CAJ | 학술논문

[目的] 建立并优化利用微阵列-比较基因组杂交技术检测单细胞非整倍体的实验方案. [方法] 纤维母细胞系GM02732(47,XY,+18)和GM00343[46,XY,4(del) (qter ＞ p14)]被用于实验.阳性对照组为上述细胞系的基因组DNA(gDNA)(A组与B组,n ＝ 6);实验组为单细胞模板的多重置换扩增(MDA)产物(C组与D组,n ＝ 10);阴性对照组为空白对照的MDA产物(E组,n ＝ 6).以上样本与10 k 2.0基因分型芯片杂交并进行染色体拷贝数分析,比较利用非扩增的正常gDNA和同法扩增的DNA(MDA-DNA)作为分析参照对C组和D组的结果准确度的影响.[结果] A→E组的芯片杂交信号判读率分别为98.7%、97.2%、86.7%、85.9%与3.2%.利用单细胞MDA-DNA作为参照时,C组与D组杂交信号的变异程度明显小于使用gDNA作为参照时(P ＜ 0.05).利用CNAT分析软件,发现以gDNA作为参照时,C组与D组部分染色体优势扩增明显,而使用MDA-DNA作为参照时则未观察到类似现象. [结论] 结合MDA和基因芯片平台对单细胞进行非整倍体检测时应使用同法扩增的DNA作为分析参照.
[목적] 건립병우화이용미진렬-비교기인조잡교기술검측단세포비정배체적실험방안. [방법] 섬유모세포계GM02732(47,XY,+18)화GM00343[46,XY,4(del) (qter ＞ p14)]피용우실험.양성대조조위상술세포계적기인조DNA(gDNA)(A조여B조,n ＝ 6);실험조위단세포모판적다중치환확증(MDA)산물(C조여D조,n ＝ 10);음성대조조위공백대조적MDA산물(E조,n ＝ 6).이상양본여10 k 2.0기인분형심편잡교병진행염색체고패수분석,비교이용비확증적정상gDNA화동법확증적DNA(MDA-DNA)작위분석삼조대C조화D조적결과준학도적영향.[결과] A→E조적심편잡교신호판독솔분별위98.7%、97.2%、86.7%、85.9%여3.2%.이용단세포MDA-DNA작위삼조시,C조여D조잡교신호적변이정도명현소우사용gDNA작위삼조시(P ＜ 0.05).이용CNAT분석연건,발현이gDNA작위삼조시,C조여D조부분염색체우세확증명현,이사용MDA-DNA작위삼조시칙미관찰도유사현상. [결론] 결합MDA화기인심편평태대단세포진행비정배체검측시응사용동법확증적DNA작위분석삼조.
[Objective] To set up an optimized protocol for aneuploidy detection from single cells through Array- Comparative Genetic Hybridization (CGH).[Method] Two cell lines,trisomy 18 (Tri-18;GM02732,47,XY,+18) and chromosome 4 segment deletion [sDel-4;GM00343,46,XY,4(del) (qter ＞ p14)],were used in the study.In combination of 10 k 2.0 SNP mapping array platform and multiple displacement amplification (MDA),the diagnostic accurate rates of MDA product from single cells of the two cell lines using gDNA and single-cell MDA product as reference were compared.[Result] An extremely lower call rate (3.2 ± 1.2)% in the negative control group was observed compared to the experiment groups.When the single-cell MDA product was used as reference,the standard deviations of Log2 (signal intensity ratio) were significantly decreased in both groups,compared with when the gDNA as reference (P ＝ 0.004).Through CNAT analytic software,some specific chromosomes (16,17,19,and 22) presented obvious preferential amplification (PA) when the gDNA was used as reference,but this PA could be eliminated when single-cell MDA product was used as reference.[Conclusion] 10 k 2.0 SNP mapping array in combination with MDA could be a quick,highly efficient and accurate method to detect aneuploidy in single cells.