Telenius H, de Vos D, Blennow E, Willat L R, Ponder B A, Carter N P
Department of Pathology, University of Cambridge, United Kingdom.
Cytometry. 1993;14(1):97-101. doi: 10.1002/cyto.990140116.
We describe a phenomenon where a second flow karyotype is superimposed on the normal metaphase flow karyotype of human lymphoblastoid chromosomes. The events of this second flow karyotype contain half the DNA content of corresponding events in the normal flow karyotype and, when sorted, show the morphology of single chromatids. DNA amplified by degenerate oligonucleotide-primed PCR of 300 chromatids sorted from a single peak and hybridized onto normal metaphase spreads painted the entire length of the corresponding chromosome type. In retrospect, we could observe this phenomenon at a low level in 37% of randomly selected, previously analyzed flow karyotypes and in several published flow karyotypes from other laboratories. Experimentally, chromatid frequency was shown to vary with the colcemid blocking conditions as well as with the cell line used. At the extreme, we obtained a 4:10 ratio of chromatids to metaphase chromosomes, whereas this could be reduced tenfold by altering the colcemid blocking conditions in the same cell line. The presence of chromatids in chromosome preparations has important implications for the purity of isolated fractions used for generating libraries and in PCR analysis.
我们描述了一种现象，即在人类淋巴母细胞染色体的正常中期流式核型上叠加了第二种流式核型。这种第二种流式核型的事件所含DNA含量是正常流式核型中相应事件的一半，分选时显示出单条染色单体的形态。从单个峰中分选的300条染色单体经简并寡核苷酸引物PCR扩增后，DNA杂交到正常中期染色体铺片上，覆盖了相应染色体类型的全长。回顾过去，我们发现在37%随机选择的、之前分析过的流式核型以及其他实验室发表的几种流式核型中，都能在低水平上观察到这种现象。实验表明，染色单体频率随秋水仙酰胺阻断条件以及所用细胞系的不同而变化。在极端情况下，我们得到染色单体与中期染色体的比例为4:10，而在同一细胞系中改变秋水仙酰胺阻断条件，这一比例可降低十倍。染色体标本中染色单体的存在对于用于构建文库和PCR分析的分离组分的纯度具有重要意义。
