Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.
Abstract: A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.
Publication Date: 1994-10-11 PubMed ID: 7937143PubMed Central: PMC331910DOI: 10.1093/nar/22.20.4167Google Scholar: Lookup
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- Journal Article
- Antibodies
- Biochemistry
- Biopsy
- Biotechnology
- Biotin
- Diagnostic Technique
- DNA
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Enzymes
- Equine Health
- Genetic Testing
- Genetic Variants
- Genetics
- Genomics
- Genotyping
- Immunology
- Laboratory Methods
- Molecular biology
- Ophthalmology
- Pedigree
- Phosphatase
- Polymerase Chain Reaction
- Thoroughbreds
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research article presents a novel method, Genetic Bit Analysis (GBA), for typing single nucleotide polymorphisms (SNPs) in DNA. Specific fragments of genomic DNA are amplified, rendered single-stranded, captured, and sensed to determine the nature of the resolved nucleotides. Applying this technique, the researchers successfully verified the parentage for thoroughbred horses.
Methodology and Techniques
- Genomic DNA containing the polymorphic sites was amplified using the Polymerase chain reaction (PCR). PCR is a standard molecular biology technique used to make multiple copies of a particular DNA segment from a less quantity. Special primers including a phosphorothioate-modified primer were used. A primer is a short DNA or RNA segment that attaches to the target DNA to start the replication process.
- The molecular technique was implemented to transform the double-stranded amplified DNA into single strands. The T7 gene 6 exonuclease enzyme was used, which selectively digests double-stranded DNA starting from the ends followed by capturing on a plate.
- The capture onto the wells of a plate was done using immobilized oligonucleotide primer. Oligonucleotide primers are small pieces of single-stranded DNA that are designed to bind to the start of the section of DNA to be amplified or studied.
- The extension of the oligonucleotide is carried out using a mixture of labeled and unlabeled dideoxynucleoside triphosphates and either the Klenow fragment of DNA polymerase I or modified T7 DNA polymerase known as Sequenase. Dideoxynucleoside triphosphate is a molecule similar to those used to construct DNA but, when incorporated into a growing DNA chain, it causes chain termination.
- To determine the nature of the extended base, ELISA (enzyme-linked immunosorbent assay), a method used to detect the presence of antibodies in a liquid sample, was implemented using enzymes such as alkaline phosphatase and horseradish peroxidase. These enzymes were conjugated to the antibodies for detection purposes.
Applications of Genetic Bit Analysis (GBA)
- This research utilized the developed GBA method for the task of parentage verification of thoroughbred horses. The process was semi-automated and employed a prior set of 26 diallelic polymorphisms from the equine genome.
- Diallelic polymorphisms represent the simplest form of polymorphisms where there are only two possible alleles, which refer to alternative forms of a gene that arise due to mutation.
- By using the GBA method, the researchers could accurately match offspring to parents based on their genetic information, which is crucial in the practice of breeding horses.
- The successful implementation of GBA in this context implies its potential application in other areas such as human genetic diseases studies, population genetics, and forensic analysis where detecting single nucleotide polymorphisms is critical.
Cite This Article
APA
Nikiforov TT, Rendle RB, Goelet P, Rogers YH, Kotewicz ML, Anderson S, Trainor GL, Knapp MR.
(1994).
Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.
Nucleic Acids Res, 22(20), 4167-4175.
https://doi.org/10.1093/nar/22.20.4167 Publication
Researcher Affiliations
- Molecular Tool, Inc., Alpha Center, Hopkins Bayview Research Campus, Baltimore, MD 21224.
MeSH Terms
- Autoanalysis
- Base Sequence
- Biotin
- Colorimetry
- DNA / analysis
- DNA / chemistry
- DNA Polymerase I / metabolism
- DNA Primers
- DNA, Single-Stranded
- DNA-Directed DNA Polymerase / metabolism
- Enzyme-Linked Immunosorbent Assay
- Escherichia coli / enzymology
- Exodeoxyribonucleases / metabolism
- Fluorescein
- Fluoresceins
- Fluorescent Dyes
- Indicators and Reagents
- Molecular Sequence Data
- Nucleic Acid Hybridization
- Polymerase Chain Reaction
- Polymorphism, Genetic
- Templates, Genetic
References
This article includes 30 references
- Wilson JT, Marotta CA, Forget BG, Weissman SM. Structure of human hemoglobin messenger RNA and its relation to hemoglobinopathies.. Trans Assoc Am Physicians 1977;90:117-26.
- Nikiforov TT, Rendle RB, Kotewicz ML, Rogers YH. The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.. PCR Methods Appl 1994 Apr;3(5):285-91.
- Conner BJ, Reyes AA, Morin C, Itakura K, Teplitz RL, Wallace RB. Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides.. Proc Natl Acad Sci U S A 1983 Jan;80(1):278-82.
- Cooper DN, Smith BA, Cooke HJ, Niemann S, Schmidtke J. An estimate of unique DNA sequence heterozygosity in the human genome.. Hum Genet 1985;69(3):201-5.
- Myers RM, Larin Z, Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes.. Science 1985 Dec 13;230(4731):1242-6.
- Prober JM, Trainor GL, Dam RJ, Hobbs FW, Robertson CW, Zagursky RJ, Cocuzza AJ, Jensen MA, Baumeister K. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides.. Science 1987 Oct 16;238(4825):336-41.
- Myers RM, Maniatis T, Lerman LS. Detection and localization of single base changes by denaturing gradient gel electrophoresis.. Methods Enzymol 1987;155:501-27.
- Cotton RG, Rodrigues NR, Campbell RD. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations.. Proc Natl Acad Sci U S A 1988 Jun;85(12):4397-401.
- Landegren U, Kaiser R, Sanders J, Hood L. A ligase-mediated gene detection technique.. Science 1988 Aug 26;241(4869):1077-80.
- Gyllensten UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus.. Proc Natl Acad Sci U S A 1988 Oct;85(20):7652-6.
- Tabor S, Richardson CC. Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis.. J Biol Chem 1989 Apr 15;264(11):6447-58.
- Wu DY, Ugozzoli L, Pal BK, Wallace RB. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.. Proc Natl Acad Sci U S A 1989 Apr;86(8):2757-60.
- 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 Apr 11;17(7):2503-16.
- Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.. Proc Natl Acad Sci U S A 1989 Aug;86(16):6230-4.
- Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui LC. Identification of the cystic fibrosis gene: genetic analysis.. Science 1989 Sep 8;245(4922):1073-80.
- Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis.. Cell 1990 Jun 1;61(5):759-67.
- Nickerson DA, Kaiser R, Lappin S, Stewart J, Hood L, Landegren U. Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay.. Proc Natl Acad Sci U S A 1990 Nov;87(22):8923-7.
- Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase.. Proc Natl Acad Sci U S A 1991 Jan 1;88(1):189-93.
- Syvänen AC, Aalto-Setälä K, Harju L, Kontula K, Söderlund H. A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E.. Genomics 1990 Dec;8(4):684-92.
- Kuppuswamy MN, Hoffmann JW, Kasper CK, Spitzer SG, Groce SL, Bajaj SP. Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes.. Proc Natl Acad Sci U S A 1991 Feb 15;88(4):1143-7.
- Sung CH, Davenport CM, Hennessey JC, Maumenee IH, Jacobson SG, Heckenlively JR, Nowakowski R, Fishman G, Gouras P, Nathans J. Rhodopsin mutations in autosomal dominant retinitis pigmentosa.. Proc Natl Acad Sci U S A 1991 Aug 1;88(15):6481-5.
- Dryja TP, Hahn LB, Cowley GS, McGee TL, Berson EL. Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa.. Proc Natl Acad Sci U S A 1991 Oct 15;88(20):9370-4.
- Ugozzoli L, Wahlqvist JM, Ehsani A, Kaplan BE, Wallace RB. Detection of specific alleles by using allele-specific primer extension followed by capture on solid support.. Genet Anal Tech Appl 1992 Aug;9(4):107-12.
- Hayashi K. PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA.. PCR Methods Appl 1991 Aug;1(1):34-8.
- Syvänen AC, Sajantila A, Lukka M. Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.. Am J Hum Genet 1993 Jan;52(1):46-59.
- Nyrén P, Pettersson B, Uhlén M. Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay.. Anal Biochem 1993 Jan;208(1):171-5.
- Goodman MF, Creighton S, Bloom LB, Petruska J. Biochemical basis of DNA replication fidelity.. Crit Rev Biochem Mol Biol 1993;28(2):83-126.
- Balaguer P, Térouanne B, Allibert P, Cros P, Boussioux AM, Mandrand B, Nicolas JC. Detection of single base substitutions in polynucleotides by capture with immobilized oligonucleotides.. Mol Cell Probes 1993 Apr;7(2):155-9.
- Bromberg LE, Klibanov AM. Detergent-enabled transport of proteins and nucleic acids through hydrophobic solvents.. Proc Natl Acad Sci U S A 1994 Jan 4;91(1):143-7.
- Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms.. Am J Hum Genet 1980 May;32(3):314-31.
Citations
This article has been cited 34 times.- Rajagopalan RM, Fujimura JH. Variations on a Chip: Technologies of Difference in Human Genetics Research.. J Hist Biol 2018 Dec;51(4):841-873.
- Tang C, Liu H, Tang Y, Guo Y, Liang X, Guo L, Pi R, Yang J. Analysis of mitochondrial transcription factor A SNPs in alcoholic cirrhosis.. Exp Ther Med 2014 Jan;7(1):73-79.
- Nadal P, Pinto A, Svobodova M, Canela N, O'Sullivan CK. DNA aptamers against the Lup an 1 food allergen.. PLoS One 2012;7(4):e35253.
- Ollitrault P, Terol J, Garcia-Lor A, Bérard A, Chauveau A, Froelicher Y, Belzile C, Morillon R, Navarro L, Brunel D, Talon M. SNP mining in C. clementina BAC end sequences; transferability in the Citrus genus (Rutaceae), phylogenetic inferences and perspectives for genetic mapping.. BMC Genomics 2012 Jan 10;13:13.
- Shen G, Tercero N, Gaspar MA, Varughese B, Shepard K, Levicky R. Charging behavior of single-stranded DNA polyelectrolyte brushes.. J Am Chem Soc 2006 Jul 5;128(26):8427-33.
- Takatsu K, Yokomaku T, Kurata S, Kanagawa T. A FRET-based analysis of SNPs without fluorescent probes.. Nucleic Acids Res 2004 Nov 8;32(19):e156.
- Wakai J, Takagi A, Nakayama M, Miya T, Miyahara T, Iwanaga T, Takenaka S, Ikeda Y, Amano M, Urata T. A novel method of identifying genetic mutations using an electrochemical DNA array.. Nucleic Acids Res 2004 Oct 21;32(18):e141.
- Vaarno J, Ylikoski E, Meltola NJ, Soini JT, Hänninen P, Lahesmaa R, Soini AE. New separation-free assay technique for SNPs using two-photon excitation fluorometry.. Nucleic Acids Res 2004 Jul 19;32(13):e108.
- Huang CY, Stíººker J, Yuryev A, Huang J, Scott KE, Kuebler J, Varde S, Alfisi S, Gelfand CA, Pohl M, Boyce-Jacino MT. Auto-validation of fluorescent primer extension genotyping assay using signal clustering and neural networks.. BMC Bioinformatics 2004 Apr 2;5:36.
- Matsuzaki H, Loi H, Dong S, Tsai YY, Fang J, Law J, Di X, Liu WM, Yang G, Liu G, Huang J, Kennedy GC, Ryder TB, Marcus GA, Walsh PS, Shriver MD, Puck JM, Jones KW, Mei R. Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array.. Genome Res 2004 Mar;14(3):414-25.
- Jungerius BJ, Veenendaal A, Van Oost BA, Te Pas MF, Groenen MA. Typing single-nucleotide polymorphisms using a gel-based sequencer: a new data analysis tool and suggestions for improved efficiency.. Mol Biotechnol 2003 Nov;25(3):283-8.
- Zhang G, Zhou Y, Wu X, Yuan J, Ren S. Covalent attachment of DNA to glass supports using a new silane coupling agent and chemiluminescent detection.. J Tongji Med Univ 2000;20(2):89-91.
- Batley J, Barker G, O'Sullivan H, Edwards KJ, Edwards D. Mining for single nucleotide polymorphisms and insertions/deletions in maize expressed sequence tag data.. Plant Physiol 2003 May;132(1):84-91.
- Yuryev A, Huang J, Pohl M, Patch R, Watson F, Bell P, Donaldson M, Phillips MS, Boyce-Jacino MT. Predicting the success of primer extension genotyping assays using statistical modeling.. Nucleic Acids Res 2002 Dec 1;30(23):e131.
- 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 Aug 1;30(15):e75.
- Gardner AF, Jack WE. Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases.. Nucleic Acids Res 2002 Jan 15;30(2):605-13.
- Waterfall CM, Cobb BD. Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis.. Nucleic Acids Res 2001 Dec 1;29(23):E119.
- Erdogan F, Kirchner R, Mann W, Ropers HH, Nuber UA. Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays.. Nucleic Acids Res 2001 Apr 1;29(7):E36.
- Pastinen T, Raitio M, Lindroos K, Tainola P, Peltonen L, Syvänen AC. A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays.. Genome Res 2000 Jul;10(7):1031-42.
- 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 Jun;10(6):853-60.
- Chen J, Iannone MA, Li MS, Taylor JD, Rivers P, Nelsen AJ, Slentz-Kesler KA, Roses A, Weiner MP. A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension.. Genome Res 2000 Apr;10(4):549-57.
- Hacia JG, Collins FS. Mutational analysis using oligonucleotide microarrays.. J Med Genet 1999 Oct;36(10):730-6.
- Maldonado-Rodriguez R, Espinosa-Lara M, Loyola-Abitia P, Beattie WG, Beattie KL. Mutation detection by stacking hybridization on genosensor arrays.. Mol Biotechnol 1999 Feb;11(1):13-25.
- Maldonado-Rodriguez R, Espinosa-Lara M, Calixto-Suárez A, Beattie WG, Beattie KL. Hybridization of glass-tethered oligonucleotide probes to target strands preannealed with labeled auxiliary oligonucleotides.. Mol Biotechnol 1999 Feb;11(1):1-12.
- Chen X, Levine L, Kwok PY. Fluorescence polarization in homogeneous nucleic acid analysis.. Genome Res 1999 May;9(5):492-8.
- Picoult-Newberg L, Ideker TE, Pohl MG, Taylor SL, Donaldson MA, Nickerson DA, Boyce-Jacino M. Mining SNPs from EST databases.. Genome Res 1999 Feb;9(2):167-74.
- Liggett SB, Wagoner LE, Craft LL, Hornung RW, Hoit BD, McIntosh TC, Walsh RA. The Ile164 beta2-adrenergic receptor polymorphism adversely affects the outcome of congestive heart failure.. J Clin Invest 1998 Oct 15;102(8):1534-9.
- Head SR, Rogers YH, Parikh K, Lan G, Anderson S, Goelet P, Boyce-Jacino MT. Nested genetic bit analysis (N-GBA) for mutation detection in the p53 tumor suppressor gene.. Nucleic Acids Res 1997 Dec 15;25(24):5065-71.
- Haff LA, Smirnov IP. Multiplex genotyping of PCR products with MassTag-labeled primers.. Nucleic Acids Res 1997 Sep 15;25(18):3749-50.
- Fahy E, Nazarbaghi R, Zomorrodi M, Herrnstadt C, Parker WD, Davis RE, Ghosh SS. Multiplex fluorescence-based primer extension method for quantitative mutation analysis of mitochondrial DNA and its diagnostic application for Alzheimer's disease.. Nucleic Acids Res 1997 Aug 1;25(15):3102-9.
- Haff LA, Smirnov IP. Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.. Genome Res 1997 Apr;7(4):378-88.
- Chen X, Kwok PY. Template-directed dye-terminator incorporation (TDI) assay: a homogeneous DNA diagnostic method based on fluorescence resonance energy transfer.. Nucleic Acids Res 1997 Jan 15;25(2):347-53.
- Tobe VO, Taylor SL, Nickerson DA. Single-well genotyping of diallelic sequence variations by a two-color ELISA-based oligonucleotide ligation assay.. Nucleic Acids Res 1996 Oct 1;24(19):3728-32.
- Delahunty C, Ankener W, Deng Q, Eng J, Nickerson DA. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay.. Am J Hum Genet 1996 Jun;58(6):1239-46.
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