High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

Abstract

Background: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation.

Results: SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe.

Conclusions: Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.

Figure 1

Schematic of RCA for SNP identification. An allele specific OCP anneals to the target sequence so that the 5' and 3' arms are adjacent to each other. The 3' terminal nucleotide of the OCP is at the SNP position. If it is complementary to the target, the OCP ends are covalently linked by a DNA ligase to form circles. The circles are then amplified in an ERCA reaction containing two primers, one of which is an Amplifluor containing a fluorophore and a non-fluorescent quencher as indicated. Incorporation of the primer into DNA product opens the primer hairpin giving a fluorescent signal.

Figure 2

Kinetics of the ligation reaction. All reactions contained an oligonucleotide target with the wild type T at the SNP position. Reactions labeled ''Match'' had the corresponding 3' A on the OCP (a). Reactions labeled ''Mismatch'' had T on the OCP 3' end (b). Reactions contained 20 unit Ampligase/reaction (closed circles), 40 unit Ampligase/reaction (open circles), or 80 unit Ampligase/reaction (triangles). OCP was labeled on the 5' end with γ-³²P-dATP and T4 polynucleotide kinase. Ligation was quantified as percent conversion to circular form on a 6% DNA sequencing gel.

Figure 3

Effect of 3' arm length and ligation temperature on allele discrimination. The 3' arm of the matching OCPs formed 12, 15, 20, or 23 basepairs, as indicated, when annealed to the oligonucleotide target. Ligation reaction temperatures were as indicated.

Figure 4

Detection of purified circles with ERCA. A 10-fold serial dilution of preformed circles ranging from 10⁶ to about 1 copy was used as substrate in an ERCA reaction. (a) Real-time profile of the amplification signal obtained with each of the circle dilutions. (b) A standard curve of threshold cycle (C_T) vs. circle number shows that the reaction is linear over six logs of the target.

Figure 5

SNP identification on human genomic DNA. Alu I digested human genomic DNA (100 ng) was used as template for annealing and ligation of the OCP specific for the SNP G1822A. The ligation products were then amplified by ERCA. Three DNA samples representing the three genotypes were each analyzed in two reactions, each reaction containing an OCP/ P1 Amplifluor/ P2 combination corresponding to one of the two possible alleles. (a) Real-time profiles of the ERCA reactions indicate that an amplification signal is obtained only from reactions where the OCP/ P1 Amplifluor/ P2 combination matched the SNP nucleotide on the genomic DNA samples. When the combination OCPinC/ P1inFAM/ P2inC was used with DNA sample homozygous for the G allele, a fluorescent FAM signal was observed (top left panel). No signal was observed with the mismatched OCPocT and the corresponding primer pair P1ocTET/ P2ocT (top right panel). Similarly, when the combination OCPocT/ P1ocTET/ P2ocT was used with DNA sample homozygous for the A allele, only a fluorescent TET signal was observed (middle right panel). No signal was observed with the mismatched OCPinC and the corresponding primer pair P1inFAM/ P2inC (middle left panel). In the case of heterozygous DNA both the OCP/ primer combinations gave a fluorescent signal (bottom panels) (b) The endpoint products of these reactions were resolved on a 2% agarose gel. The characteristic double-stranded ERCA ladder was observed only from those reactions that gave a fluorescent signal in the real-time assay. (c) Scatter plot of the ERCA reaction endpoint fluorescence values obtained by screening a set of 96 different genomic DNA samples using the above assay.

Figure 6

Genotyping of SNP G1822A on 1 ng of genomic DNA in a single-tube reaction. Ligation reactions were performed with 1 ng of Alu I digested genomic DNA in single tubes containing both of the allele-specific OCPs. ERCA reactions included both of the allele-specific P2 Amplifluor primers. Real-time amplification signal was observed only when the OCP/ P2 combination matched the SNP nucleotide on the genomic DNA. Therefore, FAM and TET P2s gave a signal with DNA homozygous for the A and G alleles respectively. DNA heterozygous for the SNP gave an amplification signal with both P2 Amplifluor primers.