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Convenient single-nucleotide polymorphism typing from whole blood by probe extension and bioluminescence detection.

The information obtained on sequence variations in the genome (1)(2) could make it possible for clinical science, based on molecular biology, to provide patient-specific medical care based on a patient’s genotype. Of these variations, single-nucleotide polymorphisms (SNPs) are the most common, and they are gradually being revealed as associated with phenotypes related to the onset or severity of diseases and to the effects of drugs. Methods for genotyping SNPs are becoming increasingly important for diagnosing diseases and determining an appropriate treatment for each patient.

We recently reported that SNP types can be determined by a new genotyping method based on the bioluminescence detection of the pyrophosphate produced in a specific probe-extension reaction. That method, called bioluminometric assay coupled with modified probe extension reactions (BAMPER) (3), requires that the single-stranded PCR products used as templates for probe-extension reactions be purified before the reaction by use of streptavidin-conjugated magnetic beads to remove the remaining PCR primers and substrates, which is a time-consuming and labor-intensive process. We also reported a BAMPER-based SNP-typing method that uses the PCR products directly and does not require single-stranded DNA (4). Before the SNP-typing probe-extension reaction, exonuclease I and shrimp alkaline phosphatase are used to remove the PCR primers, the residual deoxynucleotide triphosphates, and the residual pyrophosphate produced during the PCR reaction (5) to minimize the background intensity in the subsequent luciferin-luciferase assay. In this report we introduce a simplified and improved BAMPER method that can be used for SNP genotyping of whole blood drawn from individuals as well as for genotyping of purified genomic DNA by use blood dried on paper. The process flow for our method is shown schematically in Fig. 1 of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue8/.

The procedure for our novel BAMPER …