Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei.
Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei.
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发表时间：
1991
影响因子：
9.8
通讯作者：
B. Trask;H. Massa;Sue Kenwrickt;Jane Gitschiert
中科院分区：
文献类型：
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作者：
B. Trask;H. Massa;Sue Kenwrickt;Jane Gitschiert
我们利用间期染色质中探针杂交位点的邻近性来推导人类染色体 Xq28 的 2-3-Mbp 区域中 DNA 序列的顺序。生成的图谱将遗传和脉冲场凝胶电泳图谱的结果结合起来，产生比单独使用这些其他技术中的任何一种都更完整的 Xq28 图谱。双色荧光原位杂交（FISH）用于检测两个或多个探针在 G1 雄性间期细胞核中的位置。我们表明，可以通过两种替代方法快速对相距 50 kbp 至 2-3 Mbp 的粘粒进行排序：（1）通过比较两个探针之间的平均测量距离；（2）在用两种荧光染料检测到三个或更多探针后，简单地通过对红色和绿色荧光点的顺序进行评分。使用来自大约 800 kbp 区域的五个粘粒证明了这些方法的有效性，其中包括因子 VIII (F8)、葡萄糖-6-磷酸脱氢酶 (G6PD) 和色觉色素 (CV) 基因。从间期作图得到的粘粒图谱与通过限制性片段分析确定的图谱一致。然后使用两种间期作图方法（1）相对于两个标记 c1A1 和 st14c 来定向 F8/CV 簇，我们通过中期作图显示它们靠近 F8/CV 簇，（2）将 st14c (DXS52) 定位在 c1A1 和 F8 之间，以及（3）通过使用两个 CV 侧翼粘粒 18b41 和 18b41 来相对于 G6PD 来定向 CV 基因簇相对于 G6PD。 fr7。 Xq28 中源自相间邻近性的探针顺序为 cen-c1A1-st14c-5'F8 (p624-p542-p625)-G6PD-18b41-3' green-green-red-fr7-tel。我们还表明，要使用中期染色体确定它们的顺序，序列必须至少相距 1 Mbp，比间期染色质所需的数量级大一个数量级。数据显示，FISH 作图是一种对间隔大于或等于 50 kbp 的序列进行排序的简单方法，用于构建哺乳动物基因组的远程图谱。
We have used the proximity of probe hybridization sites in interphase chromatin to derive the order of DNA sequences in a 2-3-Mbp region of human chromosome Xq28. The map generated bridges the results of genetic and pulsed-field gel electrophoresis mapping to produce a more complete map of Xq28 than possible with either of these other techniques alone. Two-color fluorescence in situ hybridization (FISH) was used to detect the positions of two or more probes in G1 male interphase nuclei. We show that cosmids that are 50 kbp to 2-3 Mbp apart can be ordered rapidly with two alternative approaches: (1) by comparing the average measured distance between two probes and (2) simply by scoring the order of red and green fluorescent dots after detection of three or more probes with two fluorochromes. The validity of these approaches is demonstrated using five cosmids from a region spanning approximately 800 kbp that includes the factor VIII (F8), glucose-6-phosphate dehydrogenase (G6PD), and color-vision pigment (CV) genes. The cosmid map derived from interphase mapping is consistent with the map determined by restriction-fragment analysis. The two interphase mapping approaches were then used (1) to orient the F8/CV cluster relative to two markers, c1A1 and st14c, which we show by metaphase mapping to be proximal to the F8/CV cluster, (2) to position st14c (DXS52) between c1A1 and F8, and (3) to orient the CV gene cluster relative to G6PD by using two CV-flanking cosmids, 18b41 and fr7. The probe order in Xq28 derived from interphase proximity is cen-c1A1-st14c-5'F8 (p624-p542-p625)-G6PD-18b41-3' green-green-red-fr7-tel. We also show that, to determine their order by using metaphase chromosomes, sequences must be at least 1 Mbp apart, an order of magnitude greater than required in interphase chromatin. The data show that FISH mapping is a simple way to order sequences separated by greater than or equal to 50 kbp for the construction of long-range maps of mammalian genomes.