从水稻Ac/Ds插入突变体扩增Ds侧翼序列的最适TAIL-PCR引物

温度非对称交互PCR(TAIL-PCR)技术已广泛应用于从多种生物体系克隆侧翼于已知序列的DNA片段的分子操作中，并极大地促进了反向遗传学研究。但是，可能由于不同物种间基因组大小和序列存在显差异，在采用该技术进行转座元件Ds水稻插入突变体鉴定过程中，常因TAIL-PCR反应的稳定性差而影响突变体筛选效率。有鉴于此，根据Ds核苷酸序列设计了分别对应或互补于Ds插入元件两端长度不同的12个特异引物组成32个组合，在大量预试验基础上与6个不同简并性(32～256)的随机简并引物分别组合进行TAIL-PCR反应，较系统地研究了引物特性对以水稻基因组DNA为模板的TAIL-PCR反应效率的影响。结果发现，第一反应采用长序列特异引物(36～40mer)可显提高扩增特异性，随机简并引物的简并度对反应的影响显。还选择出两个适于从水稻Ds插入突变体基因组高效扩增出Ds插入侧翼片段的最优特异引物组合和最适简并引物。应用本研究结果可显地提高TAIL-PCR技术筛选水稻插入突变体的效率。

Selection of Optimal Primers for TAIL-PCR in Identifying Ds Flanking Sequences from Ac/Ds Insertion Rice Lines

Thermal asymmetric interlaced-PCR, as a PCR-based technique in identifying DNA fragments flanking known sequences,has obtained wide application in different organisms thereby greatly promotes the efficiency in reverse genetics. Unfortunately, in spite of the fact that TAIL-PCR technique has been expanded vastly and adopted in transposon mutagenesis in rice, a reliable, highly reproducible TAIL-PCR procedure especially for rice genomic DNA is still not available, mainly due to the complexity of rice genome and the lack of optimal primers for TAIL-PCR in rice. Given the current situation, we designed 12 specific primers corresponding to3' end or complimentary to 5' end of Ds insertion, which constitute 32 sets, each with 3 specific primers for three rounds of TAILPCR, for screening the optimal combinations of Ds-specific primers. Based on the massive results from pilot experiments, two optimal sets of specific primers (Ds3L1/Ds3L2/Ds3S3 at 3' end; Ds5L1/Ds5S2/Ds5S3 at 5'end) were chosen, and used together with six arbitrary degenerate (AD) primers, respectively, to comparatively investigate the effects of arbitrary degenerate primers on the specificity of TAIL-PCR. Among the tested six AD primers, AD4 (5'-NTCAGSTWTSGWGWT-3'), possessing 128 fold degeneracy,was proved to be the most efficient for TAIL-PCR with rice genomic DNA. Moreover, our results also implied that long specific primers (36～ 40mers) in the primary reaction favored the TAIL-PCR by increasing specificity, and different AD primers led to significant differences in PCR amplification, presumably due to great difference in degeneracy. Our data may provide helpful information for TAIL-PCR technique to improve the efficiency in identifying DNA fragments flanking Ds insertion in rice or other organisms.