Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Methods Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing Anders Alderborn,1 Anna Kristofferson,2 and Ulf Hammerling3 1Research & Development, Pyrosequencing AB, Uppsala, Sweden; 2Technology & Diagnostics, Eurona Medical AB, Uppsala, Sweden The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin–Angiotensin–Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5–10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run. Since the first report on molecular polymorphism in newed interest in association studies (Lander and humans describing the ABO system (Landsteiner 1900) Schork 1994; Collins et al. 1997). a very large number of genetic variations have been Even though various simplified techniques are characterized. One of the most common types of ge- presented as applicable for primary definition of SNPs, netic diversity is the Single-Nucleotide Polymorphism semiautomated sequencing of DNA remains to date (SNP), featuring a biallelic situation in which the alter- the most reliable method for this purpose (Eng and native bases occur at a frequency exceeding 1% (Scha- Vijg 1997). Emerging technologies for assessing DNA fer and Hawkins 1998). The overall occurrence of SNPs sequence differences between individuals, however, of- in the human genome is not known, but a rough esti- fer considerable promise for increasing the rate at mate of 0.1–1% of all bases has been reported (Cooper which such polymorphisms can be defined (Collins et et al. 1985; Collins et al. 1997; Schafer and Hawkins al. 1997). In the case of investigating characterized 1998). Recent findings, based on screening of many SNPs, several methods have been extensively evalu- individuals, reveal occurrences of 1 SNP per ∼220–350 ated. Except for those directly focused on polymerase base pairs in the human genome (Cargill et al. 1999; chain reaction (PCR) and ligation methods (Landegren Halushka et al. 1999). This diversity provides an excel- et al. 1988; Bottema et al. 1993; Livak 1995), most such lent tool for a wide panel of genetic analyses, including approaches can be roughly divided into techniques forensics, studies on population migration, diagnosis based on primer extension or on the recognition of of disorders with a significant genetic influence, and heteroduplex DNA. The former category includes sev- various pharmacogenetic/pharmacogenomic applica- eral assay types, such as Solid Phase Minisequencing tions. Especially in the latter areas, the rapid accumu- (SPM) and detection of dissimilarly sized extension lation of available polymorphisms has elicited a re- fragments by MALDI-TOF mass spectroscopy (Little et al. 1997; Syva¨nen 1999), whereas the latter involves a wide number of applications, including mismatch Present address: National Food Administration, SE-751 26, cleavage detection, oligoarray hybridization, molecu- Uppsala, Sweden. lar beacon signaling, fluorescence monitoring of PCR, 3Corresponding author. E-MAIL: [email protected]; FAX 46 (18) 171433. and electronic dot blot assay (Pease et al. 1994; Mashal 10:1249–1258 ©2000 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/00 $5.00; www.genome.org Genome Research 1249 www.genome.org Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Alderborn et al. et al. 1995; Southern 1996; Tyagi and Kramer 1996; counterparts and because of the low level of back- Gilles et al. 1999; Nauck et al. 1999; for review, see ground signal. Moreover, the ability to prepare and Graber et al. 1998). determine the sequence of 96 samples in parallel en- In our study design, we have approached the issue ables expedient analysis of large sample numbers. of SNP identification by using the recently described real-time pyrophosphate (PP ) detection method i RESULTS known as pyrosequencing (Nyre´n and Lundin 1985; Ronaghi et al. 1996, 1998). This technique is based on PCR Optimization and Preparation of Templates an indirect bioluminometric assay of the pyrophos- for Pyrosequencing phate (PPi) that is released from each dNTP upon DNA The target sequences employed for PCR-amplification chain elongation. Following Klenow polymerase- and ensuing SNP genotyping by pyrosequencing were mediated base incorporation, PPi is released and used derived from reports on angiotensinogen (AGT) (Fuka- Ј as a substrate, together with adenosine 5 -phos- mizu et al. 1990; Jeunemaitre et al. 1992), angiotensin phosulfate (APS), for ATP sulfurylase, which results in I-converting enzyme (ACE) (Soubrier et al. 1988; the formation of ATP. Subsequently, the ATP accom- Hubert et al. 1991), and angiotensin II type I receptor plishes the conversion of luciferin to its oxi-derivative (AT1R) (Guo et al. 1994). Among reported polymor- by the action of luciferase. The ensuing light output phisms, two in AGT (A-20C and G-6A)(Jeunemaitre et becomes proportional to the number of added bases, al. 1992), five in ACE (A-240T, T1237C, G2215A, up to about four bases. To allow processivity of the G2350A, and T3409C) (Villard et al. 1996; Low et al. method dNTP excess is degraded by apyrase, which is 1998) and one in AT1R (A1166C) (Bonnardeaux et al. also present in the starting reaction mixture, so that 1994) were selected for analysis by pyrosequencing. only dNTPs are added to the template during the se- Furthermore, two variations located in the AT1R up- quencing procedure (Nyre´n and Lundin 1985; Ronaghi stream region (A-227C and C-226G) were identified by et al. 1996). The process has been fully automated and Sanger DNA sequencing and were included in the adapted to a 96-well format, which allows rapid screen- panel of polymorphic positions studied here. Accord- ing of large SNP panels. ingly, selected motifs were separately optimized for To assess the applicability of pyrosequencing for PCR product amount and specificity, using a panel of SNP identification, we selected 10 such variations, dis- test temperature cycling protocols. As a result of such Ј tributed throughout the 5 regulatory and coding se- titrations, a single setting that enabled high-quality quences of angiotensinogen, angiotensin I-converting outputs was adopted for all 10 fragments. enzyme, and angiotensin II type I receptor. These To obtain a sequencing template from PCR one of genes represent essential components of the Renin– each PCR primer pair was covalently coupled to biotin. Angiotensin–Aldosterone System (RAAS), which plays Single-stranded DNA was generated by exposing am- a crucial role in hormonal mechanisms that regulate plified DNA fragments, immobilized onto streptavidin- blood pressure and electrolyte–blood-volume homeo- coupled beads, to alkali, followed by release of the stasis (Goodfriend et al. 1995; Sealy and Laragh 1995). complementary strand and neutralization. In this ex- Several biallelic variations in this gene cluster have perimental setting, bead-attached DNA was consis- been analyzed in various combinations for correlation tently employed as a template. The entire sample to clinical drug response data. Using large sample pan- preparation procedure, including amplification, strand els, combinations of three to six polymorphisms were separation, and annealing to the respective primer, was ultimately compiled into genetic signatures that are conducted in 96-well microtiter plates. Sample transfer indicative of patient susceptibility to a class of antihy- from various solutions in this chain of events was ex- pertensive substances (Sanders 1999). The panel of pedited by a magnetic 96-pin manifold. Subsequent to nucleotide variations selected for this study encom- these steps, samples were loaded in special microplates passes several