Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids.
Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids.
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使用含有锁定核酸的引物预先放大靶基因，通过逆PCR从环境DNA中检索整个基因。
DOI：
10.1111/j.1462-2920.2007.01518.x
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发表时间：
2008-04
影响因子：
5.1
通讯作者：
Harayama, Shigeaki
中科院分区：
文献类型：
--
作者：
Yamada, Kazutaka;Terahara, Takeshi;Kurata, Shinya;Yokomaku, Toyokazu;Tsuneda, Satoshi;Harayama, Shigeaki
我们一直未能成功地扩增所需的核苷酸序列从各种环境DNA样品通过使用反向聚合酶链反应（IPCR）技术，最有可能是因为目标DNA序列的拷贝数已经相当低。为了在IPCR之前富集靶DNA序列，使用含有锁核酸（LNA）的位点特异性引物进行滚环扩增。在大肠杆菌模型实验中，与标准IPCR相比，该方法的灵敏度提高了近10 000倍。然后，我们应用PAI-PCR方法从马蠕虫状附件和白蚁肠道提取的DNA中分离糖基水解酶基因。采用PAI-PCR成功地扩增并克隆了目的基因的侧翼序列，而标准IPCR没有扩增。
We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.
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影响因子：
4.4
作者：
Eschenfeldt, WH;Stols, L;Donnelly, MI
通讯作者：
Donnelly, MI
影响因子：
5.1
作者：
Gonzalez, JM;Portillo, MC;Saiz-Jimenez, C
通讯作者：
Saiz-Jimenez, C
影响因子：
14.9
作者：
HAYASHI, K;NAKAZAWA, M;OBAYASHI, A
通讯作者：
OBAYASHI, A
影响因子：
3.8
作者：
Laghi, L;Randolph, AE;Boland, CR
通讯作者：
Boland, CR
影响因子：
4.4
作者：
Raghunathan, A;Ferguson, HR;Lasken, RS
通讯作者：
Lasken, RS