Hairpin OCP Design
All oligomers were ordered from Integrated DNA Technologies, Inc.(Coralville, IA). Sequences of oligomers are shown in Table 1. All OCPs were 5' phosphorylated and gel purified. OCPs were designed such that the 3' terminal nucleotide annealed opposite the mutation of interest (Figure 1). The Tm for the 3' arm of the probe annealed to target was designed to be between 60–65°C. The 5' arm of all OCPs annealed to target upstream of the mutation and was designed to have a Tm of approximately 70°C. Oligo 6 (Molecular Biology Insights, Inc., Cascade, CO) was used to calculate Tm values.
To reduce or eliminate the artifactual amplification of unligated OCPs, a hairpin was designed into the 3' end of the OCP, with approximately 10 bases in the stem and 10 in the loop. The hairpin was calculated to be stable at 60°C in the context of the entire OCP using the DNA folding server at Michael Zucker's website http://bioinfo.math.rpi.edu/~zukerm/ OCP sequence was chosen such that the hairpin was the only stable structure in the OCP, with a ΔG of -0.5 to -1.5 kcal/mol.
Generic P1 and P2 design
ERCA primers P1 and P2 were chosen using Oligo 6. These were generic primer sequences, and could be used to amplify any circle sequence. First, 10–20 kb of randomly generated DNA sequence was scanned for primers using settings limiting 3' dimer ΔG to -1.0 kcal/mol, 3' dimer length to 3, 3' terminal stability range -5.5 to -8.0 kcal/mol, GC clamp stability to -10 kcal/mol, no acceptable loop, Tm range 52.1 to 56.2°C. Compatible primers were selected using the multiplex function of Oligo 6. A subset of these compatible primers was utilized as generic P1 and P2 primers (see Table 1). ERCA P1 primers were labeled with FAM (if paired with the normal OCP) or 5-Cy3 (if paired with the mutant OCP) and were RP-HPLC purified. P2 primers were purified by desalting.
Allele Specific ERCA primers
The sequence of these primers was chosen from the target specific 5' and 3'-arms of the OCP, either complementary to the OCP (5'-arm) or the exact sequence of the OCP (3'-arm). These primers annealed to both the normal and mutant OCPs. The sequences of these allele specific primers are shown in Table 1. Allele specific primers were desalted after synthesis.
Q-PNA
Exponential amplification of circularized OCP (see Figure 2) was accomplished using generic primers P1 and P2, along with allele specific primer(s). Each P1 primer contained a universal 13 base 5'-tail (see Table 1) with a fluorescent dye at the 5'-end. Fluorescence was quenched in unincorporated primers by the annealing of a 13-residue PNA molecule, Q-PNA-13 (Applied Biosystems/Boston Probes, Bedford, MA). The sequence of Q-PNA-13 is Ac-X-OO-TGA-TTG-CGA-ATG-A-Lys (Dabcyl). The C-terminal dabcyl was positioned so that it was proximal to the 5'-fluorescent moiety on the DNA reporter, quenching fluorescence when the reporter and Q-PNA-13 were annealed, as shown in Figure 2. After P1 primer is incorporated into ERCA product, Q-PNA is displaced, producing a fluorescent signal.
Genomic DNA
Genomic DNA samples were purchased from Coriell Cell Repositories, Camden, NJ. Human Variation Panel, Caucasian panel of 100, Reference # HD100CAU, was used as non-mutant controls and for general screening purposes. Human samples containing specific mutations were also purchased from Coriell: Factor V Leiden mutation (heterozygous NA14642 and homozygous NA14899); Hemochromatosis C282Y (heterozygous NA14642, homozygous NA14620); and Hemochromatosis H63D (heterozygous NA14641 and homozygous NA13591); Factor II Prothrombin mutation (heterozygous NA16028 and homozygous NA16000). Other patient DNA samples were genotyped using RFLP, and an aliquot of the purified DNA was subsequently used either directly in genotyping reactions, or used after Whole Genome Amplification (WGA).
Whole Genome Amplification (WGA) of genomic DNA samples
For some rare genotypes, it was only possible to obtain a few nanograms of sample, insufficient for more than one genotyping reaction. As a result, the rare samples were subjected to whole genome amplification (WGA), which yielded enough DNA for 200 or more reactions. WGA was performed as described previously [10]. Briefly, 10 ng of each individual genomic sample was amplified into 40 μg of product by adding 99 μl of amplification mix and incubating at 30°C for 6 hours. Quality control assays demonstrating that whole genome amplification was successful were performed for locus 979 and 1004 as described [10], and amplified DNA was used directly in genotyping reactions at the same concentration as the original genomic DNA, typically 200 ng/reaction.
Genotyping reactions
50 ng to 1 μg of either genomic DNA or WGA genomic sample was mixed with OCP (typically 0.5 nM final concentration) and 0.5 unit of Ampligase (Epicentre Technologies, Madison, WI) in 1x Ampligase buffer (Epicentre), for a total volume of 10 μl. The reaction was heated to 95°C for 10 seconds, and cooled to 63–68°C for 5–20 minutes, during which time OCP annealed to genomic target and was circularized by ligase. The reaction was subsequently heated to 95°C for 10 minutes to release ligated circles from genomic DNA. The reaction was cooled to 4°C, and 20 μl ERCA reaction mix was added (typically 16 units BST polymerase (New England Biolabs, Beverly, MA), 6 mM dNTPs, 0.5 μM P1, 0.5 μM P2, 4 μM Q-PNA, 7.5 μM TMAO in 1x ThermoPol Buffer II, all concentrations final). Reactions were incubated at 60°C for 1 hour in an I-Cycler (BioRad, Hercules, CA) reading both FAM and Cy3 channels. Signals typically appeared after 10–20 minutes.
Specific reaction component concentrations
FV ligation reaction conditions
0.5 nM wt OCP and 0.5 nM mut OCP (final concentration); 0.5 U ampligase.
FV ERCA reaction conditions
0.5 μM WT P1; 0.5 μM mutant P1; 0.75 μM P2; 0.75 μM 5'-allele specific primer; 0.75 μM 3'-allele specific primer; 4 μM PNA (final concentrations); 24–32 U DNA polymerase.
FII ligation reaction conditions
0.5 nM wt OCP and 0.5 nM mut OCP (final concentration); 0.5 U ampligase.
FII ERCA reaction conditions
0.5 μM WT P1; 0.7 μM mutant P1; 0.9 μM P2; 0.4 μM 3'-allele specific primer; 4 μM PNA (final concentrations); 16 U DNA polymerase.
Hemochromatosis H63 ligation reaction conditions
0.1 nM wt OCP and 1.2 nM mut OCP (final concentration); 0.5 U ampligase.
Hemochromatosis H63 ERCA reaction conditions
0.4 μM WT P1; 0.5 μM mutant P1; 0.75 μM P2; 0.5 μM 5'-allele specific primer; 0.5 μM 3'-allele specific primer; 4 μM PNA (final concentrations); 16 U DNA polymerase.
Hemochromatosis C282 ligation reaction conditions
0.1 nM wt OCP and 1.2 nM mut OCP (final concentration); 0.5 U ampligase.
Hemochromatosis C282 ERCA reaction conditions
0.4 μM WT P1; 0.5 μM mutant P1; 0.75 μM P2; 0.5 μM 5'-allele specific primer; 0.5 μM 3'-allele specific primer; 4 μM PNA (final concentrations); 16 U DNA polymerase.
Determination of genotype
In this retrospective study, the genotype of each sample was known. Genotyping by RFLP analysis (data not shown) was performed prior to OCP ligation/ ERCA genotyping, and the results of OCP ligation/ ERCA analysis were compared to the RFLP analysis results.
Real Time OCP ligation/ ERCA
The genotype for each sample was determined by amplitude of amplification. The average amplification threshold time for all amplified reactions was determined using the I-Cycler software. Fluorescence traces were normalized using early cycles as a baseline, and a threshold value was determined, typically at 10-fold above the average standard deviation of the baseline values. Threshold cycle for each trace was measured at the point where the trace crossed the threshold value. Threshold cycle values fell into three distinct clusters, one each for homozygous normal, heterozygous, and homozygous mutant. Reactions with times more than 2 standard deviations beyond the mean cluster value were considered failures and repeated. In general, amplification reactions with an increase of less than 300 units were scored as negative. Any reaction with amplification of 300 fluorescence units above baseline or greater was scored as positive. Reactions with fluorescent units between 101–299 above baseline were repeated. Any reaction where signal was baseline for both alleles was repeated.
End Point OCP ligation/ ERCA
ERCA reactions were allowed to incubate at 60°C until the reaction was expected to be complete (30 to 40 minutes, depending on the assay). After the reaction was complete, the results could be read at any time. Some reactions were allowed to remain at room temperature overnight, some were stored at 4°C, some were frozen at -20°C. In each case, the reaction was protected from light to prevent photobleaching of the fluorescent reporters. The genotype of each sample was determined automatically using a modified fuzzy c-means clustering algorithm [3], which groups the data into three genotypes plus a negative control, and assigns a confidence level to each genotyping call from 0 (not in cluster) to 1 (100% certainty that point belongs to cluster).