• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Cytometry: The Journal of the Society for Analytical Cytology >Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS
【24h】

Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS

机译：使用C-PRINS快速鉴定和确定按流分类的植物染色体的纯度

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Background: Flow-sorted plant chromosomes are being increasingly used in plant genome analysis and mapping. Consequently, there is a need for a rapid method for identification of sorted chromosomes and for determination of their purity. We report on optimization of procedures for primed in situ DNA labeling (PRINS) and cycling-PRINS (C-PRINS) for fluorescent labeling of repetitive DNA sequences on sorted plant chromosomes suitable for their identification. Methods: Chromosomes of barley, wheat, and field bean were sorted onto microscope slides, dried, and subjected to PRINS or C-PRINS with primers for GAA microsatellites (barley and wheat) or FokI repeat (field bean). The following parameters were optimized to achieve the highest specificity and intensity of fluorescent labeling: ratio of labeled versus unlabeled nucleotides, nucleotide concentration, and the number and concentration of primers. Results: Under optimal conditions, C-PRINS resulted in strong and specific labeling of GAA microsatellites on sorted barley and wheat chromosomes and FokI repeats on sorted held bean chromosomes. The labeling patterns were characteristic for each chromosome and permitted their unequivocal identification as well as determination of purity after sorting, which ranged from 96% to 99%. A standard polymerase chain reaction (PCR) with chromosome-specific primers was not sensitive enough to detect low-frequency contamination. Conclusions: The results indicate that a single C-PRINS assay with primers that give chromosome-specific labeling pattern is sufficient not only to determine chromosome content of peaks on flow karyotype but also to determine the purity of sorted chromosome fractions. The whole procedure can be performed in less than 3 h on the next day after sorting. Numerous applications are expected in the area of plant flow cytogenetics. Cytometry 41: 102-108, 2000. (C) 2000 Wiley-Liss, Inc. [References: 37]
机译：背景：按流分类的植物染色体正越来越多地用于植物基因组分析和作图。因此，需要一种用于鉴定排序的染色体并确定其纯度的快速方法。我们报告优化的程序的原位DNA标记（PRINS）和循环PRINS（C-PRINS）的荧光标记的适合其鉴定的植物染色体上的重复DNA序列的循环标记。方法：将大麦，小麦和大田豆的染色体分选到显微镜载玻片上，干燥，并用GAA微卫星（大麦和小麦）或FokI重复序列（大田豆）的引物进行PRINS或C-PRINS。优化以下参数以实现荧光标记的最高特异性和强度：标记与未标记核苷酸的比率，核苷酸浓度以及引物的数量和浓度。结果：在最佳条件下，C-PRINS导致在分选的大麦和小麦染色体上对GAA微卫星进行强而特异性的标记，并在分选后的固定豆染色体上进行FokI重复。标记模式是每个染色体的特征，可以明确鉴定并确定分选后的纯度，范围从96％到99％。具有染色体特异性引物的标准聚合酶链反应（PCR）不够灵敏，无法检测到低频污染。结论：结果表明，使用具有给出染色体特异性标记模式的引物的单个C-PRINS检测不仅足以确定流型核型峰的染色体含量，而且还能够确定分类的染色体级分的纯度。整个过程可以在分拣后第二天不到3小时内完成。在植物流细胞遗传学领域中有望有许多应用。细胞计数41：102-108，2000。（C）2000 Wiley-Liss，Inc。[参考：37]。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号