 20 sec, 55°C for 20 sec. 40°C for 20 sec with SYBR Gold I fluorescence collection at the 72°C step and Cy5 fluorescence collection at the 55°C and 40°C steps. The thermal cycling profile for rs858521 amplification was 95°C for 3 min; followed by 18 cycles of 95°C for 10 sec, 66°C for 10 sec, and 72°C for 20 sec; then 33 cycles of 95°C for 10 sec, 65°C for 10 sec., 72°C for 20 sec, 55°C for 20 sec. 25°C for 20 sec with SYBR Gold I fluorescence collection at the 72°C step and Cy5 fluorescence collection at the 55°C and 25°C steps. The thermal cycling profile for rs2270517 amplification was 95°C for 3 min; followed by 18 cycles of 95°C for 10 sec, 66°C for 20 sec, and 72°C for 20 sec; then 32 cycles of 95°C for 10 sec, 66°C for 20 sec., 72°C for 20 sec. In the latter case, Cy5 fluorescence signals were acquired at 57°C and 45°C after LATE-PCR amplification for two-temperature endpoint genotyping. Calibration of the ABI 7700 for use of the Cy5 ResonSense® probe and SYBR Gold I dye was performed according to the manufacturer's instructions using 1.25 μM of the G269 probe (for the ResonSense® probe) and 100 ng human genomic DNA stained with 0.24× SYBR Gold I (for SYBR Gold I).
DNA mixtures containing a 2:1 imbalance allele ratio of C and G alleles at the TSD G269 locus were constructed by mixing homozygous normal CC and heterozygous GC TSD G269 DNA samples of matched concentrations at a 1:2 ratio. The concentration of the mixture was then adjusted to 1000 genomes-equivalents/ul (6 ng/ul)
Mismatch-tolerant probe design criteria
It is important that the mismatch-tolerant ResonSense® probe has minimal secondary structure at lower temperatures to prevent the probe from acquiring any double-stranded nature that would bind any SYBR Gold and yield Cy5 fluorescence in the absence of target binding. Typically, the probe is designed to have a melting temperature (Tm) with the perfectly complementary target that is at least 10°C above the Tm of the probe with the mismatched target, and is also at least 5°C below the Tm of the limiting primer. Also, the ResonSense® probes used in this study were labelled at the 5'-end with Cy5 and phosphorylated at the 3' end to prevent them from serving as a primer as described in the original publication . An alternative, simpler design would be to use Cy5-labeled probes labelled at the 3' end instead.
Determination of optimal detection temperatures for Two-Temperature LATE-PCR genotyping
Determination of the optimal upper and lower temperatures for signal acquisition to achieve maximal allele discrimination and detection of total sequence variants with mismatch-tolerant probes respectively was done empirically for each probe-target combination by collecting probe fluorescent signals at 5°C intervals, starting 9°C–10°C below the Tm of the limiting primer down to 25°C. The optimal upper temperature shows the greatest difference between the homozygous and heterozygous genotypes of the interrogated allele, while the most preferred lower temperature shows the least difference between the homozygous forms of the interrogated and the non-interrogated allele (see Figure 3).
Probe signal normalization
Cy5 fluorescent probe signals collected at the various temperatures were exported to Microsoft Excel from the ABI Sequence Detector software built into the ABI 7700 as clipped files. Although the data presented in this paper was exported after baseline correction according to the parameters of the ABI Sequence Detector software, clipped fluorescence values with no baseline correction can be exported as well (this was the case for the analysis shown in Figures 5 and 6 where baseline could not be determined since it is an endpoint assay). For two-temperature genotyping, we calculated for each sample the ratio of Cy5 fluorescence value collected at the upper temperature at each amplification cycle to the Cy5 fluorescent signal collected at the lower temperature for the same sample. For discrimination of heterozygous and DNA mixtures containing 2:1 allele ratios, we substrated from the fluorescence signals collected at the upper and the lower temperatures at the end of the assay for each sample the background fluorescence signals of the probe collected at 70°C prior to the start of the assay for the same sample and then calculated fluorescence ratios. Maximum discrimination of allele imbalances occurs when the fluorescence signals were collected at 52°C instead of 55°C.
The variability in final fluorescent signals among replicate samples was measured by calculating the mean and standard deviation (S.D.). The spread of the fluorescent signals was expressed as the coefficient of variation or CV (i.e., the standard deviation expressed as a percentage of the mean). The assay was considered 99.7% accurate if the distribution of fluorescence signals from homozygous and heterozygous replicate samples were separated by more than three S.D. of the mean of each distribution. Statistical analysis of replicate symmetric PCR samples was done using the two-sample test for independent analysis with unequal variances. The probability value p refers to the null hypothesis (i.e., that the distribution of the means of each dataset are the same). Analysis was performed using Microsoft Excel or an online statistical calculator .