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Haplotypes determination and new variants within the ovine prion CDS region.

Haplotypes determination and new variants within the ovine prion CDS region. S. Marcos, J.H.Calvo,C. González & M.Serrano INIA. Madrid. Spain. The genotypes that control susceptibility and resistance are THE most important thing a producer wants know about scrapie genetics!.

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Haplotypes determination and new variants within the ovine prion CDS region.

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  1. Haplotypesdetermination and new variants within the ovine prion CDS region. S. Marcos, J.H.Calvo,C. González & M.Serrano INIA. Madrid. Spain

  2. The genotypes that control susceptibility and resistance are THE most important thing a producer wants know about scrapie genetics!

  3. Methods used :sequencingRFLP´sprimer extension Expensive for associations (next months)

  4. Provide a method: simple/intuitive cheeper than others easy to perform(minimum technical proficiency and instrumentation ) allows correct assignement of haplotypes

  5. A A G T SSCP´s (Orita et al 1989) Target DNA PCR denatured and separated sequence variants shift mobility

  6. 3F primer Codon 99 ORF Codon 206 136 154 171 3R primer Single Strand Conformational Polymorphisms (SSCP´s) PCR standar Ovine PRNP gene (GenBank U67922)

  7. Single Strand Conformational Polymorphisms (SSCP´s) Non-denaturant Polyacrylamide (49:1) 8% polyacrylamide 8,7% glycerol 10ºC 0,5X TBE 15 hours 8 watts

  8. ARR ARQ VRQ ARH/ AHQ/ ARH ARQ ARQ

  9. ARQ/ ARQ/ ARQ/ ARQ/ ARR/ ARH AHQ ARQ* ARQ ARR

  10. ARR/ ARR/ ARR/ ARR/ ARH AHQ ARQ* ARQ

  11. NEW VARIANTS Ovine PRNP gene (GenBank U67922) M-F primer ORF OPR primer 381R primer PCR standar: M-F and 381R Sequencing: OPR

  12. Bossers et al.2000

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