Quantification of damage in DNA recovered from highly degraded samples--a case study on DNA in faeces.
Quantification of damage in DNA recovered from highly degraded samples--a case study on DNA in faeces.
复制标题
从高度降解的样品中回收的DNA损伤的量化 - 对粪便中DNA的案例研究。
DOI：
10.1186/1742-9994-3-11
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发表时间：
2006-08-16
影响因子：
2.8
通讯作者：
Jarman SN
中科院分区：
文献类型：
--
作者：
Deagle BE;Eveson JP;Jarman SN
保存不良的生物组织已成为广泛动物学研究的重要DNA来源。通常需要测量从这些样品中获得的DNA的质量；然而，目前还没有广泛使用的技术来量化高度降解DNA样本中的损伤。我们提出了一种通用的方法，可以用来确定聚合酶阻断DNA损伤的频率在特定的基因区域在这样的样品。该方法使用定量PCR来测量样品中存在的几个片段大小的DNA量。根据随机退化模型，可用模板的数量随着受损样本中片段大小的增加呈指数下降，通过确定下降率可以估计DNA损伤的频率（λ）。通过分析从海狮粪便样本中提取的DNA来说明该方法。粪便中含有多种来源的DNA的复杂混合物，预计不同的成分会被不同地降解。我们估计了单个粪便样本中捕食者和猎物DNA中DNA损伤的频率。每个目标的片段长度分布与随机降解过程的假设很好地吻合，并且与我们的预期一致，在同一样本中，捕食者DNA的估计损伤频率总是小于猎物DNA（平均λ捕食者= 0.0106个核苷酸；平均λ猎物= 0.0176个核苷酸）。这项研究首次明确定义了从粪便中提取的任何DNA中模板损伤的数量，并首次量化了个体粪便样本中存在的捕食者和猎物DNA的数量。我们提出了一种方法来表征混合的，高度降解的PCR模板，如那些在生态研究中经常遇到的，使用非侵入性样本作为DNA来源，野生动物法医调查和古代DNA研究。这种方法将允许研究人员测量模板质量，以便评估DNA的替代来源、不同的样品保存方法和不同的DNA提取方案。这项技术也可以应用于研究DNA衰变的过程。
Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ) can be estimated by determining the rate of decline. The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide). This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will allow researchers to measure template quality in order to evaluate alternate sources of DNA, different methods of sample preservation and different DNA extraction protocols. The technique could also be applied to study the process of DNA decay.