Abstract
BackgroundSingle 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.ResultsSNP 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.ConclusionsCompatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.
Highlights
Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses
The OCPs were ∼ 90 nucleotides in length, and are designed such that regions at both the 5' and 3' ends annealed to contiguous segments of the SNP-containing target sequence
Allele specificity of circularization is dependent upon two factors: 1) favorable hybridization of the matching OCP because of the correct base pair formed at the 3' end Recently a new method for SNP detection from genomic DNA based on DNA ligase-mediated single nucleotide discrimination and signal amplification by Rolling Circle Amplification (RCA) has been described [6–10]
Summary
Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, 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. The probe with a 3' base complementary to the SNP is circularized by ligation
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