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微卫星是基因组上的特殊短重复序列,微卫星不稳定的程度与一些肿瘤的分型、治疗和预后相关。目前,微卫星长度变化的检测往往是基于大量细胞,检测灵敏度低。本文整合激光显微切割技术,基于多次退火环状循环扩增(multiple annealing and looping-based amplification cycles,MALBAC)的单细胞全基因组放大技术和毛细管电泳长度测量方法,经过关键技术点的改进,建立了一套针对组织中少量细胞的多微卫星位点检测方法。研究结果表明,基于技术改进,HE染色的组织细胞经激光显微切割分选后,可成功用MALBAC技术进行全基因组放大,并在多微卫星位点的检测上,获得了高度的准确性和重复性。利用所建立的方法,发现肠型胃癌早期病变组织-肠上皮化生(intestinal metaplasia,IM)的单个腺体内部出现多种长度变化的微卫星改变,说明该癌前病变组织中DNA错配修复系统已经出现问题。本方法所获得的全基因组放大产物,也可以用于外显子组测序和全基因组测序。另外,该方法适用于任何组织的少量细胞,甚至单细胞的多微卫星位点检测,以及全基因组的研究,为精细研究少量病变细胞的基因组特征以及组织异质性提供了有效的方法。
Microsatellites are special short repeat sequences on the genome. The degree of microsatellite instability is associated with the classification, treatment and prognosis of some tumors. Currently, the detection of microsatellite length changes is often based on a large number of cells, the detection sensitivity is low. This article integrates laser microdissection, single-cell whole-genome amplification based on multiple annealing and looping-based amplification cycles (MALBAC), and capillary electrophoresis length measurement. Through the improvement of key technical points, A set of multi-microsatellite loci detection method for a small number of cells in the tissue was established. The results show that, on the basis of technical improvement, HE-stained tissue cells can be successfully genomic amplified by MALBAC technology after being sorted by laser microdissection, and highly accurate in the detection of multi-microsatellite loci and Repeatability. Using the established method, it was found that there were many microsatellite changes in the length of a single gland of intestinal metaplasia (IM) in intestinal-type gastric cancer, indicating that DNA mismatch repair in this precancerous lesion The system has been a problem. The genome-wide amplification product obtained by the method can also be used for exome sequencing and genome-wide sequencing. In addition, this method is suitable for the detection of microsatellite loci and even single-cell microsatellite loci in any tissue, as well as genome-wide studies, which provides an effective method for studying the genomic characteristics and tissue heterogeneity of a few lesion cells.