SNP genotyping by multiplexed solid-phase amplification and fluorescent minisequencing

Abstract

The emerging role of single-nucleotide polymorphisms (SNPs) in clinical association and pharmacogenetic studies has created a need for high-throughput genotyping technologies. We describe a novel method for multiplexed genotyping of SNPs that employs PCR amplification on microspheres. Oligonucleotide PCR primers were designed for each polymorphic locus such that one of the primers contained a recognition site for BbvI (a type IIS restriction enzyme), followed by 11 nucleotides of locus-specific sequence, which reside immediately upstream of the polymorphic site. Following amplification, this configuration allows for any SNP to be exposed by BbvI digestion and interrogated via primer extension, four-color minisequencing. Primers containing 5' acrylamide groups were attached covalently to the solid support through copolymerization into acrylamide beads. Highly multiplexed solid-phase amplification using human genomic DNA was demonstrated with 57 beads in a single reaction. Multiplexed amplification and minisequencing reactions using bead sets representing eight polymorphic loci were carried out with genomic DNA from eight individuals. Sixty-three of 64 genotypes were accurately determined by this method when compared to genotypes determined by restriction-enzyme digestion of PCR products. This method provides an accurate, robust approach toward multiplexed genotyping that may facilitate the use of SNPs in such diverse applications as pharmacogenetics and genome-wide association studies for complex genetic diseases.

Figure 1

(A) Solid-phase amplification. Acrydite-containing primers are incorporated into an acrylamide bead or spot. Multiple cycles of denaturation, annealing, and extension result in a product that is covalently attached to the surface at both ends. (B) Solid-phase amplification from genomic DNA. Digested human genomic DNA was amplified using an acrylamide derivatized polyester sheet containing the INSR RsaI-specific primers. Amplification products were released from the beads with NotI and run on a 10% acrylamide TBE gel. (C) Multiplex, solid-phase PCR with human genomic DNA. Shown is a composite of six 10% Acrylamide TBE gels. Fifty-eight beads (57 beads containing PCR primers and one blank bead) were multiplexed and the resulting radiolabeled products released from the beads are shown in random order. No amplification is observed with beads lacking oligonucleotide primers. Amplification products range in size from ∼70 bp to ∼1300 bp.

Figure 1

(A) Solid-phase amplification. Acrydite-containing primers are incorporated into an acrylamide bead or spot. Multiple cycles of denaturation, annealing, and extension result in a product that is covalently attached to the surface at both ends. (B) Solid-phase amplification from genomic DNA. Digested human genomic DNA was amplified using an acrylamide derivatized polyester sheet containing the INSR RsaI-specific primers. Amplification products were released from the beads with NotI and run on a 10% acrylamide TBE gel. (C) Multiplex, solid-phase PCR with human genomic DNA. Shown is a composite of six 10% Acrylamide TBE gels. Fifty-eight beads (57 beads containing PCR primers and one blank bead) were multiplexed and the resulting radiolabeled products released from the beads are shown in random order. No amplification is observed with beads lacking oligonucleotide primers. Amplification products range in size from ∼70 bp to ∼1300 bp.

Figure 1

(A) Solid-phase amplification. Acrydite-containing primers are incorporated into an acrylamide bead or spot. Multiple cycles of denaturation, annealing, and extension result in a product that is covalently attached to the surface at both ends. (B) Solid-phase amplification from genomic DNA. Digested human genomic DNA was amplified using an acrylamide derivatized polyester sheet containing the INSR RsaI-specific primers. Amplification products were released from the beads with NotI and run on a 10% acrylamide TBE gel. (C) Multiplex, solid-phase PCR with human genomic DNA. Shown is a composite of six 10% Acrylamide TBE gels. Fifty-eight beads (57 beads containing PCR primers and one blank bead) were multiplexed and the resulting radiolabeled products released from the beads are shown in random order. No amplification is observed with beads lacking oligonucleotide primers. Amplification products range in size from ∼70 bp to ∼1300 bp.

Figure 2

Single-nucleotide polymorphism (SNP) minisequencing. (A) Diagram of the components and products of solid-phase amplification and SNP minisequencing using 5HT_2A-specific primers. The polymorphic site (C/T) is shown in parentheses. The fluorescently labeled nucleotides (G* or A*) are depicted following BbvI digestion and nucleotide incorporation. (B) Fluorescent image of 10% acrylamide gel with single-color FAM-ddNTP minisequencing of 5-HT_2A solid-phase PCR product. (C) Sybr green I staining of same gel from panel B. (D) Four-color minisequencing of 5HT_2A locus.

Figure 2

Single-nucleotide polymorphism (SNP) minisequencing. (A) Diagram of the components and products of solid-phase amplification and SNP minisequencing using 5HT_2A-specific primers. The polymorphic site (C/T) is shown in parentheses. The fluorescently labeled nucleotides (G* or A*) are depicted following BbvI digestion and nucleotide incorporation. (B) Fluorescent image of 10% acrylamide gel with single-color FAM-ddNTP minisequencing of 5-HT_2A solid-phase PCR product. (C) Sybr green I staining of same gel from panel B. (D) Four-color minisequencing of 5HT_2A locus.

Figure 2

Single-nucleotide polymorphism (SNP) minisequencing. (A) Diagram of the components and products of solid-phase amplification and SNP minisequencing using 5HT_2A-specific primers. The polymorphic site (C/T) is shown in parentheses. The fluorescently labeled nucleotides (G* or A*) are depicted following BbvI digestion and nucleotide incorporation. (B) Fluorescent image of 10% acrylamide gel with single-color FAM-ddNTP minisequencing of 5-HT_2A solid-phase PCR product. (C) Sybr green I staining of same gel from panel B. (D) Four-color minisequencing of 5HT_2A locus.

Figure 3

(A) An image of released solid-phase PCR/four-color minisequencing products separated on a 6%, 8M urea acrylamide gel. Lanes 1–8 contain products from eight unrelated individuals. Lane 9 is an internal reference standard, and lanes labeled MW contain the GENESCAN-500 ROX size standards. The identity of each amplified locus is shown on the left, and the sizes of the ROX-labeled standards are shown on the right. (B) Electropherograms for representative genotypes of each locus are shown. The Y axis denotes relative fluorescent intensity, and the X axis denotes scan number. The numerical genotype designations are indicated in Table 1.

Figure 3

(A) An image of released solid-phase PCR/four-color minisequencing products separated on a 6%, 8M urea acrylamide gel. Lanes 1–8 contain products from eight unrelated individuals. Lane 9 is an internal reference standard, and lanes labeled MW contain the GENESCAN-500 ROX size standards. The identity of each amplified locus is shown on the left, and the sizes of the ROX-labeled standards are shown on the right. (B) Electropherograms for representative genotypes of each locus are shown. The Y axis denotes relative fluorescent intensity, and the X axis denotes scan number. The numerical genotype designations are indicated in Table 1.