Haymes Kenneth M, Ibrahim Ibrahim A, Mischke Sue, Scott David L, Saunders James A
Alternate Crops and Systems Laboratory, Plant Sciences Institute, Beltsville Agricultural Research Center, Building 50, Room 100, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705, USA.
J Agric Food Chem. 2004 Aug 25;52(17):5456-62. doi: 10.1021/jf0497962.
DNA isolation from plants is sometimes difficult due to the existence of high levels of endogenous phenolics, polysaccharides, or other substances that may interfere with DNA extraction. Theobroma cacao produces high levels of anthocyanins in young leaves. These plant polyphenols can interfere with DNA isolation. After examination of various procedures for DNA isolation, two commercial isolation procedures have proved to be repeatedly successful using these types of plants, the D(2) BioTechnologies DNA X-tract Plus kit and the Qiagen DNeasy Plant System DNA kit. These commercial kits were chosen for their speed and ease over the CTAB procedure, which is more labor intensive. All protocols assessed yielded DNA suitable for AFLP or SSR procedures. An additional factor in DNA extraction efficiency is the degree of cell breakage, which may be more difficult with the highly fibrous leaf tissue that is found in many monocots, including date palm. Two commercially produced pieces of equipment were tested and, for cacao, both resulted in template DNA yielding amplification product in AFLP or SSR fingerprinting. However, for the fibrous date palm leaf, the larger FastPrep homogenizer consistently yielded DNA that generated higher signals when amplified than did the smaller Disruptor Genie.
由于植物中存在高水平的内源性酚类、多糖或其他可能干扰DNA提取的物质，从植物中分离DNA有时会很困难。可可树在幼叶中产生高水平的花青素。这些植物多酚会干扰DNA分离。在对各种DNA分离程序进行检查后，两种商业分离程序已被证明在使用这类植物时反复取得成功，即D(2)生物技术公司的DNA X-tract Plus试剂盒和Qiagen公司的DNeasy植物系统DNA试剂盒。选择这些商业试剂盒是因为它们比CTAB程序更快且更简便，而CTAB程序劳动强度更大。所有评估的方案都产生了适用于AFLP或SSR程序的DNA。DNA提取效率的另一个因素是细胞破碎程度，对于许多单子叶植物（包括枣椰树）中发现的高度纤维化的叶片组织来说，这可能更困难。测试了两种商业生产的设备，对于可可树，两者都能产生在AFLP或SSR指纹分析中产生扩增产物的模板DNA。然而，对于纤维化的枣椰树叶片，较大的FastPrep匀浆器始终能产生在扩增时比小的Disruptor Genie产生更高信号的DNA。
