We have demonstrated a solution-based, efficient, homogeneous and robust assay for genotyping SNPs directly from human genomic DNA utilizing ligation of open circle probes and rolling circle amplification. The design of the assay is fairly straightforward and the assay can be carried out in three hours (30 min for the denaturation / annealing / ligation reaction and 2 1/2 h for the amplification reaction) in a 96-well format. SpecifiCity of the OCP to its target sequence is achieved by the complementarity of the two ends of the OCP to the target and the requirement of these ends to be adjacent for ligation. Single nucleotide discrimination is achieved at the ligation step by the use of the thermostable DNA ligase, Ampligase, which has a high affinity for a perfectly matched substrate at the 3' end of a DNA molecule . We were able to enhance allele discrimination at the ligation step by designing the OCP such that the 5' complementary region is firmly hybridized to the target sequence whereas the 3' region is in equilibrium with its target at the ligation temperature . This would result in increased specifiCity since the correctly matched OCP will have a greater chance to act as substrate for the ligase. It has been previously reported that 3'-T/G and 3' G/T mismatches are not good substrates for single nucleotide discrimination . However, we found that the G1822A SNP, which would result in a 3'-T/G mismatch, was efficient for allele discrimination. The ability of this assay to genotype any SNP regardless of the base pair involved is an important advantage over assays based on primer extension such as PCR.
Allele discrimination achieved at the ligation step results in small circular DNA molecules topologically linked to the target DNA strand. These DNA circles are amplified in the powerful homogeneous ERCA reaction capable of 1012-fold signal amplification , similar to that achieved with PCR technology, the current gold standard in genetic analysis and quantitation. However, PCR involves exponential target amplification, thereby increasing the risk of amplicon cross-contamination. Even though this shortcoming can be overcome, it increases the cost and complexity of the assay, making it less attractive for high-throughput analysis. Since ERCA is a signal (circle) amplification method, it does not have the problems associated with PCR. In addition, ERCA is an isothermal reaction and the reaction endpoint can be used as the assay readout. Even though the present study was conducted on a real-time ABI 7700 Sequence Detector instrument the strategy can be easily adapted for a simple fluorescence plate reader coupled to an adjustable heating block. These properties make it an ideal system for high-throughput analysis.
The assay was tested for 10 SNPS on two sets of 96 different DNA samples (Table II). Results were compared to the known genotypes that we determined by RFLP or single nucleotide sequencing reactions. The assay had an average genotyping accuracy of 93% when samples were screened initially. When the mis-scored samples were repeated in triplicate, the genotyping accuracy jumped to over 99%. The majority of the mis-scoring involved a homozygous sample being called heterozygous, i.e., an amplification signal was observed with both sets of OCP/primer combinations. A DNA sample homozygous for one allele was never genotyped as homozygous for the other allele. This implied that there is a low frequency of DNA synthesis artifacts resulting in a fluorescence signal.
Indeed when these reactions were analyzed on an agarose gel, the size of the characteristic ERCA DNA ladder was different from that obtained with amplification of the OCP (data not shown). We have used abasic residues at the 5' ends of P2 primers in order to reduce these artifacts. Other nucleotide modifications that have been reported to reduce primer-dimer formation will be tested to improve the accuracy of SNP genotyping. In addition, reagents that have been shown to reduce primer-dimer formation in PCR will be tested in the ERCA reaction.
Background signal is sometimes related to the amount of OCP used in the assay. For each SNP, the optimal OCP concentration needs to be determined before screening. Excess OCP concentrations result in an increase in non-specific fluorescence signal, therefore lowering the accuracy rate of genotyping. Potentially, un-ligated OCP can act as template or primer giving rise to a low level of non-specific DNA synthesis and subsequent fluorescent signal. We are currently developing a modified OCP design to overcome the need for concentration optimization. In this design, the 3' region of the OCP forms a stable hairpin-loop structure and opens up only to anneal to it target sequence (O. Alsmadi, unpublished). As with molecular beacons, this may also improve target specifiCity [19, 20]. Any unused OCP is self primed and extended to form an inert double stranded molecule, thus eliminating OCP as a source for non-specific amplification. Initial experiments with this design have been encouraging.