Improvement of the dot-blot-SNP technique for efficient and cost-effective genotyping
Improvement of the dot-blot-SNP technique for efficient and cost-effective genotyping
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DOI：
10.1007/s11032-009-9315-6
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发表时间：
2010-01-01
影响因子：
3.1
通讯作者：
Nishio, Takeshi
中科院分区：
文献类型：
--
作者：
Shiokai, Sachiko;Shirasawa, Kenta;Nishio, Takeshi
尽管斑点印迹-SNP技术是用于大量植物的SNP基因分型的节省劳动力、成本有效的方法，但用作探针的5 ′-地高辛（DIG）标记的寡核苷酸的合成仍然是昂贵的。我们为此技术开发了两种探针标记方法，一种是通过PCR进行洋地黄毒苷标记寡核苷酸（PCR-DIG标记），另一种是使用桥探针和5 '-DIG标记的寡核苷酸进行杂交（桥杂交）。在与5 ′-DIG标记的寡核苷酸和生物素标记的寡核苷酸类似的杂交条件下，桥式杂交检测到等位基因特异性信号，而通过PCR-DIG标记仅在较低严格条件下检测到信号。作为一次使用多个标记的基因分型方法，可以使用两种方法，即，采用混合引物对的PCR和采用混合探针的杂交，均获得成功的结果。设计了85个SNP标记用于水稻品种的基因分型，检测到等位基因特异性信号，基因分型结果与以前报道的结果一致。
Although the dot-blot-SNP technique is a laborsaving, cost-effective method for SNP genotyping of a large number of plants, the synthesis of 5'-digoxigenin (DIG)-labeled oligonucleotides for use as probes is still costly. We developed two probe-labeling methods for this technique, one being digoxigenin labeling of oligonucleotides by PCR (PCR-DIG labeling) and the other being hybridization using a bridge probe and a 5'-DIG-labeled oligonucleotide (bridge hybridization). Bridge hybridization detected allele-specific signals under hybridization conditions similar to those for the 5'-DIG-labeled oligonucleotides and biotin-labeled oligonucleotides, while signals were detected only under a lower stringency condition by PCR-DIG labeling. As a method for genotyping using many markers at one time, two methods, i.e., PCR using mixed primer pairs and hybridization using mixed probes, were examined with successful results. Eighty-five SNP markers designed for genotyping of rice cultivars detected allele-specific signals, the genotyping results corresponding to the previously reported ones.