Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine PRNP gene using linear fluorescent probes and melting curve analysis

نویسندگان

  • David J French
  • Dominic Jones
  • David G McDowell
  • Jim A Thomson
  • Paul G Debenham
چکیده

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.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Genotyping common SNP and a microsatellite sequence closely linked to waxy gene in rice by DNA based markers

The potential of different DNA based molecular markers was examined for the detection of single nucleotide polymorphism (SNP) in the waxy gene and a microsatellite (SSR) sequence closely linked to it in a collection of rice varieties. DNA was extracted from leaf samples of 68 different rice cultivars by the CTAB method and specific primers were designed for the amplification of waxy gene and SS...

متن کامل

Association of IGF-I Gene Polymorphisms with Carcass Traits in Iranian Mehraban Sheep Using SSCP Analysis

Molecular genetics selection on individual genes is a promising method to genetically improve economically important traits in livestock. The insulin like growth factor-I (IGF-I) gene may play important roles in growth of multiple tissues, including muscle cells, cartilage and bone. The objectives of the present study were the estimate the haplotype frequencies of the IGF-I gene polymorphisms i...

متن کامل

DNA Polymorphisms at Candidate Gene Loci and Their Relation with Milk Production Traits in Murrah Buffalo (Bubalus bubalis)

DNA polymorphism within diacylglycerol transferase 2 (DGAT2) / monoacyl glycerol transferases 2 (MOGAT2), leptin and butyrophilin genes were analysed using PCR-SSCP in Murrah buffalo. The single strand conformation polymorphism (SSCP) analysis of amplified gene fragment in exon 5 of MOGAT2, exon 3 of leptin and intron 1 of butyrophilin gene revealed different patterns. A, B and C showed the fol...

متن کامل

The miR526b-5p-Related Single Nucleotide Polymorphisms, rs72618599, Located in 3\'-UTR of TCF3 Gene, is Associated with the Risk of Breast and Gastric Cancers

Introduction: Single nucleotide polymorphisms result in dysregulation of the proto-oncogene TCF3 gene, which is associated with the development, metastasis, and chemoresistance of different malignancies. Methods: GSE10810 microarray dataset and GEPIA2 online software were used to find differentially expressed genes and the TCF3 status in breast cancer (BC) and gastric cancer (GC), respectively....

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • BMC Infectious Diseases

دوره 7  شماره 

صفحات  -

تاریخ انتشار 2007