Ovine reference materials and assays for prion genetic testing
Ovine reference materials and assays for prion genetic testing
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DOI：
10.1186/1746-6148-6-23
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发表时间：
2010-04-30
影响因子：
2.6
通讯作者：
Laegreid, William W.
中科院分区：
文献类型：
--
作者：
Heaton, Michael P.;Leymaster, Kreg A.;Laegreid, William W.
背景：绵羊瘙痒病的遗传易感性与朊病毒蛋白基因（PRNP）肽序列的多种变异有关。基于 DNA 的 PRNP 密码子评分测试是根除瘙痒症和评估罕见等位基因增强抗病能力的重要工具。除了与痒病相关的基因外，不同鸡群中还可能出现数十种 PRNP 多态性。如果不加以考虑，这些位点可能会导致 DNA 测试中使用的寡核苷酸碱基对不匹配。因此，通过了解目标羊群中 PRNP 多态性的位置和频率，可以提高痒病基因检测的保真度。 结果：开发了一种自适应 DNA 测序策略，以确定任何绵羊的 771 bp PRNP 编码序列，从而为目标羊群产生共有序列。该策略最初考虑了 43 个已知的多态性，并通过重叠的扩增子设计促进未知多态性的检测。该策略应用于美国多个品种的 953 只绵羊 DNA。样本包括两组参考羊：一组用于标准化 PRNP 基因测试，另一组用于发现多态性、估计等位基因频率和确定单倍型阶段。 DNA 测序揭示了 16 个先前未报道的多态性，包括 F-141 单倍型的 L237P 变体。开发了两种质谱多重检测方法来对美国绵羊的 5 个感兴趣的密码子进行评分：112、136、141、154 和 171。该项目的参考组织、DNA、跟踪文件和基因型可公开使用，不受限制。结论：识别目标羊群中的绵羊 PRNP 多态性对于设计有效的痒病基因检测系统至关重要。与参考 DNA 组合一起，这些信息有助于培训、认证以及新测试和知识的开发，从而加快根除羊瘙痒症的速度。
Background: Genetic predisposition to scrapie in sheep is associated with several variations in the peptide sequence of the prion protein gene (PRNP). DNA-based tests for scoring PRNP codons are essential tools for eradicating scrapie and for evaluating rare alleles for increased resistance to disease. In addition to those associated with scrapie, there are dozens more PRNP polymorphisms that may occur in various flocks. If not accounted for, these sites may cause base-pair mismatching with oligonucleotides used in DNA testing. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of PRNP polymorphisms in targeted flocks.Results: An adaptive DNA sequencing strategy was developed to determine the 771 bp PRNP coding sequence for any sheep and thereby produce a consensus sequence for targeted flocks. The strategy initially accounted for 43 known polymorphisms and facilitates the detection of unknown polymorphisms through an overlapping amplicon design. The strategy was applied to 953 sheep DNAs from multiple breeds in U. S. populations. The samples included two sets of reference sheep: one set for standardizing PRNP genetic testing and another set for discovering polymorphisms, estimating allele frequencies, and determining haplotype phase. DNA sequencing revealed 16 previously unreported polymorphisms, including a L237P variant on the F-141 haplotype. Two mass spectrometry multiplex assays were developed to score five codons of interest in U. S. sheep: 112, 136, 141, 154, and 171. Reference tissues, DNA, trace files, and genotypes from this project are publicly available for use without restriction.Conclusion: Identifying ovine PRNP polymorphisms in targeted flocks is critical for designing efficient scrapie genetic testing systems. Together with reference DNA panels, this information facilitates training, certification, and development of new tests and knowledge that may expedite the eradication of sheep scrapie.