Cseko Y M, Stone J, Sederoff R
Biochim Biophys Acta. 1979 Dec 17;565(2):253-64. doi: 10.1016/0005-2787(79)90203-x.
We have developed and characterized a method for the rapid detection and quantitation of specific DNAs in partially purified extracts of single Drosophila. While the method should be applicable to a number of repetitious DNA sequences, we have used the polypyrimidine DNA sequences (TCTCT)n to develop this technique. Using hydroxyapatite chromatography, we were able to measure the amount of nucleic acid hybrid formed and to obtain a thermal elution profile of the hybrid formed in extracts of single flies. Under a variety of conditions, purified DNA and DNA in partially purified extracts gave essentially identical results. The procedure can be used to detect the presence of rare sequences, or to measure the relative abundance of a prevalent DNA species. 40 different wild type strains of Drosophila melanogaster were examined using this technique and all contain similar amounts of the same polypyrimidine/polypurine sequence. From a small scale screening of different laboratory stocks of D. melanogaster, a variant was found which formed more DNA-DNA hybrid with labelled polypyrimidine tracts than did wild type. The additional hybrid was distinguished by a lower thermal stability than the hybrid formed in wild type.
我们已经开发并鉴定了一种用于快速检测和定量单个果蝇部分纯化提取物中特定DNA的方法。虽然该方法应适用于许多重复DNA序列，但我们已使用聚嘧啶DNA序列（TCTCT）n来开发此技术。通过羟基磷灰石色谱法，我们能够测量形成的核酸杂交体的量，并获得单个果蝇提取物中形成的杂交体的热洗脱图谱。在各种条件下，纯化的DNA和部分纯化提取物中的DNA给出了基本相同的结果。该程序可用于检测稀有序列的存在，或测量普遍存在的DNA物种的相对丰度。使用该技术检查了40种不同的黑腹果蝇野生型菌株，所有菌株都含有相似数量的相同聚嘧啶/聚嘌呤序列。通过对不同黑腹果蝇实验室菌株的小规模筛选，发现了一个变体，它与标记的聚嘧啶片段形成的DNA-DNA杂交体比野生型更多。额外的杂交体的热稳定性低于野生型中形成的杂交体。
