Chromosome copy analysis by single-cell comparative genomic hybridization technique based on primer extension preamplification and degenerate oligonucleotide primed-PCR / 中华医学遗传学杂志
Chinese Journal of Medical Genetics
;
(6): 387-392, 2010.
Artigo
em Chinês
| WPRIM
| ID: wpr-234399
ABSTRACT
<p><b>OBJECTIVE</b>To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD).</p><p><b>METHODS</b>Twelve single-cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed.</p><p><b>RESULTS</b>The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR, and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods.</p><p><b>CONCLUSION</b>PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell. Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneously, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.</p>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Oligonucleotídeos
/
Química
/
Testes Genéticos
/
Primers do DNA
/
Diagnóstico Pré-Implantação
/
Técnicas de Amplificação de Ácido Nucleico
/
Hibridização Genômica Comparativa
/
Cariotipagem
/
Métodos
/
Hibridização de Ácido Nucleico
Tipo de estudo:
Estudo diagnóstico
Limite:
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Medical Genetics
Ano de publicação:
2010
Tipo de documento:
Artigo
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