Gutaker Rafal M, Reiter Ella, Furtwängler Anja, Schuenemann Verena J, Burbano Hernán A
Research Group for Ancient Genomics and Evolution, Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.
Institute of Archaeological Sciences, University of Tübingen, Tübingen, Germany.
Biotechniques. 2017 Feb 1;62(2):76-79. doi: 10.2144/000114517.
DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths. Extraction with N-phenacylthiazolium bromide (PTB) buffer decreased median fragment length by 35% when compared with cetyl-trimethyl ammonium bromide (CTAB); modifying the binding conditions of DNA to silica allowed for an additional decrease of 10%. We did not observe a further decrease in length for single-stranded DNA (ssDNA) versus double-stranded DNA (dsDNA) library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens, which will help to unlock the genetic information stored in herbaria.
从植物标本中提取的DNA高度碎片化；因此，使用能够提取短DNA分子的提取方案至关重要。动物遗骸的提取和DNA文库制备方案的改进使得能够高效提取短于50 bp的分子。在此，我们将这些改进应用于植物标本的DNA提取方案，并通过鸟枪法测序评估提取性能，鸟枪法测序能够准确估计DNA片段长度的分布。与十六烷基三甲基溴化铵（CTAB）相比，用N-苯甲酰基噻唑溴化物（PTB）缓冲液提取可使中位片段长度降低35%；改变DNA与二氧化硅的结合条件可使长度进一步降低10%。我们没有观察到单链DNA（ssDNA）与双链DNA（dsDNA）文库制备方法在长度上的进一步降低。我们的方案能够从植物标本中提取超短分子，这将有助于解锁植物标本中存储的遗传信息。
