| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ANIMAL GENETICS |
Cell Biology Group, Agriculture and Life Sciences Division, Lincoln University, Canterbury, New Zealand
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionLiterature Cited |
|---|
Key Words: Polymorphism • Prion Protein Gene (PRNP) • Sheep • Single-Strand Conformational Polymorphism
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionLiterature Cited |
|---|
, 1996). Genetic susceptibility to scrapie seems to be influenced by variation in the prion gene (PRNP; Goldmann et al., 1994; Hunter et al., 1996). In sheep, variation in the PRNP gene has been identified at a number of codons, but polymorphism at some codons is rare and only three codons (136, 154, and 171) have a reported linkage with the incidence of scrapie (reviewed by Baylis and Goldman, 2004). The association between ovine PRNP polymorphism at codons 136, 154, and 171 and scrapie susceptibility is the basis for the marker-assisted breeding for decreased scrapie susceptibility now underway in many countries (Tranulis, 2002). Various PCR-based approaches have been used to determine ovine PRNP sequences at codons 136, 154, and 171. These include direct DNA sequencing (Tranulis et al., 1999; Vaccari et al., 2001; Baylis et al., 2002; Sipos et al., 2002), RFLP (Hunter et al., 1993; Ikeda et al., 1995), allele-specific oligonucleotide hybridization (Ishiguro et al., 1998), and primer extension assay (Vaccari et al., 2004). However, none of these methods allows for accurate determination of the PRNP haplotype, as they either do not profile more than a fragment of the gene or require sequencing, which can be confounded in heterozygous sheep. In addition, the recent identification of the novel haplotypes A136H154R171 and VRR in sheep (Kutzer et al., 2002) makes the results from the current typing methods difficult or sometimes impossible to interpret. Accordingly, a technique that is able to determine accurately and rapidly the PRNP genotype of sheep is needed. We investigate allelic polymorphism of the ovine PRNP gene using PCR–single-strand conformational polymorphism (SSCP) analysis, with the aim of developing an accurate typing method for the ovine PRNP gene.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionLiterature Cited |
|---|
A total of 400 sheep from six different breeds (Awassi, Borderdale, Corriedale, Finnish Landrace, Merino, and Romney) in New Zealand were used to screen for genetic variation in the PRNP gene. The DNA was isolated from blood using a high-salt procedure as described by Montgomery and Sise (1990), or was extracted from blood collected on FTA Classic cards (Whatman BioScience, Middlesex, U.K.), following the manufacturer’s protocol.
PCR Amplification
Two PCR primers were designed based on published GenBank sequences to amplify a 173-bp fragment of exon 3 of the ovine PRNP gene covering the three codons considered important for scrapie susceptibility. These primers were PRNP-up (5'-GGTGGCTACATGCTGG-GAAGT-3') and PRNP-dn (5'-GTGATGTTGACACAGT-CATGCAC-3'), corresponding to codons 129 to 135 and codons 179 to 186, respectively. Primers were synthesized by Proligo (Boulder, CO).
Amplifications were performed in a 20-µL reaction containing 50 ng of genomic DNA extracted from whole blood or genomic DNA on a 1.2-mm diameter circular punch from blood on an FTA card, 0.25 µM of each primer, 150 µM nucleotides (Eppendorf, Hamburg, Germany), 0.5 U Taq DNA polymerase, and 1x the reaction buffer supplied with the enzyme (containing 1.5 mM MgCl2) (Qiagen, Hilden, Germany). Amplification was carried out in an iCycler (Bio-Rad Laboratories, Hercules, CA) and consisted of denaturation at 94°C for 2 min, followed by 32 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. This was followed by a final extension step at 72°C for 5 min. Amplimers were visualized by electrophoresis in 1% Seakem LE agarose (BioWhittaker Molecular Applications, Rockland, ME) gels, using 1x TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA) containing 200 ng of ethidium bromide/mL.
Single-Strand Conformational Polymorphism Analysis
A 0.7-µL aliquot of each amplimer was mixed with 7 µL of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene-cyanol), and after denaturation at 95°C for 5 min, samples were rapidly cooled on wet ice and then loaded on 16 cm x 18 cm, 14.5% acrylamide:bisacrylamide (37.5:1; Bio-Rad Laboratories) gels containing 0.2% glycerol. Electrophoresis was performed using Protean II xi cells (Bio-Rad Laboratories), at 420 V for 18 h at 4°C in 0.5x TBE buffer, and gels were silver-stained according to the method of Bassam et al. (1991).
Cloning of PCR Amplimers and Screening of Clones
Amplimers representative of the unique SSCP patterns were cloned into a pGEM T-Easy vector (Promega, Madison, WI), and transformed into competent Escherichia coli cells, following the protocol recommended by the manufacturer. For each transformation, 6 or 12 clones, for single or double SSCP patterns respectively, were selected and incubated overnight in Terrific broth (Invitrogen, Carlsbad, CA) at 37°C in a shaking rotary incubator (225 rpm).
Plasmids were recovered from bacterial cells by boiling for 10 min in 0.8% Trixton X-100 solution and 1 µL of the supernatant fraction was used as a template for PCR under the conditions described previously. Amplimers from these clones and the corresponding genomic DNA were run adjacent to each other on SSCP gels for comparison of the banding patterns, and only those clones for which the banding patterns matched those of the corresponding genomic DNA were selected for subsequent DNA sequencing.
DNA Sequencing and Sequence Analysis
Plasmids from the selected clones were extracted using a QIAprep Spin Miniprep kit (Qiagen), and were then sequenced in both directions using M13 forward and reverse primers at the Waikato DNA Sequencing Facility at the University of Waikato, New Zealand. Identical sequences obtained from at least three clones from different sheep, or independent PCR amplifications from the same sheep, were subjected to further sequence analysis.
Sequence analysis was performed using DNAMAN (v. 4.0, Lynnon BioSoft, Vaudreuil, Canada). The BLAST algorithm was used to search the NCBI Gen-Bank databases (http://www.ncbi.nlm.nih.gov/) for homologous sequences.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionLiterature Cited |
|---|
). One or two patterns were observed for each animal, which is consistent with homozygous and heterozygous genotypes.
|
). The nucleotide differences between these sequences were small, and only one or two nucleotide differences were typically detected. This indicates that SSCP analysis is an accurate tool for identifying small sequence variations in DNA fragments.
|
). Of the nine ovine PRNP allelic sequences identified, one sequence (ARQ-T142) was new. This allelic sequence had the uncharged polar AA threonine (T) at position 142, compared with the nonpolar AA isoleucine in other alleles. This polymorphism has not been reported previously. The effect of this polymorphism on scrapie susceptibility is currently unknown. The sequence of this allele has been deposited into the NCBI GenBank and assigned the Accession No. AY730557.
Individual ovine PRNP alleles exhibited unique SSCP patterns and could be readily differentiated by SSCP analysis. This suggests that this could be used as an alternative method for typing the gene. This was assessed by using the method to genotype 10 animals from one three-generation mixed-breed full-sib family belonging to the International Mapping Flock (Crawford et al., 1995). The SSCP patterns of the PRNP gene segregated in a manner consistent with Mendelian inheritance (Figure 3).
|
). Second, the resultant SSCP profiles are uncomplicated and can be easily interpreted either manually or automatically with a gel reader. Third, the technique allows for the visualization of individual amplimers, and thus can readily identify erroneous results, including the presence of contaminating DNA sequences or unexpected amplification products. Fourth, because the PCR-SSCP technique scans the whole sequence of the gene region amplified, it has the potential to identify unknown or new allelic variation. Finally, the PCR-SSCP approach is typically less expensive, particularly when large numbers of samples require analysis. To our knowledge, this is the first report that shows the use of SSCP in differentiating sheep PRNP alleles. This approach will be useful for genotyping this gene both in terms of accuracy and efficiency.
|
Footnotes |
|---|
2 Correspondence: P. O. Box 84 (phone: +64-3-325 2811; fax: +64-3-325 3851; e-mail: hickford{at}lincoln.ac.nz).
Received for publication November 30, 2004. Accepted for publication January 19, 2005.
|
Literature Cited |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionLiterature Cited |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. G. H. Hickford, H. Zhou, Q. Fang, S. O. Byun, and H. Gong Frequency of PRNP genotypes in common New Zealand sheep breeds Vet Rec., October 11, 2008; 163(15): 453 - 454. [Full Text] [PDF]
|
|
|
|
 |
|
 G. Vaccari, C. D'Agostino, R. Nonno, F. Rosone, M. Conte, M. A. Di Bari, B. Chiappini, E. Esposito, L. De Grossi, F. Giordani, et al. Prion Protein Alleles Showing a Protective Effect on the Susceptibility of Sheep to Scrapie and Bovine Spongiform Encephalopathy J. Virol., July 1, 2007; 81(13): 7306 - 7309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. H. Hickford, H. Zhou, and Q. Fang Haplotype analysis of the DQA genes in sheep: Evidence supporting recombination between the loci J Anim Sci, March 1, 2007; 85(3): 577 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Kunkle, J. M. Miller, D. P. Alt, R. C. Cutlip, N. E. Cockett, S. Wang, J. A. Richt, B. V. Thomsen, and S. Hall Determination of Sheep Prion Gene Polymorphisms from Paraffin-embedded Tissue J Vet Diagn Invest, September 1, 2006; 18(5): 443 - 447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Alvarez, J. J. Arranz, and F. S. Primitivo Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep J Anim Sci, February 1, 2006; 84(2): 259 - 265. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), ARQ-R143 (black box with white ring), ARQ-F141 (black box with white circle), and AHQ (
) were identified in this family. All these patterns segregated in a Mendelian fashion.