Bloomquist B T, Johnson R C, Mains R E
Neuroscience Department, Johns Hopkins Medical School, Baltimore, MD 21205.
DNA Cell Biol. 1992 Dec;11(10):791-7. doi: 10.1089/dna.1992.11.791.
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.
本文描述了一种通过聚合酶链反应（PCR）快速且可重复地分离与已知DNA序列相邻的基因组DNA的方法。使用生物素化的序列特异性引物与通用杂交引物相结合来分离侧翼基因组DNA，该通用杂交引物可与人工添加到基因组DNA末端的脱氧寡核苷酸序列结合。通过与固相链霉亲和素基质结合，从非生物素化的扩增产物中纯化出包含生物素化引物的扩增序列。对生物素化的扩增产物进行第二轮扩增，之后可进行亚克隆和分析。该技术应用于三个内含子-外显子连接的分离。通过基于生物素-RAGE分离的内含子序列设计引物、使用这些内含子引物跨外显子进行扩增以及对PCR产生的产物进行测序，完成了对这些连接序列身份的确证。
