Wehausen J D, Ramey R R, Epps C W
University of California, White Mountain Research Station, 3000 East Line St., Bishop, CA 93514, USA.
J Hered. 2004 Nov-Dec;95(6):503-9. doi: 10.1093/jhered/esh068.
Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet materials, different amounts of fecal pellet material, and the effects of eliminating two DNA extraction steps for four microsatellite loci and four samples heterozygous at each locus. We evaluated 192 PCR outcomes for each treatment using indices of PCR success and peak height (signal strength) developed from analysis output of sequencer chromatograms. Outermost pellet material produced PCR results almost equivalent to DNA extracted from blood. Where any inner pellet material was used for DNA extraction, PCR results were poorer and inconsistent among samples. PCR success was not sensitive to amount of pellet material used until it was decreased to 15 mg from 60 mg. Our PCR index provides considerably more information relative to potential genotyping errors than simply comparing genotypes derived from paired fecal and blood or tissue samples. Our DNA extraction method probably has wide applicability to herbivores that produce pelleted feces where samples dry rapidly after deposition.
对于使用非侵入性DNA来源的研究而言，基因分型的可靠性是一个问题。我们强调优化DNA提取方法对于最大化此类DNA来源基因分型的可靠性和效率的重要性。我们提出了一种简单通用的方法，用于定量比较各种DNA提取技术和所用样品材料的基因分型可靠性。对于大角羊（加拿大盘羊）粪便样本，我们比较了不同的粪便颗粒材料、不同量的粪便颗粒材料，以及去除四个微卫星位点和每个位点杂合的四个样本的两个DNA提取步骤的效果。我们使用从测序仪色谱图分析输出中得出的PCR成功率和峰高（信号强度）指标，对每种处理的192个PCR结果进行了评估。最外层的颗粒材料产生的PCR结果几乎与从血液中提取的DNA相当。若使用任何内部颗粒材料进行DNA提取，PCR结果较差且样本间不一致。直到颗粒材料用量从60毫克降至15毫克，PCR成功率才对所用颗粒材料的量敏感。相对于简单比较来自配对粪便、血液或组织样本的基因型，我们的PCR指标能提供更多有关潜在基因分型错误的信息。我们的DNA提取方法可能广泛适用于产生颗粒状粪便且样本在沉积后迅速干燥的食草动物。
