Data from: Molecular profiling of diatom assemblages in tropical lake sediments using taxon-specific PCR and Denaturing High-Performance Liquid Chromatography (PCR-DHPLC)

Here we present a protocol to genetically detect diatoms in sediments of the Kenyan tropical Lake Naivasha, based on taxon-specific PCR amplification of short fragments (approx. 100 bp) of the small subunit ribosomal (SSU) gene and subsequent separation of species-specific PCR products by PCR-based DHPLC. An evaluation of amplicons differing in primer specificity to diatoms and length of the fragments amplified demonstrated that the number of different diatom sequence types detected after cloning of the PCR products critically depended on the specificity of the primers to diatoms and the length of the amplified fragments whereby shorter fragments yielded more species of diatoms. The DHPLC was able to discriminate between very short amplicons based on the sequence difference, even if the fragments were of identical length. and if the amplicons differed only in a small number of nucleotides. Generally the method identified the dominant sequence types from mixed amplifications. A comparison with microscopic analysis of the sediment samples revealed that the sequence types identified in the molecular assessment corresponded well with the most dominant species. In summary, the PCR-based DHPLC protocol offers a fast, reliable, and cost-efficient possibility to study DNA from sediments and other environmental samples with unknown organismic content, even for very short DNA fragments.本研究提出了一种针对肯尼亚热带奈瓦沙湖沉积物中硅藻的遗传检测方案，该方案基于小亚基核糖体（SSU）基因的短片段（约100 bp）的类群特异性聚合酶链式反应（PCR）扩增，以及后续通过基于PCR的变性高效液相色谱（DHPLC）分离物种特异性PCR产物。本研究针对引物对硅藻的特异性及扩增片段长度存在差异的扩增子开展评估，结果显示，对PCR产物进行克隆后，所检测到的不同硅藻序列类型数量，主要取决于引物对硅藻的特异性以及扩增片段的长度，其中较短的片段可鉴定出更多硅藻物种。即便片段长度完全一致，DHPLC也可基于序列差异区分极短的扩增子；若扩增子仅在少数核苷酸位点存在差异，同样可实现有效区分。总体而言，该方法可从混合扩增产物中识别出占主导地位的序列类型。将该方法与沉积物样本的显微镜分析结果进行对比后发现，分子检测中鉴定出的序列类型与样本中最优势的硅藻物种吻合度极佳。综上，基于PCR的DHPLC方案为研究沉积物及其他未知生物组成的环境样本中的DNA提供了一种快速、可靠且经济高效的途径，即便针对极短的DNA片段亦适用。