Engelen J J, Albrechts J C, Hamers G J, Geraedts J P
Department of Molecular Cell Biology and Genetics, University of Limburg, Maastricht, The Netherlands.
J Med Genet. 1998 Apr;35(4):265-8. doi: 10.1136/jmg.35.4.265.
A simple and efficient method for the dissection of (marker) chromosomes, (micro)nuclei, and chromosome regions is presented. Before microdissection, metaphases are overlaid with milli-Q water to rehydrate the chromosomes, which makes them soft and sticky. The dissected chromosome fragments are dissolved without proteinase-K or topoisomerase treatment and directly amplified using a degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). The advantages of this microFISH method over previously reported methods are: (1) microdissection in this way is very fast; (2) a chromosome, marker, (micro)nucleus, or chromosome region is collected as a whole using only one microneedle; (3) the dissected material sticks tightly to the needle without the risk of getting lost; (4) no Sequenase is used in the DOP-PCR reaction which reduces the risk of contamination.
本文介绍了一种简单有效的方法，用于解剖（标记）染色体、（微）核和染色体区域。在显微切割之前，用超纯水覆盖中期染色体，使染色体复水，从而使其变得柔软且具有粘性。切割得到的染色体片段无需蛋白酶K或拓扑异构酶处理即可溶解，并直接使用简并寡核苷酸引物聚合酶链反应（DOP-PCR）进行扩增。这种微荧光原位杂交（microFISH）方法相对于先前报道的方法具有以下优点：（1）以这种方式进行显微切割非常快速；（2）仅使用一根微针即可将整条染色体、标记、（微）核或染色体区域完整收集；（3）切割的材料紧密粘附在针上，没有丢失的风险；（4）DOP-PCR反应中不使用测序酶，降低了污染风险。
