Hybridization probe size control: optimized 'oligolabelling'.
Hybridization probe size control: optimized 'oligolabelling'.
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DOI：
10.1093/nar/15.15.6295
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发表时间：
1987-08
影响因子：
14.9
通讯作者：
C. Hodgson;R. Z. Fisk
中科院分区：
文献类型：
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作者：
C. Hodgson;R. Z. Fisk
DNA杂交探针可以通过切口平移（1 - 2）或寡核苷酸引发（寡核苷酸标记，3-4）用32 P进行内部放射性标记。后一种方法的优点是消除了两种核酸酶活性来源（DNA酶I和5 '-3'核酸外切酶），从而可以更好地控制终产物的大小。在寡聚标记方法的进一步改进中，我们最近发现新生单链rRNA DNA的平均大小是引物浓度的反函数：长度= k/nP（其中P是引物浓度）。此外，我们还从pH值、酶、温度、DNA、引物和缓冲液组分等方面对寡聚化方法进行了优化。在此呈现的所得制剂能够在1小时反应后将78-90%的标记物掺入探针中，输出比活性高达5 X 10 μ g/g。
DNA hybridization probes can be radiolabelled internally with 32P either by nick-translation (1 2) or by oligonucleotide-priming (oligolabelling, 3-4). The latter method has the advantage that two sources of nuclease activity (DNAse I and 5'-3' exonuclease) are eliminated, allowing greater control over the size of the end product. in a further refinement of the oligolabelling method we have recently discovered that the average size of nascent single-strand rrole DNA is an inverse function of primer concentration: length = k/n P (where P is the primer concentration). In addition, we optimized the oligolabe ling meth with respect to pH, enzyme, temperature, DNA, rimer and buffer components. The resulting formulation presented here enables 78-90% incorporation avter a 1 hour reaction) of label into probe with output specific activity as high as 5 X 10 DPM//"g.