Nichols R, Dixon J E
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Anal Biochem. 1988 Apr;170(1):110-5. doi: 10.1016/0003-2697(88)90096-6.
A simple and rapid strategy for distinguishing between positively hybridizing colonies and false positive-hybridization signals is described. The isolation of a specific DNA sequence depends on the ability to distinguish between a clone that contains the correct sequence and a false hybridization-positive or background signal. This procedure utilizes the same oligonucleotide mixture both as a screening probe and as a sequencing primer. The mixture of oligonucleotides is used as a primer to obtain sequence information directly from double-stranded DNA. Conditions for sequencing with oligonucleotides having up to 64-fold degeneracy are described. Since the sequence information obtained is directly adjacent to the site of oligonucleotide:DNA hybridization, it is necessary to know only a minimal length of DNA or peptide sequence to both design oligonucleotide probes and confirm the authenticity of the hybridization positives. The advantages of the degenerate oligonucleotide sequencing method include the rapid, reliable identification of authentic versus false hybridization positives made directly without subcloning into single-stranded M13 phage, without sequencing large regions of DNA, or without synthesizing sequence-specific primers.
本文描述了一种区分阳性杂交菌落和假阳性杂交信号的简单快速策略。特定DNA序列的分离取决于区分包含正确序列的克隆与假杂交阳性或背景信号的能力。该方法使用相同的寡核苷酸混合物作为筛选探针和测序引物。寡核苷酸混合物用作引物直接从双链DNA中获取序列信息。文中描述了使用具有高达64倍简并性的寡核苷酸进行测序的条件。由于获得的序列信息直接毗邻寡核苷酸与DNA杂交的位点，因此在设计寡核苷酸探针和确认杂交阳性的真实性时，只需要知道最短长度的DNA或肽序列。简并寡核苷酸测序方法的优点包括快速、可靠地直接鉴定真实与假杂交阳性，无需亚克隆到单链M13噬菌体中，无需对大片段DNA进行测序，也无需合成序列特异性引物。
