Cloning and mapping of variety-specific rice genomic DNA sequences: Amplified fragment length polymorphisms (AFLP) from silver-stained polyacrylamide gels
Cloning and mapping of variety-specific rice genomic DNA sequences: Amplified fragment length polymorphisms (AFLP) from silver-stained polyacrylamide gels
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DOI：
10.1139/g96-048
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发表时间：
1996-04-01
期刊：
影响因子：
3.1
通讯作者：
McCouch, SR
中科院分区：
文献类型：
--
作者：
Cho, YG;Blair, MW;McCouch, SR
开发了一种从银染变性聚丙烯酰胺凝胶中克隆 DNA 的有效技术，可以分离从选择性限制性片段扩增 (SRFA) 中获得的特定条带。事实证明，该方法与从同一凝胶中克隆放射性标记的 SRFA 条带一样可靠。使用相同的 PCR 曲线，使用带有或不带有 [P-32]dCTP 的水稻 DNA 作为模板。使用变性聚丙烯酰胺凝胶电泳分离扩增产物，并通过凝胶银染色或通过 P-32 标记产物的放射自显影来可视化。我们通过一轮 PCR 扩增直接从变性聚丙烯酰胺凝胶中克隆了特定的多态性 SRFA 条带，并确认银染凝胶中的条带序列与相应的 P-32 标记条带相同。所选条带代表粳稻和籼稻品种之间的扩增片段长度多态性 (AFLP)。我们通过将两个克隆的 AFLP 带映射为种间水稻群体中的 RFLP，研究了它们作为可遗传遗传标记的能力，并发现它们代表了水稻基因组中独特位点的单拷贝 DNA。
An efficient technique for cloning DNA from silver-stained denaturing polyacrylamide gels was developed to allow the isolation of specific bands obtained from selective restriction fragment amplification (SRFA). This method proved as reliable as cloning radioactively labelled SRFA bands from the same gels. Rice DNA was used as a template, both with and without [P-32]dCTP, using the same PCR profiles. Amplified products were separated using denaturing polyacryamide gel electrophoresis and visualized either by silver staining of gels or by autoradiography of P-32-labelled products. We cloned specific polymorphic SRFA bands directly from the denaturing polyacrylamide gels with one round of PCR amplification and confirmed that the sequences of the bands from silver-stained gels were identical to the corresponding P-32-labelled bands. The bands that were chosen represented amplified fragment length polymorphisms (AFLPs) between japonica and indica rice varieties. We studied the ability of two cloned AFLP bands to serve as heritable genetic markers by mapping them as RFLPs in an interspecific rice population and found that they represented single-copy DNA at unique loci in the rice genome.