一个低简并引物库提高了高效热不对称交错PCR在扩增……中T-DNA侧翼序列的效率。
A low degenerate primer pool improved the efficiency of high-efficiency thermal asymmetric interlaced PCR to amplify T-DNA flanking sequences in .
作者信息
Zhang Huchen, Xu Weiwei, Feng Zhiyang, Hong Zhi
机构信息
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023 China.
College of Food Science and Technology, Nanjing Agriculture University, Nanjing, 210095 China.
出版信息
3 Biotech. 2018 Jan;8(1):14. doi: 10.1007/s13205-017-1032-y. Epub 2017 Dec 11.
Abstract
We employed hi-TAIL-PCR to identify T-DNA loci in our 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 .
摘要
我们采用热不对称交错PCR（hi-TAIL-PCR）来鉴定激活标签文库中的T-DNA位点，当使用推荐的引物池C1和C2时，72个样本中总共只有28个（39%）插入位点得到了表征。相比之下，我们发现简并度相对较低的C1比C2更有效地扩增T-DNA插入的侧翼序列。我们用一段最初用于TAIL-PCR的16个碱基对的简并序列替换了长任意简并（LAD）引物中的简并序列，该序列简并度相对较低。我们的结果表明，新的LAD引物池N增加了有效扩增，共鉴定出37个（51%）T-DNA位点，表明其在扩增T-DNA侧翼序列方面更有效。
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本文引用的文献
1
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Plant J. 2017 Aug;91(3):361-370. doi: 10.1111/tpj.13582. Epub 2017 Jun 21.
2
Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes in T-DNA tagged mutants.利用热不对称交错PCR（TAIL-PCR）克隆T-DNA标签突变体中基因时的常见问题及其解决方法
Biotechnol Biotechnol Equip. 2015 Mar 4;29(2):260-267. doi: 10.1080/13102818.2014.998161. Epub 2015 Jan 14.
3
Effort and contribution of T-DNA Insertion mutant library for rice functional genomics research in China: review and perspective.中国利用 T-DNA 插入突变体库进行水稻功能基因组学研究的努力和贡献：回顾与展望。
J Integr Plant Biol. 2012 Dec;54(12):953-66. doi: 10.1111/j.1744-7909.2012.01171.x. Epub 2012 Nov 29.
4
High-throughput characterization of plant gene functions by using gain-of-function technology.利用功能获得技术高通量鉴定植物基因功能。
Annu Rev Plant Biol. 2010;61:373-93. doi: 10.1146/annurev-arplant-042809-112143.
5
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6
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