HIGH-RESOLUTION IN SITU HYBRIDIZATION TECHNIQUE FOR THE DIRECT VISUALIZATION OF LABELED G-BANDED EARLY METAPHASE AND PROPHASE CHROMOSOMES
HIGH-RESOLUTION IN SITU HYBRIDIZATION TECHNIQUE FOR THE DIRECT VISUALIZATION OF LABELED G-BANDED EARLY METAPHASE AND PROPHASE CHROMOSOMES
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DOI：
10.1159/000130970
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发表时间：
1978-01-01
期刊：
影响因子：
--
通讯作者：
YUNIS, JJ
中科院分区：
文献类型：
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作者：
CHANDLER, ME;YUNIS, JJ
一种改进的原位杂交技术现在使得直接观察放射性标记的核酸探针和人淋巴细胞的早期中期和前期染色体成为可能。鉴于以前哺乳动物的原位杂交研究是用未显带的中期染色体进行的，这些染色体表现出相对较差的形态，或者依赖于杂交前显带的染色体和放射自显影后再染色的染色体的照片的比较，这种技术更简单，并大大提高了精确定位染色体特异性碱基序列的准确性。特别重要的是：培养的淋巴细胞与10-7 M氨甲喋呤的同步化以及暴露于低水平的秋水仙胺10- 15分钟（0.06 μ g/ml）以获得大量的早期有丝分裂细胞; DNA用0.2N盐酸在室温下变性20分钟，或用70%甲酰胺在2 × SSC中在70 ℃下变性2分钟，然后在乙醇中快速脱水，其允许充分保存染色体结构;与125 I标记的RNA在50%甲酰胺中在2 × SSC中在37 ℃杂交2-9小时以使RNA和染色体降解最小化7;以及直接G显带方法，其使用0.25/o Wright染色剂以1：3用0.06M磷酸盐缓冲液处理，4在杂交后染色体上显示出G-和C-联合带型。
An improved technique for in situ hybridization now makes possible the direct visualization of both the radioactively labeled nucleic acid probe and sharply banded and elongated early metaphase and prophase chromo somes of human lymphocytes. Whereas previous mammalian in situ hybridization studies have been carried out with unbanded metaphase chromosomes exhibiting relatively poor morphology, or relied on the comparison of photographs of chromosomes banded before hybridization and restained after autoradiography,’-3 this technique is simpler and results in greatly improved accuracy in the refined localization of specific base sequences to chromosomes. Of particular importance are: synchroni zation of cultured lymphocytes with 10-7 M amethopterin and a 10-to 15-min exposure to a low level of Colcemid (0.06¿¿ g/ml) to obtain high numbers of early mitotic cells’-3; DNA denaturation with 0.2N hydro chloric acid at room temperature for 20 min0 or 70% formamide in 2 X SSC at 70 C for 2 min, followed by rapid dehydration in ethanol, which allow sufficient preservation of chromosome structure; hybridization with 125I-labeled RNA in 50% formamide in 2 X SSC at 37 C for 2-9 h to minimize RNA and chromosome degradation7; and a direct G-banding method utilizing 0.25/o Wright stain diluted 1: 3 with 0.06 M phosphate buffer, 4 which elicits a combined G-and C-banding pattern in post hybridization chromosomes.