Design factors that influence PCR amplification success of cross-species primers among 1147 mammalian primer pairs
Design factors that influence PCR amplification success of cross-species primers among 1147 mammalian primer pairs
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DOI：
10.1186/1471-2164-7-253
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发表时间：
2006-10-09
期刊：
影响因子：
4.4
通讯作者：
Venta, Patrick J.
中科院分区：
文献类型：
--
作者：
Housley, Donna J. E.;Zalewski, Zachary A.;Venta, Patrick J.
背景资料：跨物种引物已被用于解决有关基因组结构，进化和基因功能的各种问题，并取得了一定的成功。然而，影响其成功的因素从未得到充分解决，特别是在产生一致的方法以实现高通量方面。使用1，147个哺乳动物跨物种引物对（1089个以前未报道），我们测试了几个因素，以确定它们对给定靶标在单一扩增条件下在给定物种中扩增的概率的影响。这些因素包括：两个物种之间的错配数量用于鉴定引物设计的保守区域、基因和扩增区域的GC含量、引物区域中的CpG二核苷酸、编码蛋白的保守程度、引物长度以及目标物种与两个指示物种之间的进化距离。两个指示种之间的错配数量显著影响跨种引物的扩增成功率（引物对中每个错配降低6-8%），扩增区域内的GC含量（狗的GC >= 50%，56.9%扩增; GC <50%，74.2%扩增）、蛋白质保守程度（R-2 = 0.14）和目标种与指示种的亲缘关系。对于狗，对930个引物对的598个产物（64.3%）（不包括以狗为指标物种的引物）进行了测序，结果显示是预期产物，另外3%产生了错误序列。当仓鼠DNA与单一扩增条件下使用的微量滴定板为基础的格式，510 1087引物对（46.9%）产生扩增产物。引物对在人类基因组中以2.3 Mb的平均距离间隔，并且可用于产生高达数十万bp的物种特异性序列。影响成功扩增比例的最重要因素是指标物种错配的数量、目标扩增物的GC丰富度以及目标物种与指标物种的相关性，至少在所用的单一PCR条件下。1147个跨物种引物对可以以高通量的方式用于产生用于未测序的哺乳动物基因组的遗传学和基因组学研究的数据。
Background: Cross-species primers have been used with moderate success to address a variety of questions concerning genome structure, evolution, and gene function. However, the factors affecting their success have never been adequately addressed, particularly with respect to producing a consistent method to achieve high throughput. Using 1,147 mammalian cross-species primer pairs (1089 not previously reported), we tested several factors to determine their influence on the probability that a given target will amplify in a given species under a single amplification condition. These factors included: number of mismatches between the two species (the index species) used to identify conserved regions to which the primers were designed, GC-content of the gene and amplified region, CpG dinucleotides in the primer region, degree of encoded protein conservation, length of the primers, and the degree of evolutionary distance between the target species and the two index species.Results: The amplification success rate for the cross-species primers was significantly influenced by the number of mismatches between the two index species (6-8% decrease per mismatch in a primer pair), the GC-content within the amplified region (for the dog, GC >= 50%, 56.9% amplified; GC < 50%, 74.2% amplified), the degree of protein conservation (R-2 = 0.14) and the relatedness of the target species to the index species. For the dog, 598 products of 930 primer pairs (64.3%) ( excluding primers in which dog was an index species) were sequenced and shown to be the expected product, with an additional three percent producing the incorrect sequence. When hamster DNA was used with the single amplification condition in a microtiter plate-based format, 510 of 1087 primer pairs (46.9%) produced amplified products. The primer pairs are spaced at an average distance of 2.3 Mb in the human genome and may be used to produce up to several hundred thousand bp of species-specific sequence.Conclusion: The most important factors influencing the proportion of successful amplifications are the number of index species mismatches, GC-richness of the target amplimer, and the relatedness of the target species to the index species, at least under the single PCR condition used. The 1147 cross-species primer pairs can be used in a high throughput manner to generate data for studies on the genetics and genomics of non-sequenced mammalian genomes.