Duell T, Nielsen L B, Jones A, Young S G, Weier H U
Life Sciences Division, Lawrence Berkeley National Laboratory, CA 94720, USA.
Cytogenet Cell Genet. 1997;79(1-2):64-70. doi: 10.1159/000134685.
Quantitative DNA fiber mapping (QDFM) is a high-resolution technique for physical mapping of DNA. The method is based on hybridization of fluorescently labeled DNA probes to individual DNA molecules stretched on a chemically modified glass surface. We now demonstrate and validate a rapid QDFM-based approach for the mapping of multiple restriction sites and precise localization of restriction fragments in large genomic clones. Restriction fragments of a 70-kb P1 clone (P1-70) containing the 5' region of the human apolipo-protein B gene (APOB) were subcloned and mapped along straightened P1-70 DNA molecules. Multicolor fluorescence in situ hybridization (FISH) and digital image analysis allowed us to rapidly position 29 restriction fragments, ranging in size from 0.5 kb to 8 kb, and to map 43 restriction sites. The restriction map obtained by QDFM was in excellent agreement with information obtained by RecA-assisted restriction endonuclease (RARE) cleavage, long-range PCR, and DNA sequence analyses of the P1-70 clone. These data demonstrate that QDFM is a rapid, reliable method for detailed restriction site-mapping of large DNA clones.
定量DNA纤维图谱分析（QDFM）是一种用于DNA物理图谱绘制的高分辨率技术。该方法基于荧光标记的DNA探针与拉伸在化学修饰玻璃表面上的单个DNA分子的杂交。我们现在展示并验证了一种基于QDFM的快速方法，用于在大型基因组克隆中绘制多个限制性酶切位点并精确定位限制性片段。将包含人类载脂蛋白B基因（APOB）5'区域的70-kb P1克隆（P1-70）的限制性片段进行亚克隆，并沿着拉直的P1-70 DNA分子进行图谱绘制。多色荧光原位杂交（FISH）和数字图像分析使我们能够快速定位29个大小从0.5 kb到8 kb不等的限制性片段，并绘制43个限制性酶切位点的图谱。通过QDFM获得的限制性图谱与通过RecA辅助限制性内切酶（RARE）切割、长距离PCR以及P1-70克隆的DNA序列分析获得的信息高度一致。这些数据表明，QDFM是一种用于大型DNA克隆详细限制性酶切位点图谱绘制的快速、可靠方法。
