高效、新T-DNA侧翼序列分离技术--Actail-PCR

Actail-PCR—A New and Efficient Procedure for Isolation of Unknown Target Sequences Adjacent to T-DNA Border

【目的】建立一种有效分离T-DNA插入突变体侧翼基因组序列的方法。【方法】分离侧翼的目标基因组序列是利用T-DNA标签法研究植物功能基因组学的一个关键步骤,笔者发展稳定高效的Actail-PCR分离技术。该技术一侧引物来自于T-DNA载体,另一侧引物为一个复性控制引物。复性控制引物在3'端为一个14bp的随机简并引物,在5'端非目标尾部序列接上一个通用引物,中间由5个多聚脱氧次黄嘌呤核苷(poly(dI))连接。【结果】Actail-PCR技术将随机简并引物重新编辑成复性控制引物,在40℃的低严谨条件下,3'端的随机简并引物可以与T-DNA插入突变体的侧翼目标区段随机的结合,扩增得到目标片段,随后利用5'端的通用引物与T-DNA载体上的巢式引物依次组合,在65℃(10个循环后逐步降温至58℃)的高严谨条件下逐步扩增特异片段,同时抑制非特异性扩增,可以显著提高目标片段的扩增效率,减少假阳性。【结论】相对传统的TAIL-PCR,Actail-PCR技术可以更高效地分离T-DNA插入突变体的侧翼基因组序列。

【Objective】 This study aimed at establishing a protocol to identify unknown target sequences adjacent to T-DNA borders. 【Method】 DNA tagging by T-DNA insertions has become an important approach for study of functional genomics in plants. To identify the genes tagged by T-DNA insertions, a novel and efficient procedure,named as annealing control thermal asymmetric interlaced PCR （Actail-PCR）, was developed to isolate genomic sequences flanking the insertion tags. In this procedure, four nested sequence-specific primers from T-DNA were utilized. The other side primer was a annealing control primer （ACP）, which comprises a tripartite structure with a polydeoxyinosine （poly （dI）） linker between the 3＇ end shorter arbitrary degenerated primer sequence （AD） and the 5＇ end nontarget universal sequence. 【Result】 Annealing control primers were designed for Actail-PCR instead of shorter arbitrary degenerated primers of TAIL-PCR. At 40℃ low stringency, PCR cycle was conducted to create one or more annealing sites for the AD primer along the target sequence like TAIL-PCR, and then, target products were preferentially amplified over 5＇ end nontarget universal primer and nested sequence-specific primers from T-DNA at 65℃ （after 10 cycles, annealing temperature droped to 58℃） high-stringency, so the efficiency and specificity of PCR amplification were greatly improved. 【Conclusion】 An novel procedure for isolation of unknown target sequences adjacent to T-DNA in the rice has been established. Actail-PCR is more efficient and useful for identification of the genes tagged by T-DNA insertions