Pyrosequencing protocol requiring a unique biotinylated primer
-
Jose Luis Royo
Abstract
Background: DNA sequencing has markedly changed the nature of biomedical research. Large-scale sequencing projects have generated several millions of potential polymorphisms widespread in the human genome requiring validation and incorporation into screening panels. As a consequence, high-throughput analysis of these variants in different populations of interest is now the cornerstone of structural genomics. Pyrosequencing is a versatile technique allowing an easy 96-well typing format. However, every polymorphism requires a specific labeled primer to generate a single-stranded DNA fragment containing the region of interest.
Methods: We describe how with an adjusted primer stoichiometry we can standardize the labeling of every amplicon with a single biotinylated universal primer (BM13S).
Results: We circumvent the need for specific biotinylated primers for each single-nucleotide polymorphism (SNP) under study. As an example, we assessed this novel protocol by genotyping three SNPs mapping calpain-10, caveolin-1 and CYP19A1.
Conclusion: The present approach represents an alternative to standard pyrosequencing protocols, since it requires a single biotinylated primer that is suitable for each SNP under study.
References
1. Hinds DA, Stuve LL, Nilsen GB, Halperin E, Eskin E, Ballinger DG, et al. Whole-genome patterns of common DNA variation in three human populations. Science 2005; 307:1072–9.10.1126/science.1105436Search in Google Scholar PubMed
2. Tyagi S, Bratu DP, Kramer FR. Multicolor molecular beacons for allele discrimination. Nat Biotechnol 1998; 16:49–53.10.1038/nbt0198-49Search in Google Scholar PubMed
3. Ronaghi M, Uhlen M, Nyren P. A sequencing method based on real-time pyrophosphate. Science 1998; 281:363–5.10.1126/science.281.5375.363Search in Google Scholar PubMed
4. Hardenbol P, Baner J, Jain M, Nilsson M, Namsaraev EA, Karlin-Neumann GA, et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat Biotechnol 2003; 21:673–8.10.1038/nbt821Search in Google Scholar PubMed
5. O'Meara D, Ahmadian A, Odeberg J, Lundeberg J. SNP typing by a pyrase-mediated allele-specific primer extension on DNA microarrays. Nucleic Acids Res 2002; 30:e75.10.1093/nar/gnf074Search in Google Scholar PubMed PubMed Central
6. Kruckeberg KE, Thibodeau SN. Pyrosequencing technology as a method for the diagnosis of multiple endocrine neoplasia type 2. Clin Chem 2004; 50:522–9.10.1373/clinchem.2003.027128Search in Google Scholar PubMed
7. Frey UH, Nuckel H, Dobrev D, Manthey I, Sandalcioglu IE, Eisenhardt A, et al. Quantification of G protein Gαs sub-unit splice variants in different human tissues and cells using pyrosequencing. Gene Expr 2005; 12:69–81.10.3727/000000005783992124Search in Google Scholar PubMed PubMed Central
8. Soderback E, Zackrisson AL, Lindblom B, Alderborn A. Determination of CYP2D6 gene copy number by pyrosequencing. Clin Chem 2005; 51:522–31.10.1373/clinchem.2004.043182Search in Google Scholar PubMed
9. Pielberg G, Day AE, Plastow GS, Andersson L. A sensitive method for detecting variation in copy numbers of duplicated genes. Genome Res 2003; 13:2171–7.10.1101/gr.1188203Search in Google Scholar PubMed PubMed Central
10. Sivertsson A, Platz A, Hansson J, Lundeberg J. Pyrosequencing as an alternative to single-strand conformation polymorphism analysis for detection of N-ras mutations in human melanoma metastases. Clin Chem 2002; 48:2164–70.10.1093/clinchem/48.12.2164Search in Google Scholar
11. Aquilante CL, Lobmeyer MT, Langaee TY, Johnson JA. Comparison of cytochrome P450 2C9 genotyping methods and implications for the clinical laboratory. Pharmacotherapy 2004; 24:720–6.10.1592/phco.24.8.720.36074Search in Google Scholar
12. Nordfors L, Jansson M, Sandberg G, Lavebratt C, Sengul S, Schalling M, et al. Large-scale genotyping of single nucleotide polymorphisms by Pyrosequencing™ and validation against the 5′-nuclease (Taqman) assay. Hum Mutat 2002; 19:395–401.10.1002/humu.10062Search in Google Scholar
13. Gharizadeh B, Eriksson J, Nourizad N, Nordstrom T, Nyren P. Improvements in pyrosequencing technology by employing Sequenase polymerase. Anal Biochem 2004; 330:272–80.10.1016/j.ab.2004.03.018Search in Google Scholar
14. Nordstrom T, Nourizad K, Ronaghi M, Nyren P. Method enabling pyrosequencing on double-stranded DNA. Anal Biochem 2000; 282:186–93.10.1006/abio.2000.4603Search in Google Scholar
15. Fakhrai-Rad H, Pourmand N, Ronaghi M. Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms. Hum Mutat 2002; 19:479–85.10.1002/humu.10078Search in Google Scholar
16. Pacey-Miller T, Henry R. Single-nucleotide polymorphism detection in plants using a single-stranded pyrosequencing protocol with a universal biotinylated primer. Anal Biochem 2003; 317:166–70.10.1016/S0003-2697(03)00089-7Search in Google Scholar
©2006 by Walter de Gruyter Berlin New York
Articles in the same Issue
- Natriuretic peptides and evidence-based quality specifications
- Preanalytical variability: the dark side of the moon in laboratory testing
- Clinical relevance of biological variation: the lesson of brain natriuretic peptide (BNP) and NT-proBNP assay
- Hepatorenal syndrome
- Modified Levey-Jennings charts for calculated laboratory tests
- Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate: a pilot study
- The exponentially weighted moving average (EWMA) rule compared with traditionally used quality control rules
- Intermethod calibration of alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) results: application to Fibrotest® and Actitest® scores
- Comparison of TEST 1 with SRS 100 and ICSH reference method for the measurement of the length of sedimentation reaction in blood
- Multicenter evaluation of the interference of hemoglobin, bilirubin and lipids on Synchron LX-20 assays
- Technical evaluation of the Beckman Coulter OV-Monitor (CA 125 antigen) immunoassay
- Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C→T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine
- Matrix metalloproteinases and their inhibitors in different acute stroke subtypes
- Pyrosequencing protocol requiring a unique biotinylated primer
- Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA
- Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry
- Time-level relationship between indicators of oxidative stress and Glasgow Coma Scale scores of severe head injury patients
- Stepwise strategies in analysing haematuria and leukocyturia in screening
- Elevation of serum cerebral injury markers correlates with serum choline decline after coronary artery bypass grafting surgery
- Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study
- Release of anandamide from blood cells
- Rapid decrease in plasma D-lactate as an early potential predictor of diminished 28-day mortality in critically ill septic shock patients
- Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry
- Sample processing and its preanalytical impact on the measurement of circulating matrix metalloproteinases
- Physiological matrix metalloproteinase (MMP) concentrations: comparison of serum and plasma specimens
- Importance of the functional sensitivity determination of a serum hyaluronic acid assay for the prediction of liver fibrosis in patients with features of the metabolic syndrome
- The dilemma of invasive and non-invasive investigations for adult and paediatric non-alcoholic fatty liver disease: has the time come for a new biochemical marker?
- Is cystatin C a reliable renal marker in trauma?
- On the independence of intraindividual reference values
- Sixth International Symposium on Molecular Diagnostics, Graz, Austria, May 25-27, 2006
Articles in the same Issue
- Natriuretic peptides and evidence-based quality specifications
- Preanalytical variability: the dark side of the moon in laboratory testing
- Clinical relevance of biological variation: the lesson of brain natriuretic peptide (BNP) and NT-proBNP assay
- Hepatorenal syndrome
- Modified Levey-Jennings charts for calculated laboratory tests
- Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate: a pilot study
- The exponentially weighted moving average (EWMA) rule compared with traditionally used quality control rules
- Intermethod calibration of alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) results: application to Fibrotest® and Actitest® scores
- Comparison of TEST 1 with SRS 100 and ICSH reference method for the measurement of the length of sedimentation reaction in blood
- Multicenter evaluation of the interference of hemoglobin, bilirubin and lipids on Synchron LX-20 assays
- Technical evaluation of the Beckman Coulter OV-Monitor (CA 125 antigen) immunoassay
- Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C→T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine
- Matrix metalloproteinases and their inhibitors in different acute stroke subtypes
- Pyrosequencing protocol requiring a unique biotinylated primer
- Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA
- Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry
- Time-level relationship between indicators of oxidative stress and Glasgow Coma Scale scores of severe head injury patients
- Stepwise strategies in analysing haematuria and leukocyturia in screening
- Elevation of serum cerebral injury markers correlates with serum choline decline after coronary artery bypass grafting surgery
- Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study
- Release of anandamide from blood cells
- Rapid decrease in plasma D-lactate as an early potential predictor of diminished 28-day mortality in critically ill septic shock patients
- Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry
- Sample processing and its preanalytical impact on the measurement of circulating matrix metalloproteinases
- Physiological matrix metalloproteinase (MMP) concentrations: comparison of serum and plasma specimens
- Importance of the functional sensitivity determination of a serum hyaluronic acid assay for the prediction of liver fibrosis in patients with features of the metabolic syndrome
- The dilemma of invasive and non-invasive investigations for adult and paediatric non-alcoholic fatty liver disease: has the time come for a new biochemical marker?
- Is cystatin C a reliable renal marker in trauma?
- On the independence of intraindividual reference values
- Sixth International Symposium on Molecular Diagnostics, Graz, Austria, May 25-27, 2006
