Rapid isolation of flanking genomic DNA using biotin-RAGE, a variation of single-sided polymerase chain reaction.
Rapid isolation of flanking genomic DNA using biotin-RAGE, a variation of single-sided polymerase chain reaction.
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DOI：
10.1089/dna.1992.11.791
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发表时间：
1992-12
影响因子：
3.1
通讯作者：
B. Bloomquist;Richard C. Johnson;R. E. Mains
中科院分区：
文献类型：
--
作者：
B. Bloomquist;Richard C. Johnson;R. E. Mains
描述了一种利用聚合酶链反应（PCR）快速、可重复地分离与已知DNA序列相邻的基因组DNA的方法。使用生物素化序列特异性引物与人工添加到基因组DNA末端的脱氧寡核苷酸序列结合的通用杂交引物分离侧翼基因组DNA。包含生物素化引物的扩增序列通过结合固相链亲和素基质从非生物素化扩增产物中纯化。将生物素化的扩增产物进行进一步的扩增，然后进行亚克隆和分析。该技术被应用于三个内含子-外显子连接的分离。验证这些连接序列的鉴定是通过基于Biotin-RAGE分离的内含子序列设计引物，使用这些内含子引物在外显子上扩增，并对pcr生成的产物进行测序来完成的。
A method is described for quickly and reproducibly isolating genomic DNA contiguous with known DNA sequence by means of the polymerase chain reaction (PCR). Flanking genomic DNA is isolated using a biotinylated sequence-specific primer in combination with a generic hybrid primer that binds to a deoxyoligonucleotide sequence artificially added to the ends of the genomic DNA. Amplified sequences that include the biotinylated primer are purified from nonbiotinylated amplification products by binding to a solid-phase streptavidin matrix. The biotinylated amplification product(s) are subjected to a further round of amplification, after which they can be subcloned and analyzed. This technique was applied to the isolation of three intron-exon junctions. Verification of the identify of these junction sequences was accomplished by designing primers based on the intron sequences isolated by Biotin-RAGE, amplifying across the exon using these intron primers, and sequencing the PCR-generated product.