CAJ | 학술논문

目的探讨不同扩增方法和探针长度对植入前单个卵裂球比较基因组杂交(comparative genomic hybridization,CGH)检测结果的影响,为植入前产前诊断奠定基础.方法随机将20枚植入前6～8细胞期胚胎卵裂球分为A和B组(各10枚),取1名正常男性外周血20枚淋巴细胞,分为C和D组(各10枚)作为对照.A和C组经简并寡聚核苷酸聚合酶链反应(degenerate oligonucleotide primed polymerase chain reaction,DOP-PCR)、B和D组经多重置换扩增(multiple displacement amplification,MDA)分别进行全基因组扩增(whole genome amplification,WGA),用上述全基因组扩增产物进行PCR扩增TBX1基因2号外显子,验证其特异性.将获得的WGA产物制备成250～750 bp和750～2000 bp 2种不同长度的探针与正常男性中期染色体核型进行杂交,Y染色体性别决定区(sex-determining region of Y,SRY)基因验证CGH检测结果.结果 (1)用DOP-PCR扩增时,A组10枚卵裂球中有9枚WGA成功,C组10枚淋巴细胞WGA均成功;但其扩增产物进一步扩增TBX1基因2号外显子时,出现较多非特异性条带.CGH检测中.5例卵裂球用长度250～750 bp的探针检测时,1例失败,1例CGH软件核型分析结果与SRY结果不一致.(2)经MDA的B和D组,均WGA成功;其产物进一步扩增TBX1基因2号外显子时,非特异性条带少.CGH检测中,探针长度为250～750 bp的5例卵裂球均检测成功,且与SRY验证结果一致.(3)用长度为750～2000 bp探针检测时,杂交效果不好.结论单卵裂球全基因组扩增,MDA较DOP-PCR方法稳定,特异性强,CGH检测中,扩增产物酶切至250～750 bp制备的探针杂交图像均匀、清晰,软件分析核型结果稳定、准确.
목적 탐토불동확증방법화탐침장도대식입전단개란렬구비교기인조잡교(comparative genomic hybridization,CGH)검측결과적영향,위식입전산전진단전정기출.방법 수궤장20매식입전6～8세포기배태란렬구분위A화B조(각10매),취1명정상남성외주혈20매림파세포,분위C화D조(각10매)작위대조.A화C조경간병과취핵감산취합매련반응(degenerate oligonucleotide primed polymerase chain reaction,DOP-PCR)、B화D조경다중치환확증(multiple displacement amplification,MDA)분별진행전기인조확증(whole genome amplification,WGA),용상술전기인조확증산물진행PCR확증TBX1기인2호외현자,험증기특이성.장획득적WGA산물제비성250～750 bp화750～2000 bp 2충불동장도적탐침여정상남성중기염색체핵형진행잡교,Y염색체성별결정구(sex-determining region of Y,SRY)기인험증CGH검측결과.결과 (1)용DOP-PCR확증시,A조10매란렬구중유9매WGA성공,C조10매림파세포WGA균성공;단기확증산물진일보확증TBX1기인2호외현자시,출현교다비특이성조대.CGH검측중.5례란렬구용장도250～750 bp적탐침검측시,1례실패,1례CGH연건핵형분석결과여SRY결과불일치.(2)경MDA적B화D조,균WGA성공;기산물진일보확증TBX1기인2호외현자시,비특이성조대소.CGH검측중,탐침장도위250～750 bp적5례란렬구균검측성공,차여SRY험증결과일치.(3)용장도위750～2000 bp탐침검측시,잡교효과불호.결론 단란렬구전기인조확증,MDA교DOP-PCR방법은정,특이성강,CGH검측중,확증산물매절지250～750 bp제비적탐침잡교도상균균、청석,연건분석핵형결과은정、준학.
Objective To investigate the effect of different amplification methods and probes with various length on the results of comparative genomic hybridization (CGH) analysis of pre-implanted single blastomere and to establish the basis for preimplantation genetic diagnosis.Methods Twenty blastomeres of embryo at 6-8 cells stage were randomly divided into A and B group with 10 in each.Twenty peripheral blood lymphocytes from a healthy man were similarly divided into C and D group with 10 in each.Degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) was used to amplify whole genomic DNA in group A and C,and multiple displacement amplification (MDA) was used in group B and D for whole genome amplification (WGA).The specificity of resultant products was confirmed by amplification of TBX1 gene exon 2.CGH was performed respectively with 250-750 bp and 750-2000 bp probes prepared from the amplified whole genomic DNA.The result of CGH was verified by sex-determining region of Y (SRY).Results (1) Nine of the 10 samples in group A and all in group C were amplifiable by DOP-PCR,but there were multiple non-specific bands in the amplification of TBX1 exon 2 when WGA products were used as templates.When 250～750 bp probe was used in CGH,1 of the 5 blastomeres was failed and another one had different karyotype from that analyzed by SRY.(2) All samples in group B and D were successfully amplified by MDA,and the non-specific bands were significantly less in the amplification of TBX1 exon 2.All 5 blastomeres were successful in CGH with the 250～750 bp probe.Moreover,the karyotype was in agreement with that of SRY.(3) When 750 ～ 2000 bp probe was used,the CGH results were suboptimal.Conclusions In WGA of single blastomere,MDA is superior to DOP-PCR in the stability and specificity.The karyotype image detected by CGH with the 250～750 bp probe is clear and homogenous.