Comparison of PrASE and Pyrosequencing for SNP Genotyping
© Käller et al; licensee BioMed Central Ltd. 2006
Received: 29 August 2006
Accepted: 16 November 2006
Published: 16 November 2006
There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers.
The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated.
The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.
It is now a common belief that single nucleotide variations in the human genome are responsible for influencing traits such as differences in drug metabolism and disease risk. These variations are referred to as single nucleotide polymorphisms (SNPs) and several large-scale technologies have recently been developed for scoring of thousands of SNPs and approaching whole-genome genotyping [1–5].
However, for smaller scale projects where potential genes are already known, technologies for genotyping of many samples instead of SNPs and in addition retain high accuracy and throughput, are more attractive compared to assays that are cost effective per SNP. A flexible choice of SNPs is also important instead of a pre-defined set of SNPs. There are several technologies already used in academic contexts but the earliest paralleled assays relied upon hybridization of short allele-specific probes to the target DNA [6, 7]. However, improvements in microarray-based technologies in terms of accuracy have been achieved by enzymatic means [8–10]. One of these technologies involves allele-specific extension (ASE) which utilizes the ability of DNA polymerase to distinguish matched and mismatched 3'-termini of primers. However, a number of reports have shown that some mismatched 3'-ends can be elongated, giving false positive signals [11–13]. Nevertheless, as previously described, by exploiting the fact that the mismatched primers have slower reaction kinetics, the problems associated with ASE can be circumvented by including a protease (Proteinase K) that degrades the polymerase . In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers.
Gene (alternative name)
Polymorphism position (nt)
PrASE amplicon lenght
PrASE amplicon GC content
serpin peptidase inhibitor, clade E (plasminogen activator inhibitor type 1), member 1
nitric oxide synthase 3
integrin, beta 3 (platelet glycoprotein IIIa)
fibrinogen beta chain
coagulation factor XIII, A1 polypeptide
coagulation factor VII
coagulation factor V
coagulation factor II (thrombin)
matrix metallopeptidase 3 (stromelysin 1)
Results and discussion
To investigate the variability between tag sequences, each allele-specific extension primer was designed with two alternative tag sequences. The cluster diagrams for each of the primer pair combinations were compared (data not shown) and all combinations gave similar clusters as compared to the diagrams presented in Figure 2, indicating that the cluster distributions were mainly related to the extension rather than the hybridization properties of the tag sequence. However, for ITGB3 the clusters were shifted towards the left but functional when using one of the primer pairs. This can be due to either differences in hybridization efficiency or failure in the primer synthesis.
In addition, in order to investigate the effect of protease on genotyping calls, eight samples were genotyped in the presence and absence of protease. Without protease (ASE), correct clustering could be obtained for 8 out of the 10 SNPs whereas with protease (PrASE) correct clustering was obtained for all SNPs. The SNPs that did not render 3 distinguishable clusters by ASE are located in the ITGB3 and FGB genes (Figure S1 from Additional File 1). In these cases, the mismatch primer was mistakenly extended for one of the homozygous types, making these samples appear as heterozygotes. The Pyrosequencing assay was employed on these SNPs, confirming the PrASE results. In addition, in the remaining 8 SNPs, the inclusion of protease renders complete partitioning of the clusters by increasing the distance between clusters, indicating the higher robustness of PrASE. These findings are consistent with previous reports indicating lack of specificity of the ASE assay [9, 13, 21, 22].
Sanger DNA sequencing
In addition to accuracy and robustness, the reproducibility of the method was investigated by analyzing 24 samples. The investigated samples were all derived from the same PCR reactions and divided into two PrASE reactions followed by hybridization to one microarray slide. Standard deviations (SDs) were calculated between the two allelic fractions for each sample. The mean SD was 0.018 for all SNPs while for the individual SNPs, the mean SD ranged between 0.0047 and 0.030. Furthermore, 12 samples were assayed twice on separate dates (four months apart and with different inner PCR reactions, batches of microarray slides, enzymes and reagents). A mean SD of 0.023 was obtained for the two separate runs and for the individual SNPs the SD ranged between 0.0054 and 0.039. The results here show that there is very little inter and intra chip variability proving the reproducibility of the assay. In addition, low SDs reflects tightly held clusters (see Figure 1).
As a complement to whole-genome SNP typing technologies, where a large number of SNPs are examined in each sample, there is an important niche for technologies that accurately can type a large number of samples in not as many SNPs. In this work, genotyping of ten polymorphisms associated with thrombosis formation was performed with PrASE and 99.8% concordance was met when data was compared to Pyrosequencing. However, the PrASE assay proved to be considerably less labor intensive due to its multiplexing capability in both PCR amplification and genotyping. Yet, the number of investigated SNPs per sample may be further increased by design and addition of more signature tags on the arrays.
There is a plentitude of genotyping technologies with similar multiplexing and sample capabilities as PrASE. Some have been commercialized and are available in with specialized instruments and kits which naturally reduce the complexity for the user but at the same time increases costs and reduces the degrees of freedom for the researcher. Some such as PrASE have only been described academically and it is therefore difficult to get a simple price quote but in this particular case running costs is in the range of 0.15 USD per SNP.
Some other techniques in the same applicaton niche as PrASE are limited in multiplexing capacity by the technique itself, such as Pyrosequencing and various real time PCR assays (5' nuclease assay or TaqMan  and molecular beacons ), whereas others are limited by the amplification method, such as single-base extension (SBE)  with microarray  or MALDI-TOF MS  detection and PrASE. With MS detection, SBE has been limited to 30-plex detection due to a limited number of mass tags available or the resolution of the system . The similar microarray platforms used for SBE and PrASE would most likely be of similar multiplexing levels except that PrASE uses the double amount of primers (a negligible cost in the case for many samples and moderate number of SNPs) and thus uses double the amount of spots on the microarray whereas SBE instead uses a two or four color detection hence a more expensive scanner. The multiplexing level for PrASE or conventional allele-specific extension (ASE) and SBE seems to be much larger than previously anticipated; the same researchers have compared 650 SNPs with ASE and SBE  and both methods are scalable to hundreds of thousands of SNPs in a single reaction . The premises upon which these were chosen are not clear and it is our belief that PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increase the number of typeable SNPs, i.e. a more flexible choice in SNPs. This is especially important since the most common biallelic variations in the human genome is the C-T and the G-A transitions that are also the most difficult polymorphisms to type by allele specific extensions if not the PrASE technology is employed.
Ten SNPs and single base insertions/deletions in as many genes were selected that have been suggested as prothrombotic genetic variations. Gene names, abbreviations and GenBank accession numbers as well as polymorphism positions and types can be found in Table 1. Note that the polymorphisms in SERPINE1 and MMP3 are single base insertions/deletions. The SERPINE1 variation is a 4 or 5 deoxyguanosine residues while the MMP3 variation is a 5 or 6 deoxythymidine residues.
DNA was extracted from blood from unrelated individuals of Caucasian/Scandinavian origin (from a cohort of patients presenting with symptoms of acute chest pain) . The patients were included in the Carlscrona Heart Attack Prognosis Study approved by the ethics committee at the University of Lund, Sweden in compliance with the Declaration of Helsinki. Each 96-well PCR plate also contained five negative water controls and one positive control (Clontech Laboratories, Palo Alto, CA, USA). To prevent contamination problems three semi-clean rooms with limitations to the DNA allowed in the rooms were used.
A nested multiplex amplification of the genomic regions was performed. The same outer PCR was used as template both for 10 separate inner PCRs for Pyrosequencing as well as an inner multiplex PCR, used for PrASE. All primers for PCR were designed from GenBank entries and searched for specificity and were synthesized by MWG-Biotech (Ebersberg, Germany) (Table S1 from Additional File 1). The outer PCR was optimized by running gradient PCRs and simplex inner PCRs. An equivalent of 1–5 ng genomic DNA was used for each 25 μl reaction with 0.1 μM of each primer (except for the MTHFR-, F5- and F2-regions which needed 0.14 μM). The PCR contained 2 mM MgCl2, 0.2 mM dNTP (Amersham Biosciences, Uppsala, Sweden) and 0.5 U AmpliTaq Gold with 1× PCR Gold buffer (Applied Biosystems, Foster City, CA). The amplification program was 94°C for 12 min followed by 35 cycles at 94°C 50 s, 65°C 30 s and 72°C 2 min and finally 72°C for 10 min and it was performed on a GeneAMP thermocycler (PE Biosystems, Foster City, CA).
Inner Simplex PCRs for Pyrosequencing
0.5 μl of the outer PCR was used as template to separately amplify each SNP region in inner PCRs with the same concentrations as above but using 0.2 U polymerase. One primer in each pair was biotinylated for later immobilization. Amplification program were as above with the exceptions of 30 s of denaturation in each cycle and annealing temperatures of 64.5°C for all SNPs but FGB which annealed at 60°C and it was performed on a MWG multi block thermocyclers (MWG-Biotech).
Inner Multiplex PCR for PrASE
0.5 μl of the outer PCR was used as template to amplify all 10 loci in 50 μl inner PCR reaction with the same concentrations as above except 0.04 μM of each of the 20 primers and using 1 U of Platinum Taq DNA polymerase with 1× PCR buffer (Invitrogen AB, Lidingö, Sweden). Primers are indicated in Table S1 from Additional File 1 and one primer in each pair was biotinylated for immobilization. The amplification program was 94°C for 5 min followed by 45 cycles at 94°C 30 s, 60°C 30 s and 72°C 30 s and finally 72°C for 10 min and it was performed on a GeneAMP thermocycler (PE Biosystems).
Single stranded DNA was generated by the use of immobilization of the biotinylated PCR products to 50 μg of streptavidin coated super paramagnetic beads (Dynabeads M-270, Dynal Biotech, Oslo, Norway) and 1.65 pmol Pyrosequencing primer (Table S1 from Additional File 1) was hybridized by the use of a Magnatrix 1200 pipetting robot (Magnetic Biosolutions, Stockholm, Sweden) according to the manufacturers' instructions. Pyrosequencing was performed according to manufacturer's instructions on a PSQ™ 96 HS instrument (Biotage, Uppsala, Sweden) and analyzed with the accompanying SNP software.
The PrASE assay was automated by the use of a Magnatrix 1200 pipetting robot (Magnetic Biosolutions) that handles magnetic beads used for streptavidin immobilization of the biotinylated PCR products. The robot is capable of handling 48 samples in parallel, which is the same number as can be hybridized to one microarray slide. 200 μg streptavidin-coated super paramagnetic beads (Dynabeads M-280, Dynal Biotech) were used for each inner multiplex PCR product. Immobilization and washes between steps were made according to the manufacturer's instructions and as described before . Single-stranded DNA was prepared by alkali treatment and annealed to allele-specific extension primers (0.08 μM in 60 μl) (Table S2 from Additional File 1). The PrASE reaction was performed at 37°C in a total volume of 60 μl. containing 1× extension buffer (42.5 mM Tris-HCl pH 8, 5 mM MgCl2 and 1 mM DTT), 0.25 % bovine serum albumin and 10 U DNA polymerase (3'-5' exonuclease deficient Klenow fragment, Fermentas, Helsingborg, Sweden). The PrASE reaction was started by simultaneous addition of 1.5 μM of each dNTP (Amersham Biosciences) and 20 μg Proteinase K (Invitrogen). 50 % of the dCTP and dUTP were Cy5 labeled to allow fluorescence detection of extended primers. Strand-specific alkali elution of the primers was made before hybridization to the tag-microarray.
Tag microarrays were prepared as previously reported . Forty-eight oligonucleotides (MWG-Biotech) were spotted (Q-array, Genetix, Hampshire, United Kingdom) in triplicates onto glass slides (Code Link, Amersham Biosceinces, Uppsala, Sweden). The oligonucleotide pattern was repeated on each slide and these sub-arrays were separated during hybridization using a silicone mask to facilitate parallel analysis of 48 samples . Hybridization of the extended allele-specific primers was performed at 50°C for 1 h. Each primer contained a specific tag at its 5'-end complementary to one of the 48 spotted oligonucleotides. The slides were washed according to the manufacturer before scanning (Agilent scanner, Agilent Technologies, Palo Alto, CA, USA). Data was extracted with GenePix 5.0 software (Axon instruments, USA) and analyzed with a custom Microsoft Excel script.
Sanger DNA sequencing
Conflicting results were resolved using Sanger dideoxy sequencing with BigDye terminator chemistry (Applied Biosystems, Foster City, CA) and an ABI 3700 Analyzer instrument (Applied Biosystems). The same PCR setups as for Pyrosequencing were used and the inner PCR primers were used as sequencing primers.
This work was supported by grants from the Swedish Research Council, the Swedish Medical Research Council, the Knut and Alice Wallenberg Foundation and the Wallenberg Consortium North, and The Magnus Bergvall Foundation.
- Fan JB, Oliphant A, Shen R, Kermani BG, Garcia F, Gunderson KL, Hansen M, Steemers F, Butler SL, Deloukas P, Galver L, Hunt S, McBride C, Bibikova M, Rubano T, Chen J, Wickham E, Doucet D, Chang W, Campbell D, Zhang B, Kruglyak S, Bentley D, Haas J, Rigault P, Zhou L, Stuelpnagel J, Chee MS: Highly parallel SNP genotyping. Cold Spring Harb Symp Quant Biol. 2003, 68: 69-78. 10.1101/sqb.2003.68.69.PubMedView ArticleGoogle Scholar
- Gunderson KL, Steemers FJ, Lee G, Mendoza LG, Chee MS: A genome-wide scalable SNP genotyping assay using microarray technology. Nat Genet. 2005, 37 (5): 549-554. 10.1038/ng1547.PubMedView ArticleGoogle Scholar
- Hardenbol P, Yu F, Belmont J, Mackenzie J, Bruckner C, Brundage T, Boudreau A, Chow S, Eberle J, Erbilgin A, Falkowski M, Fitzgerald R, Ghose S, Iartchouk O, Jain M, Karlin-Neumann G, Lu X, Miao X, Moore B, Moorhead M, Namsaraev E, Pasternak S, Prakash E, Tran K, Wang Z, Jones HB, Davis RW, Willis TD, Gibbs RA: Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res. 2005, 15 (2): 269-275. 10.1101/gr.3185605.PubMedPubMed CentralView ArticleGoogle Scholar
- Hinds DA, Stuve LL, Nilsen GB, Halperin E, Eskin E, Ballinger DG, Frazer KA, Cox DR: Whole-genome patterns of common DNA variation in three human populations. Science. 2005, 307 (5712): 1072-1079. 10.1126/science.1105436.PubMedView ArticleGoogle Scholar
- Matsuzaki H, Dong S, Loi H, Di X, Liu G, Hubbell E, Law J, Berntsen T, Chadha M, Hui H, Yang G, Kennedy GC, Webster TA, Cawley S, Walsh PS, Jones KW, Fodor SP, Mei R: Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat Methods. 2004, 1 (2): 109-111. 10.1038/nmeth718.PubMedView ArticleGoogle Scholar
- Drmanac R, Labat I, Brukner I, Crkvenjakov R: Sequencing of megabase plus DNA by hybridization: theory of the method. Genomics. 1989, 4 (2): 114-128. 10.1016/0888-7543(89)90290-5.PubMedView ArticleGoogle Scholar
- Lysov Iu P, Florent'ev VL, Khorlin AA, Khrapko KR, Shik VV: [Determination of the nucleotide sequence of DNA using hybridization with oligonucleotides. A new method]. Dokl Akad Nauk SSSR. 1988, 303 (6): 1508-1511.PubMedGoogle Scholar
- Landegren U, Kaiser R, Sanders J, Hood L: A ligase-mediated gene detection technique. Science. 1988, 241 (4869): 1077-1080. 10.1126/science.3413476.PubMedView ArticleGoogle Scholar
- Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF: Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 1989, 17 (7): 2503-2516.PubMedPubMed CentralView ArticleGoogle Scholar
- Syvanen AC, Aalto-Setala K, Harju L, Kontula K, Soderlund H: A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics. 1990, 8 (4): 684-692. 10.1016/0888-7543(90)90255-S.PubMedView ArticleGoogle Scholar
- Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ: Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 1990, 18 (4): 999-1005.PubMedPubMed CentralView ArticleGoogle Scholar
- Ahmadian A, Gharizadeh B, O'Meara D, Odeberg J, Lundeberg J: Genotyping by apyrase-mediated allele-specific extension. Nucleic Acids Res. 2001, 29 (24): E121-10.1093/nar/29.24.e121.PubMedPubMed CentralView ArticleGoogle Scholar
- Kaller M, Ahmadian A, Lundeberg J: Microarray-based AMASE as a novel approach for mutation detection. Mutat Res. 2004, 554 (1-2): 77-88.PubMedView ArticleGoogle Scholar
- Hultin E, Kaller M, Ahmadian A, Lundeberg J: Competitive enzymatic reaction to control allele-specific extensions. Nucleic Acids Res. 2005, 33 (5): e48-10.1093/nar/gni048.PubMedPubMed CentralView ArticleGoogle Scholar
- Ronaghi M, Uhlen M, Nyren P: A sequencing method based on real-time pyrophosphate. Science. 1998, 281 (5375): 363, 365-10.1126/science.281.5375.363.PubMedView ArticleGoogle Scholar
- Ahmadian A, Gharizadeh B, Gustafsson AC, Sterky F, Nyren P, Uhlen M, Lundeberg J: Single-nucleotide polymorphism analysis by pyrosequencing. Anal Biochem. 2000, 280 (1): 103-110. 10.1006/abio.2000.4493.PubMedView ArticleGoogle Scholar
- Holmberg K, Persson ML, Uhlen M, Odeberg J: Pyrosequencing analysis of thrombosis-associated risk markers. Clin Chem. 2005, 51 (8): 1549-1552. 10.1373/clinchem.2005.049932.PubMedView ArticleGoogle Scholar
- Endler G, Mannhalter C: Polymorphisms in coagulation factor genes and their impact on arterial and venous thrombosis. Clin Chim Acta. 2003, 330 (1-2): 31-55. 10.1016/S0009-8981(03)00022-6.PubMedView ArticleGoogle Scholar
- Humphries SE, Morgan L: Genetic risk factors for stroke and carotid atherosclerosis: insights into pathophysiology from candidate gene approaches. Lancet Neurol. 2004, 3 (4): 227-235. 10.1016/S1474-4422(04)00708-2.PubMedView ArticleGoogle Scholar
- Lane DA, Grant PJ: Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease. Blood. 2000, 95 (5): 1517-1532.PubMedGoogle Scholar
- O'Meara D, Ahmadian A, Odeberg J, Lundeberg J: SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays. Nucleic Acids Res. 2002, 30 (15): e75-10.1093/nar/gnf074.PubMedPubMed CentralView ArticleGoogle Scholar
- Ayyadevara S, Thaden JJ, Shmookler Reis RJ: Discrimination of primer 3'-nucleotide mismatch by taq DNA polymerase during polymerase chain reaction. Anal Biochem. 2000, 284 (1): 11-18. 10.1006/abio.2000.4635.PubMedView ArticleGoogle Scholar
- Holland PM, Abramson RD, Watson R, Gelfand DH: Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991, 88 (16): 7276-7280. 10.1073/pnas.88.16.7276.PubMedPubMed CentralView ArticleGoogle Scholar
- Tyagi S, Kramer FR: Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996, 14 (3): 303-308. 10.1038/nbt0396-303.PubMedView ArticleGoogle Scholar
- Fan JB, Chen X, Halushka MK, Berno A, Huang X, Ryder T, Lipshutz RJ, Lockhart DJ, Chakravarti A: Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays. Genome Res. 2000, 10 (6): 853-860. 10.1101/gr.10.6.853.PubMedPubMed CentralView ArticleGoogle Scholar
- Tang K, Fu DJ, Julien D, Braun A, Cantor CR, Koster H: Chip-based genotyping by mass spectrometry. Proc Natl Acad Sci U S A. 1999, 96 (18): 10016-10020. 10.1073/pnas.96.18.10016.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim S, Ulz ME, Nguyen T, Li CM, Sato T, Tycko B, Ju J: Thirtyfold multiplex genotyping of the p53 gene using solid phase capturable dideoxynucleotides and mass spectrometry. Genomics. 2004, 83 (5): 924-931. 10.1016/j.ygeno.2003.11.012.PubMedView ArticleGoogle Scholar
- Steemers FJ, Chang W, Lee G, Barker DL, Shen R, Gunderson KL: Whole-genome genotyping with the single-base extension assay. Nat Methods. 2006, 3 (1): 31-33. 10.1038/nmeth842.PubMedView ArticleGoogle Scholar
- Gunderson KL, Kuhn KM, Steemers FJ, Ng P, Murray SS, Shen R: Whole-genome genotyping of haplotype tag single nucleotide polymorphisms. Pharmacogenomics. 2006, 7 (4): 641-648. 10.2217/14622422.214.171.1241.PubMedView ArticleGoogle Scholar
- Gharizadeh B, Kaller M, Nyren P, Andersson A, Uhlen M, Lundeberg J, Ahmadian A: Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays. Nucleic Acids Res. 2003, 31 (22): e146-10.1093/nar/gng147.PubMedPubMed CentralView ArticleGoogle Scholar
- Kaller M, Hultin E, Zheng B, Gharizadeh B, Wallin KL, Lundeberg J, Ahmadian A: Tag-array based HPV genotyping by competitive hybridization and extension. J Virol Methods. 2005, 129 (2): 102-112. 10.1016/j.jviromet.2005.05.015.PubMedView ArticleGoogle Scholar
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