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检测结果的影响  

Influence of different amplification methods and probes with various lengths on the results of comparative genomic hybridization analysis of preimplanted single blastomere

作　　者：石青青[1] 孙海翔[1] 朱海燕[1] 朱湘玉[1] 盛敏[1] 胡娅莉[1] 

机构地区：[1]南京大学医学院附属鼓楼医院妇产科,210008

出　　处：《中华围产医学杂志》2011年第5期277-282,共6页Chinese Journal of Perinatal Medicine

摘　　要：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（comparativegenomic 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 oligonueleotide primedpolymerase chainreaction，DOPPCR）、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（wholegenomeamplification，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250～750bp和750～2000bp2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—determiningregionofY，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250～750bp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250～750bp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750～2000bp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DOPPcR方法稳定，特异性强，CGH检测中，扩增产物酶切至250～750bp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。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 iymphocytes 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.

关 键 词：分裂球 核酸杂交 核酸扩增技术 核酸探针 聚合酶链反应 植入前诊断 

分 类 号：R714.8[医药卫生—妇产科学]