Directional genome walking using PCR
Directional genome walking using PCR
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DOI：
10.2144/02334st07
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发表时间：
2002-10-01
期刊：
影响因子：
2.7
通讯作者：
Reddy, MK
中科院分区：
文献类型：
--
作者：
Mishra, RN;Singla-Pareek, SL;Reddy, MK
我们在这里描述了一个基于PCR的“定向基因组步行”协议。扩增的基本程序由两轮PCR组成。使用对基因组中已知序列具有特异性的生物素化引物沿着设计为具有部分简并性的四种通用步行者引物对基因组DNA进行初级PCR。将生物素化的初级PCR产物固定在链霉亲和素连接的顺磁珠上。该步骤去除了所有非特异性扩增产物，并将纯化的模板用于第二次PCR，使用巢式引物和步行者引物-2以增加特异性。该技术对克隆启动子区具有潜在的应用价值，并已成功地用于分离我们以前分离的许多cDNA克隆的5个侧翼基因组区域。
We describe here a PCR-based "directional genome walking " protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a biotinylated primer specific to a known sequence in the genome along with four universal walker primers that were designed with partial degeneracy. The biotinylated primary PCR products were immobilized on streptavidin-linked paramagnetic beads. This step removed all nonspecific amplification products, and the purified template was used for the second PCR using a nested primer and the walker primer-2 to increase specificity. This technique is potentially useful for cloning promoter regions and has been successfully used to isolate 5 flanking genomic regions of many cDNA clones previously isolated by us.