Single-Nucleotide Polymorphism Analysis by Pyrosequencing Afshin Ahmadian, Baback Gharizadeh, Anna C. Gustafsson, Fredrik Sterky, Pål Nyre ´n, Mathias Uhle ´n, and Joakim Lundeberg 1 Department of Biotechnology, The Royal Institute of Technology (KTH), SE-100 44 Stockholm, Sweden Received October 6, 1999 There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel se- quencing approach, pyrosequencing, for such pur- poses. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incor- porated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pat- tern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal deter- mination of SNPs, various protocols of nucleotide dis- pensing order were investigated. Here, we demon- strate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs. © 2000 Academic Press Key Words: SNP; DNA sequencing; pyrosequencing. Genetic variation is the basis for human diversity and plays an important role in human diseases. Meth- ods to screen and map genetic variability have, for more than two decades, been based on restriction frag- ment length polymorphism and microsatellite mark- ers. More recent efforts have focused on the most com- mon type of human genetic variation, single-nucleotide polymorphisms (SNPs). 2 A position is referred to as a SNP when it exists in at least two variants with a frequency of more than 1% for the least common alter- native (1). SNPs are distributed across the human genome by an approximate average of 1 SNP per 1000 base pairs (1, 2). As for microsatellite markers, SNPs can be used in linkage studies for identifying disease genes, in clinical genetic testing, in forensics, and for determination of loss of heterozygosity (LOH). The properties that make SNP analysis preferable com- pared to microsatellites are that SNPs are more prev- alent than microsatellites and that many SNPs are located within the genes, directly affecting the gene product (protein). As the number of identified SNPs increases, there will be an increasing demand for efficient methods to type and assess the biological impact of this kind of genetic variation. The golden standard method for SNP scoring has been conventional Sanger DNA sequencing. How- ever, sequencing based on gel electrophoresis generates more information than necessary, is time-consuming, is laborious, and requires labeling. Significant efforts have been made to improve SNP analysis with alternative techniques. Many of these techniques use allele-specific oligonucleotide (ASO) hybridization to discriminate be- tween allelic variants such as high-density microarray chips (3– 6), padlock probes (7), or allelic discrimination during PCR (8 –12). Minisequencing approaches are an attractive alternative because of the direct interrogation of the variable position by DNA polymerase extension using different means of detection of the extended prod- uct (i.e., fluorescent and radioactive dyes or by mass spectrometry) (13–16). Recently, an alternative technique for DNA sequenc- ing, called pyrosequencing, was described (17). Using a four-enzyme mixture, this sequencing-by-synthesis method relies on the luminometric detection of pyro- phosphate (PP i ) released upon nucleotide incorpora- tion. Here, we have investigated the possible use of pyrosequencing for SNP analysis. The technique has 1 To whom correspondence should be addressed: Fax: +46 8 245452. E-mail: joakim.lundeberg@biochem.kth.se. 2 Abbreviations used: SNP, single-nucleotide polymorphism; LOH, loss of heterozygosity; ASO, allele-specific oligonucleotide; PP i , pyro- phosphate; ATP, adenosine triphosphate; APS, adenosine 5'-phos- phosulfate. 0003-2697/00 $35.00 103 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 280, 103–110 (2000) doi:10.1006/abio.2000.4493, available online at http://www.idealibrary.com on