Analysis of Multiple Single Nucleotide Polymorphisms Closely Positioned in the Ovine PRNP Gene Using Linear Fluorescent Probes and Melting Curve Analysis

Overview

Journal BMC Infect Dis

Publisher Biomed Central

Specialty Infectious Diseases

Date 2007 Aug 9

PMID 17683552

Citations 1

Authors

David J French

Dominic Jones

David G McDowell

Jim A Thomson

Paul G Debenham

Affiliations

Soon will be listed here.

Abstract

Background: Resistance and susceptibility to scrapie has been associated with single nucleotide polymorphisms located within codons 136, 154 and 171 of the ovine prion protein gene (PRNP). Dual-labelled HyBeacon probes were developed to analyse single and clustered polymorphisms within these and neighbouring codons.

Methods: Extracted DNAs and unpurified blood samples were genotyped with respect to polymorphisms in PRNP codons 136, 141, 154 and 171. PCR amplicons were investigated using a LightTyper instrument, measuring the stability of probe/target hybridisation through peak melting temperatures and determining the sequence of nucleotides at polymorphic sites.

Results: The performance of HyBeacon assays was evaluated in a validation study comparing genotypes with those obtained using a primer extension assay (Sequenom MassEXTEND) analysed on a MALDI-ToF mass spectrometer. Over 12,000 sheep samples were successfully genotyped, reliably detecting A136, V136, T136, T137, L141, F141 R154, H154, L168, R171, Q171, H171 and K171 sequence variants using only 4 HyBeacon probes.

Conclusion: HyBeacon assays provide an extremely robust and accurate method for the analysis of single and clustered PRNP polymorphisms in a high-throughput format. The flexibility of the diagnostic tests ensures that samples are correctly genotyped even in the presence of additional sequence variations that flank the polymorphisms of interest. Such sequence variations may also be neutralised using universal bases such as 5-nitroindole if required.

Citing Articles

Ovine reference materials and assays for prion genetic testing.

Heaton M, Leymaster K, Kalbfleisch T, Freking B, Smith T, Clawson M BMC Vet Res. 2010; 6:23.

PMID: 20433741 PMC: 2876147. DOI: 10.1186/1746-6148-6-23.

References

Vaccari G, Conte M, Morelli L, Di Guardo G, Petraroli R, Agrimi U . Primer extension assay for prion protein genotype determination in sheep. Mol Cell Probes. 2004; 18(1):33-7. DOI: 10.1016/j.mcp.2003.06.001. View

Prusiner S . Prions. Proc Natl Acad Sci U S A. 1998; 95(23):13363-83. PMC: 33918. DOI: 10.1073/pnas.95.23.13363. View

Billinis C, Psychas V, Leontides L, Spyrou V, Argyroudis S, Vlemmas I . Prion protein gene polymorphisms in healthy and scrapie-affected sheep in Greece. J Gen Virol. 2004; 85(Pt 2):547-554. DOI: 10.1099/vir.0.19520-0. View

Buitkamp J, Semmer J . A robust, low- to medium-throughput prnp genotyping system in sheep. BMC Infect Dis. 2004; 4:30. PMC: 517712. DOI: 10.1186/1471-2334-4-30. View

Alvarez L, Arranz J, San Primitivo F . Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep. J Anim Sci. 2006; 84(2):259-65. DOI: 10.2527/2006.842259x. View

Hunter N, Goldmann W, Foster J, Cairns D, Smith G . Natural scrapie and PrP genotype: case-control studies in British sheep. Vet Rec. 1997; 141(6):137-40. DOI: 10.1136/vr.141.6.137. View

Goldmann W, Baylis M, Chihota C, Stevenson E, Hunter N . Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes. J Appl Microbiol. 2005; 98(6):1294-302. DOI: 10.1111/j.1365-2672.2005.02568.x. View

French D, Archard C, Andersen M, McDowell D . Ultra-rapid DNA analysis using HyBeacon probes and direct PCR amplification from saliva. Mol Cell Probes. 2002; 16(5):319-26. DOI: 10.1006/mcpr.2002.0425. View

Orge L, Galo A, Machado C, Lima C, Ochoa C, Silva J . Identification of putative atypical scrapie in sheep in Portugal. J Gen Virol. 2004; 85(Pt 11):3487-3491. DOI: 10.1099/vir.0.80246-0. View

10.

Belt P, Muileman I, Schreuder B, Gielkens A, Smits M . Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J Gen Virol. 1995; 76 ( Pt 3):509-17. DOI: 10.1099/0022-1317-76-3-509. View

11.

Van Poucke M, Vandesompele J, Mattheeuws M, Van Zeveren A, Peelman L . A dual fluorescent multiprobe assay for prion protein genotyping in sheep. BMC Infect Dis. 2005; 5:13. PMC: 1274271. DOI: 10.1186/1471-2334-5-13. View

12.

Gombojav A, Ishiguro N, Horiuchi M, Serjmyadag D, Byambaa B, Shinagawa M . Amino acid polymorphisms of PrP gene in Mongolian sheep. J Vet Med Sci. 2003; 65(1):75-81. DOI: 10.1292/jvms.65.75. View

13.

Vallone P, Benight A . Melting studies of short DNA hairpins containing the universal base 5-nitroindole. Nucleic Acids Res. 1999; 27(17):3589-96. PMC: 148605. DOI: 10.1093/nar/27.17.3589. View

14.

Margraf R, Mao R, Wittwer C . Masking selected sequence variation by incorporating mismatches into melting analysis probes. Hum Mutat. 2006; 27(3):269-78. DOI: 10.1002/humu.20290. View

15.

Moum T, Olsaker I, Hopp P, Moldal T, Valheim M, Moum T . Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases. J Gen Virol. 2004; 86(Pt 1):231-235. DOI: 10.1099/vir.0.80437-0. View

16.

Storm N, Van Den Boom D, Rodi C . MALDI-TOF mass spectrometry-based SNP genotyping. Methods Mol Biol. 2002; 212:241-62. DOI: 10.1385/1-59259-327-5:241. View

17.

Benestad S, Sarradin P, Thu B, Schonheit J, Tranulis M, Bratberg B . Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec. 2003; 153(7):202-8. DOI: 10.1136/vr.153.7.202. View

18.

Goldmann W, Hunter N, Foster J, Salbaum J, Beyreuther K, Hope J . Two alleles of a neural protein gene linked to scrapie in sheep. Proc Natl Acad Sci U S A. 1990; 87(7):2476-80. PMC: 53712. DOI: 10.1073/pnas.87.7.2476. View

19.

French D, McDowell D, Debenham P, Gale N, Brown T . HyBeacon probes for rapid DNA sequence detection and allele discrimination. Methods Mol Biol. 2008; 429:171-85. DOI: 10.1007/978-1-60327-040-3_12. View

20.

Schutz E, Scharfenstein M, Brenig B . Genotyping of ovine prion protein gene (PRNP) variants by PCR with melting curve analysis. Clin Chem. 2006; 52(7):1426-9. DOI: 10.1373/clinchem.2006.069666. View