ARTICLES The availability of large collections of SNPs along with recent large- scale linkage disequilibrium mapping efforts 1 have brought the promise of personalized whole-genome association studies to the field of human genetics. To achieve this goal, methodologies that permit screening of hundreds of thousands of SNPs will be needed to implement such large-scale association studies on a routine basis. These methods not only will have to be inexpensive per SNP screened, but will need to consume very little genomic DNA—that is, no more than is typically obtained from a patient’s blood sam- ple. In addition, such technologies should ideally require minimal investment in infrastructure so that the technology can be made broadly available. The challenge of genotyping the approximately 150 molecules of a given SNP locus present in 1 ng of genomic DNA is commonly met by PCR amplification of the locus before genotyping is done 2 . However, an increase in the number of target sequences for simul- taneous amplification by PCR quickly leads to unmanageable levels of cross-reaction among primer pairs 3,4 , whereas parallel hybridization on microarrays 5,6 lacks the specificity and sensitivity required to genotype large genomes directly. There are only a limited number of genotyping technologies with sufficient specificity to identify an SNP from genomic DNA with- out prior PCR amplification. Flap endonucleases have been used to generate a sequence-specific endonuclease cascade in an isothermal fashion that can be assessed with FRET probes 7,8 . However, this technology is not readily multiplexed for high-throughput applica- tions. Padlock probes are linear oligonucleotides, whose two ends can be joined by ligation when they hybridize to immediately adja- cent target sequences 9 . As shown before 10–12 , padlock probes pro- vide sufficient specificity analyze SNPs directly, without previous amplification of the target sequences. Unlike amplification strategies such as PCR and the Invader assay that require two specific primers, cross-reactive padlock probes can easily be distinguished from the desired circular products by meth- ods such as exonucleolysis 9 . This offers the opportunity to add a complex pool of padlock probes to individual DNA samples to investigate large sets of genes in parallel, without a concomitant increase in the risk of cross-reactivity between different probes. Here we present a strategy that combines DNA detection speci- ficity and sensitivity with the potential to analyze large numbers of target sequences in parallel. Sets of padlock probes with universal tag sequences were reacted with target DNA, molecularly inverted, amplified together and identified in a multiplex analysis yielding more than 1,000 genotypes simultaneously. Using molecular inver- sion probes, the information content of the SNPs was reformatted into tag sequences that could be detected using a universal oligonu- cleotide detection array 13 . We report the application of this tech- nique at unprecedented levels of multiplexing, resulting in a lowering of the scale, cost and sample requirements of high- throughput genotyping. The approach retained high accuracy through multiple hybridization and enzymatic processing events, and provided inherent quality control checking. RESULTS Selection for circularized probes using exonucleases Most genotyping methods require PCR amplification of the region spanning the sequence variation. However, when sets of n PCR primer pairs are combined in one reaction to evaluate n target sequences, any of the 2n 2 + n possible pairwise primer combinations may give rise to nonspecific amplification products 3 . With padlock probes the corresponding cross-reactive ligation products create linear dimeric molecules, easily distinguished from circularized 1 Stanford Genome Technology Center, Stanford University, 855 California Avenue, Palo Alto, California 94304, USA. 2 The Beijer Laboratory, Department of Genetics and Pathology, Rudbeck Laboratory, Se-751 85 Uppsala, Sweden. 3 Present address: ParAllele BioScience 384 Oyster Point Blvd Suite 8, S. San Francisco, California 94080, USA. Correspondence should be addressed to M.R. (mostafa@stanford.edu). Multiplexed genotyping with sequence-tagged molecular inversion probes Paul Hardenbol 1,3 , Johan Banér 2 , Maneesh Jain 1,3 , Mats Nilsson 2 , Eugeni A Namsaraev 1,3 , George A Karlin-Neumann 1,3 , Hossein Fakhrai-Rad 1,3 , Mostafa Ronaghi 1 , Thomas D Willis 1,3 , Ulf Landegren 2 & Ronald W Davis 1 We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing. NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 6 JUNE 2003 673 © 2003 Nature Publishing Group http://www.nature.com/naturebiotechnology