Becker M M, Wang Z, Grossmann G, Becherer K A
Department of Biological Sciences, University of Pittsburgh, PA 15260.
Proc Natl Acad Sci U S A. 1989 Jul;86(14):5315-9. doi: 10.1073/pnas.86.14.5315.
A simple and accurate genomic primer extension method has been developed to detect ultraviolet footprinting patterns of regulatory protein-DNA interactions in mammalian genomic DNA. The technique can also detect footprinting or sequencing patterns introduced into genomic DNA by other methods. Purified genomic DNA, containing either damaged bases or strand breaks introduced by footprinting or sequencing reactions, is first cut with a convenient restriction enzyme to reduce its molecular weight. A highly radioactive single-stranded DNA primer that is complementary to a region of genomic DNA whose sequence or footprint one wishes to examine is then mixed with 50 micrograms of restriction enzyme-cut genomic DNA. The primer is approximately 100 bases long and contains 85 radioactive phosphates, each of specific activity 3000 Ci/mmol (1 Ci = 37 GBq). A simple and fast method for preparing such primers is described. Following brief heat denaturation at 100 degrees C, the solution of genomic DNA and primer is cooled to 74 degrees C and a second solution containing Taq polymerase (Thermus aquaticus DNA polymerase) and the four deoxynucleotide triphosphates is added to initiate primer extension of genomic DNA. Taq polymerase extends genomic hybridized primer until its polymerization reaction is terminated either by a damaged base or strand break in genomic DNA or by the addition of dideoxynucleotide triphosphates in the polymerization reaction. The concurrent primer hybridization-extension reaction is terminated after 5 hr and unhybridized primer is digested away by mung bean nuclease. Primer-extended genomic DNA is then denatured and electrophoresed on a polyacrylamide sequencing gel, and radioactive primer extension products are revealed by autoradiography. By using this method we demonstrate that it is possible to footprint with ultraviolet light, in intact monkey cells, regulatory protein--DNA interactions along a single copy of a simian virus 40 viral genome integrated into the monkey genome.
已开发出一种简单而准确的基因组引物延伸方法，用于检测哺乳动物基因组DNA中调节蛋白与DNA相互作用的紫外线足迹模式。该技术还可以检测通过其他方法引入基因组DNA的足迹或测序模式。首先用一种方便的限制性内切酶切割纯化的基因组DNA，该DNA含有通过足迹或测序反应引入的受损碱基或链断裂，以降低其分子量。然后将与希望检查其序列或足迹的基因组DNA区域互补的高放射性单链DNA引物与50微克经限制性内切酶切割的基因组DNA混合。该引物约100个碱基长，含有85个放射性磷酸基团，每个磷酸基团的比活为3000 Ci/mmol（1 Ci = 37 GBq）。描述了一种制备此类引物的简单快速方法。在100℃短暂热变性后，将基因组DNA和引物溶液冷却至74℃，并加入含有Taq聚合酶（嗜热水生菌DNA聚合酶）和四种脱氧核苷三磷酸的第二种溶液，以启动基因组DNA的引物延伸。Taq聚合酶延伸基因组杂交引物，直到其聚合反应因基因组DNA中的受损碱基或链断裂或聚合反应中加入双脱氧核苷三磷酸而终止。5小时后终止同时进行的引物杂交-延伸反应，未杂交的引物被绿豆核酸酶消化掉。然后将引物延伸的基因组DNA变性并在聚丙烯酰胺测序凝胶上进行电泳，通过放射自显影显示放射性引物延伸产物。通过使用这种方法，我们证明了在完整的猴细胞中，用紫外线对整合到猴基因组中的猿猴病毒40病毒基因组的单拷贝进行调节蛋白与DNA相互作用的足迹分析是可行的。
