Inexpensive, high throughput microplate format for plant nucleic acid extraction: Suitable for multiplex southern analyses of transgenes
Inexpensive, high throughput microplate format for plant nucleic acid extraction: Suitable for multiplex southern analyses of transgenes
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DOI：
10.2135/cropsci2004.0657
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发表时间：
2005-09-01
期刊：
影响因子：
2.3
通讯作者：
Matthews, PD
中科院分区：
文献类型：
--
作者：
Flagel, L;Christensen, JR;Matthews, PD
为了从少量植物组织中获得基因组DNA的高通量分析，通过连接一系列新的互锁板，将标准核酸提取格式化为微孔板。该方案将钢颗粒组织均质化和独特的液体处理和相分离技术与最小的特殊设备相结合。该方法已在啤酒花（Humulus lupulus L.）、大豆（Glycine mar (L.)）等难识别的植物dna中得到验证。稳定。]和烟草（Nicotiana tabacum L.），用琼脂糖凝胶分离限制性片段和Southern blot分析大豆转基因整合体。Southern印迹的成功率范围为每个样品（n = 2016个样品）的73%至92%。用PicoGreen微孔板荧光法定量测定绝对产量的变化。每板样品之间的产率变化很小，足以在Southern分析之前避免单个样品浓度调整。基于荧光法的大麦（Hordeum vulgare L.）定位群体的平均绝对产量为6.8 μ g +/- 0.85 SE, n = 96；为许多单独的聚合酶链式反应（PCR）提供了足够纯净、稳定的DNA。通过对先前鉴定的大麦定位群体进行简单序列重复（SSR）展示，验证了样品间交叉污染和PCR分析的适用性。根据材料细节计算的成本为每次样品提取0.17美元。虽然实现需要一些技巧和实践，但我们认为它是一个健壮的、可适应的、通用的昂贵商业套件的替代品。
To achieve high throughput analysis of genomic DNA from small amounts of plant tissue, a standard nucleic acid extraction was formatted to microplates by linking a novel series of interlocking plates. The protocol integrates steel pellet tissue homogenization and unique liquid handling and phase separation techniques with minimal special equipment. The method was validated for difficult plant DNAs, such as hop (Humulus lupulus L.), soybean [Glycine mar (L.) Merr.], and tobacco (Nicotiana tabacum L.), with agarose gel separations of restriction fragments and Southern blot analysis of transgene integrants in soybean. The range of success rates for Southern blots was 73 to 92% per sample per plate (n = 2016 samples). Variation in absolute yield was quantified by PicoGreen microplate flourimetry. The variation in yield among samples per plate was small enough to obviate individual sample concentration adjustment before Southern analysis. Average absolute yield for the barley (Hordeum vulgare L.) mapping population based on flourimetry was 6.8 mu g +/- 0.85 SE, n = 96; providing enough pure, stable DNA for many individual polymerase chain reaction (PCR) reactions. Intersample cross-contamination and suitability for PCR analysis were validated by simple sequence repeat (SSR) display of a previously characterized barley mapping population. Cost calculation from a materials detail was U.S. $0.17 per sample extraction. Although the implementation requires some skill and practice, we rind it to be a robust, adaptable, and versatile alternative to expensive commercial kits.