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首页> 美国卫生研究院文献>3 Biotech >A low degenerate primer pool improved the efficiency of high-efficiency thermal asymmetric interlaced PCR to amplify T-DNA flanking sequences in Arabidopsis thaliana
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A low degenerate primer pool improved the efficiency of high-efficiency thermal asymmetric interlaced PCR to amplify T-DNA flanking sequences in Arabidopsis thaliana

机译：低简并引物池提高了拟南芥中高效热不对称交错PCR扩增T-DNA侧翼序列的效率

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We employed hi-TAIL-PCR to identify T-DNA loci in our Arabidopsis activation tagging library and only a total of 28 (39%) insertion sites from 72 samples were characterized when the recommended primer pools, C1 and C2 were used. By comparison, we found C1 harboring relatively low degeneracy was more efficient to amplify the flanking sequences of T-DNA insertion than C2. We replaced the degenerate sequences in long arbitrary degenerate (LAD) primers with a piece of 16-bp degenerate sequence originally used in TAIL-PCR, which had the relatively low degeneracy. Our results showed that the new LAD primer pool N increased the valid amplifications and a total of 37 (51%) T-DNA loci were identified, indicating a more effective amplification of T-DNA flanking sequences in A. thaliana.Electronic supplementary materialThe online version of this article (10.1007/s13205-017-1032-y) contains supplementary material, which is available to authorized users.
机译：我们使用hi-TAIL-PCR在拟南芥激活标签文库中鉴定T-DNA基因座，当使用推荐的引物池C1和C2时，仅对来自72个样品的28个（39％）插入位点进行了表征。相比之下，我们发现具有相对较低的简并性的C1比C2更有效地扩增T-DNA插入的侧翼序列。我们用较长的简并序列（LAD）引物中的简并序列替换为最初用于TAIL-PCR的16 bp简并序列，其简并度较低。我们的结果表明，新的LAD引物库N增加了有效扩增，并且鉴定出总共37个（51％）T-DNA位点，表明拟南芥中T-DNA侧翼序列的扩增更有效。本文的版本（10.1007 / s13205-017-1032-y）包含补充材料，可供授权用户使用。

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