Detection of plant genes using a rapid, nonorganic DNA purification method
Detection of plant genes using a rapid, nonorganic DNA purification method
复制标题
DOI：
10.2144/00282pf01
复制
发表时间：
2000-02-01
期刊：
影响因子：
2.7
通讯作者：
Saunders, JA
中科院分区：
文献类型：
--
作者：
Lin, JJ;Fleming, R;Saunders, JA
我们开发了一种使用 FTA 纸制备植物基因组 DNA 的简单程序。将植物叶子压碎在 FTA 纸上；使用简单的无机试剂纯化基因组 DNA。通过 FTA 纸上的 DNA PCR 扩增检测叶绿体基因组中的 18S rRNA 基因和编码核酮糖-1、5-二磷酸羧化酶/加氧酶大亚基 (rbcL) 的基因：使用 16 种双子叶植物和单子叶植物的提取物成功进行了 DNA 扩增。对特定植物提取物的研究表明，可以收集叶子样品的提取物并在室温下在 FTA 纸上凝视一个多月，DNA 扩增成功率不会降低。在以这种方式储存的各种大豆品种中分离的基因组 DNA 中均检测到了 18S RNA 基因和 rbcL 基因：此外，通过适度增加 DNA 扩增循环数，我们能够使用 FTA 纸上分离的基因组 DNA 检测转基因烟草和水稻叶片中的 uidA 基因以及与胞囊线虫小种 3 的抗性基因连锁的单拷贝基因。这些结果表明，使用 FTA 纸分离的基因组 DNA 可用于检测植物基因，从广泛的角度具有高或低基因拷贝数以及核或细胞质起源的一系列植物。
We have developed a simple procedure for the preparation of plant genomic DNA using FTA paper Plant leaves were crushed against FTA paper; and the genomic DNA was purified using simple, nonorganic reagents. The 18S rRNA gene and the gene encoding the ribulose-l, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome were detected by PCR amplification of DNA on FTA paper: DNA amplification was successful using extracts from 16 dicot and monocot plants. studies of specific plant extracts revealed that extracts of leaf samples could be collected and stared at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. Both the 18S RNA gene and the rbcL gene were detected in the genomic DNA isolated from various soybean cultivars stored in this manner: Furthermore, by modestly increasing the number of cycles of DNA amplification, we were able to detect the uidA gene in transgenic tobacco and rice leaves as well as a single copy gene linked to the resistance gene of cyst nematode race 3 using genomic DNA isolated on FTA paper These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plant genes, from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.