Restriction site extension PCR: a novel method for high-throughput characterization of tagged DNA fragments and genome walking.
Restriction site extension PCR: a novel method for high-throughput characterization of tagged DNA fragments and genome walking.
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DOI：
10.1371/journal.pone.0010577
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发表时间：
2010-05-11
期刊：
影响因子：
3.7
通讯作者：
Braam J
中科院分区：
文献类型：
--
作者：
Ji J;Braam J
插入突变体的分离和鉴定对于将基因与生理功能联系在一起是非常有价值的。一旦确定了插入突变表型，挑战就是分离出负责的基因。已有多种策略被用于分离突变插入两侧的未知基因组DNA，然而，由于连接步骤效率低下、在目标DNA中包含限制位点以及非特异性产物产生，所有这些方法都受到限制。当目标是以高通量方式识别插入部位时，这些限制变得几乎无法克服。我们设计了一种新的策略，称为限制位点延伸PCR(RSE-PCR)，以有效地大规模分离与DNA插入相关的未知基因组DNA片段。该策略是一种改进的适配器介导的聚合酶链式反应，而不需要连接。与限制性核酸内切酶(KpnI、NsiI、PstI或SacI)限制性DNA片段的3‘端互补的接头在初级RSE-PCR的第一个循环中延伸DNA片段的3’端。在随后的PCR循环和第二次半套式PCR(二次RSE-PCR)中，降落法和两步法相结合以提高目的片段的扩增特异性。在我们对拟南芥37个tex突变体的鉴定中，证明了该方法的有效性和特异性。RSE-PCR的所有步骤均可在96孔PCR板上进行。最后，RSE-PCR是基因组沃克的成功替代品，玉米的基因分离证明了这一点，玉米是一种比拟南芥具有更复杂基因组的植物。RSE-PCR在鉴定标记(T-DNA或转座子)序列或从大基因组植物中的已知DNA走向未知区域方面具有很高的应用潜力，在其他生物中也有可能应用。
Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner. We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR) to efficiently conduct large-scale isolation of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 3′ overhang of the endonuclease (KpnI, NsiI, PstI, or SacI) restricted DNA fragments, extends the 3′ end of the DNA fragments in the first cycle of the primary RSE-PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR), touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis. All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demonstrated by gene isolation from maize, a plant with a more complex genome than Arabidopsis. RSE-PCR has high potential application in identifying tagged (T-DNA or transposon) sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well.
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影响因子：
46.9
作者：
Dai, SM;Chen, HH;Flanagan, SD
通讯作者：
Flanagan, SD
影响因子：
6.9
作者：
Koiwa, H;Bressan, RA;Hasegawa, PM
通讯作者：
Hasegawa, PM
影响因子：
14.9
作者：
UPCROFT, P;HEALEY, A
通讯作者：
HEALEY, A
影响因子：
2.7
作者：
Spertini, D;Béliveau, C;Bellemare, G
通讯作者：
Bellemare, G
影响因子：
11.6
作者：
XU, W;PURUGGANAN, MM;BRAAM, J
通讯作者：
BRAAM, J