Fransz P F, Alonso-Blanco C, Liharska T B, Peeters A J, Zabel P, de Jong J H
Department of Genetics, Wageningen Agricultural University, Netherlands.
Plant J. 1996 Mar;9(3):421-30. doi: 10.1046/j.1365-313x.1996.09030421.x.
A technique to detect DNA sequences on extended DNA fibres (EDF) prepared from interphase nuclei from tomato (Lycopersicon esculentum) and Arabidopsis thaliana leaves by fluorescence in situ hybridization (FISH) is described. Three nuclear lysis procedures have been tested for their ability to decondense chromatin and to generate highly extended intact DNA fibres on microscopic slides. DNA probes of various sizes have been used in FISH experiments to EDFs to establish the resolution and sensitivity of the technique. The fluorescent signals of a 5S rDNA probe hybridized to tomato EDFs revealed continuous strings of about 200 microns, that corresponded to a molecular size of about 660 kb. In A. thaliana, a contig of three cosmids spanning a genomic region with a total length of about 89 kb was analysed. By means of multicolour hybridization the physical positions of the cosmids were visualized as red and green fluorescence strings with overlapping regions in yellow. Comparison of the length of the fluorescent signals with the molecular data revealed a stretching degree of the DNA fibres at 3.27 kb microns-1, which is close to the Watson-Crick DNA length estimate of 2.9 kb microns-1. Other experiments on small size molecular probes with both lambda clones (13.5-17 kb insert sizes) and plasmids (4.2 and 5 kb) in a contig of A. thaliana, and the 5S rDNA region in tomato showed close agreement with molecular data. The lower limit of the detection, which was established in a hybridization experiment with two DNA probes from the 45S ribosomal gene on extended fibres of tomato, was about 0.7 kb. Consistent patterns of alternating fluorescent red and green spots were obtained reflecting the tandemly repeated arrangement of the 18S and 25S ribosomal sequences. On the basis of the microscopic distance between these hybridization spots the size of the ribosomal unit was estimated at 8.2 kb. This implies a drastic improvement of high-resolution physical mapping of DNA sequences by FISH on plant DNA.
本文描述了一种通过荧光原位杂交（FISH）技术检测从番茄（Lycopersicon esculentum）和拟南芥叶片间期核制备的伸展DNA纤维（EDF）上DNA序列的方法。测试了三种核裂解程序在使染色质解聚并在显微镜载玻片上生成高度伸展的完整DNA纤维方面的能力。在FISH实验中，将各种大小的DNA探针用于EDF，以确定该技术的分辨率和灵敏度。与番茄EDF杂交的5S rDNA探针的荧光信号显示出约200微米的连续条带，这对应于约660 kb的分子大小。在拟南芥中，分析了跨越约89 kb基因组区域的三个黏粒的重叠群。通过多色杂交，黏粒的物理位置被可视化为红色和绿色荧光条带，重叠区域为黄色。将荧光信号的长度与分子数据进行比较，发现DNA纤维的拉伸程度为3.27 kb/微米-1，接近沃森-克里克DNA长度估计值2.9 kb/微米-1。对拟南芥重叠群中插入大小为13.5 - 17 kb的λ克隆和质粒（4.2和5 kb）以及番茄5S rDNA区域的小尺寸分子探针进行的其他实验，与分子数据显示出密切一致。在番茄伸展纤维上用来自45S核糖体基因的两个DNA探针进行杂交实验确定的检测下限约为0.7 kb。获得了交替出现的荧光红色和绿色斑点的一致模式，反映了18S和25S核糖体序列的串联重复排列。根据这些杂交斑点之间的微观距离，核糖体单位大小估计为8.2 kb。这意味着通过FISH对植物DNA进行DNA序列的高分辨率物理图谱绘制有了显著改进。
