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Inexpensive, high-throughput genotyping methods are needed for analyzing human genetic variations. We have successfully applied the regular bioluminometric assay coupled with modified primer extension reactions (BAMPER) method to single-nucleotide polymorphism (SNP) typing as well as the allele frequency determination for various SNPs. This method includes the production of single-strand target DNA from a genome and a primer extension reaction coupled with inorganic pyrophosphate (PPi) detection by a bioluminometric assay. It is an efficient way to get accurate allele frequencies for various SNPs, while single-strand DNA preparation is labor intensive. The procedure can be simplified in the typing of SNPs. We demonstrate that a modified BAMPER method in which we need not prepare a single-strand DNA can be carried out in one tube. A PCR product is directly used as a template for SNP typing in the new BAMPER method. Generally, tremendous amounts of PPi are produced in a PCR process, as well as many residual dNTPs, and residual PCR primers remain in the PCR products, which cause a large background signal in a bioluminometric assay. Here, shrimp alkaline phosphatase (SAP) and E. coli exonuclease I were used to degrade these components prior to BAMPER detection. The specific primer extension reactions in BAMPER were carried out under thermocycle conditions. The primers were extended to produce large amounts of PPi only when their bases at 3'-termini were complementary to the target. The extension products, PPis, were converted to ATP to be analyzed using the luciferin-luciferase detection system. We successfully demonstrated that PCR products can be directly genotyped by BAMPER in one tube for SNPs with various GC contents. As all reactions can be carried out in a single tube, the method will be useful for realizing a fully automated genotyping system.

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precipitation step, which is identical in both methods. In contrast, in the MS method, such loss of analyte during sample preparation is fully compensated by the highly accurate principle of ID.The ID-LC-TMS method described here is proposed for evaluation as a reference method for the quantification of 25-hydroxyvitamin D 3 . The semiautomated sample preparation protocol and excellent practicability of LC-TMS allow the method to be used with large validation series, but the method is also applicable in routine laboratory settings. A multicenter validation of the method is currently planned with the goal of implementing an international reference system for 25-hydroxyvitamin D 3 measurement. 3 Hitachi Science Systems Ltd., Tokyo, Japan; 4 Naka Division, Hitachi High-Technologies Corporation, Ibaraki, Japan;5 Department of Cardiovascular Medicine, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan; * address correspondence to this author at: Advanced Research Laboratory, Hitachi Ltd., Tokyo 185-8601, Japan; e-mail okano_kazunori@bpx.c.u-tokyo.ac.jp)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 singlestranded PCR products used as templates for probeextension reactions be purified before the reaction by use of streptavidin-conjugated magnetic beads to remove the remaining PCR primers and substrates, which is a timeconsuming 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 luciferinluciferase 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