Slade R W, Moritz C, Heideman A, Hale P T
Department of Zoology, University of Queensland, Brisbane, Australia.
Mol Ecol. 1993 Dec;2(6):359-73. doi: 10.1111/j.1365-294x.1993.tb00029.x.
We investigated the use of PCR primers designed to conserved exons within nuclear DNA to amplify potentially variable regions such as introns or hypervariable exons from a wide range of species. We then explored various approaches to assay population-level variation in these PCR products. Primers designed to amplify regions within the histone H2AF, myoglobin, MHC DQA, and aldolase (ALD) genes gave clean amplifications in diverse mammals (DQA), and in birds, reptiles and mammals (aldolase, H2AF, myoglobin). The sequenced PCR products generally, but not always, confirmed that the correct locus had been amplified. Several primer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequencing seal and reptile H2AF PCR products. Digestion with randomly selected four-base recognizing enzymes detected variation in some cases but not in others. In species/gene combinations with either low (e.g. seal H2AF, ALD-A) or high (e.g. skink ALD-1) nucleotide diversity it was more efficient to sequence a small number of distantly related individuals (e.g. one per geographic population) and from these data to identify informative or potentially informative restriction enzymes for 'targeted' digestion. We conclude that for studies of population-level variation, the optimal approach is to use a battery of primers for initial PCR of both mtDNA and scnDNA loci, select those that give clean amplifications, and sequence one sample from each population to (i) confirm gene identity, (ii) estimate the amount of variation and, (iii) search for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study.
我们研究了使用针对核DNA内保守外显子设计的PCR引物，以扩增来自广泛物种的潜在可变区域，如内含子或高变外显子。然后，我们探索了各种方法来检测这些PCR产物中的群体水平变异。设计用于扩增组蛋白H2AF、肌红蛋白、MHC DQA和醛缩酶（ALD）基因内区域的引物，在不同的哺乳动物（DQA）以及鸟类、爬行动物和哺乳动物（醛缩酶、H2AF、肌红蛋白）中均能得到清晰的扩增产物。测序的PCR产物通常（但并非总是）证实扩增的是正确的位点。几组引物产生了较小的片段，这与无内含子假基因的优先扩增一致；这一点通过对海豹和爬行动物H2AF PCR产物的测序得到了证实。用随机选择的识别四碱基的酶进行消化，在某些情况下能检测到变异，但在其他情况下则不能。在核苷酸多样性低（如海豹H2AF、ALD - A）或高（如石龙子ALD - 1）的物种/基因组合中，对少数远缘个体（如每个地理群体一个）进行测序，并根据这些数据识别用于“靶向”消化的信息性或潜在信息性限制酶，效率更高。我们得出结论，对于群体水平变异的研究，最佳方法是使用一组引物对mtDNA和scnDNA位点进行初始PCR，选择那些能得到清晰扩增产物的引物，并对每个群体的一个样本进行测序，以（i）确认基因身份，（ii）估计变异量，以及（iii）寻找诊断性限制位点。这将有助于确定大规模研究的最有效方法。
