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Establishment of an assay for cloning and sequencing the full-length HLA-Cw gene / 中华医学遗传学杂志
Chinese Journal of Medical Genetics ; (6): 258-262, 2009.
Artigo em Chinês | WPRIM | ID: wpr-287412
ABSTRACT
<p><b>OBJECTIVE</b>To establish a reliable assay for cloning and sequencing the full-length HLA-Cw gene.</p><p><b>METHODS</b>In this study, a fragment of 4.5 kb full-length HLA-Cw gene was amplified using the self-designed PCR primer pair by long template PCR, purified PCR products was cloned into the pGEM-Teasy plasmid vector and the plasmid DNA isolated from positive clones was subjected to haplotype sequencing by both directions. A total of 12 samples having been previously-genotyped by PCR sequence-based-typing (PCR-SBT) were amplified by using the TaKaRa LA Taq and Stratagene Pfu polymerase, respectively. PCR products of full length HLA-Cw gene were subjected to cloning and sequencing and the obtained haplotype sequence were compared with the PCR-SBT results.</p><p><b>RESULTS</b>The specific target fragment of HLA-Cw gene could be amplified and the full-length HLA-Cw allele sequence covering from nucleotide position -962 in 5'untranslated region (5'-UTR) to nucleotide position 3576 in downstream area of 3'-UTR region could be obtained using our method. The results of cloning and sequencing analysis indicated that the Stratagene Pfu polymerase had better fidelity than the TaKaRa LA Taq polymerase in this experiment. By comparing the sequences of Cw*07020101 with Cw*010201, 11 SNPs as well as 2 insertions/deletions in nt-962--284 of 5'-UTR, and 11 SNPs as well as 1 insertion/deletion in nt3067-3576 downstream of 3'-UTR were identified.</p><p><b>CONCLUSION</b>Our results indicate that the technique for cloning and sequencing full-length HLA-Cw gene has been established, it has a broad application in full-length HLA-Cw gene polymorphism study and the regulation and expression of HLA-Cw gene.</p>
Assuntos
Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Dados de Sequência Molecular / Sequência de Bases / China / Reação em Cadeia da Polimerase / Alinhamento de Sequência / Sequência de Aminoácidos / Clonagem Molecular / Análise de Sequência de DNA / Primers do DNA / Alelos Limite: Humanos País/Região como assunto: Ásia Idioma: Chinês Revista: Chinese Journal of Medical Genetics Ano de publicação: 2009 Tipo de documento: Artigo

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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Dados de Sequência Molecular / Sequência de Bases / China / Reação em Cadeia da Polimerase / Alinhamento de Sequência / Sequência de Aminoácidos / Clonagem Molecular / Análise de Sequência de DNA / Primers do DNA / Alelos Limite: Humanos País/Região como assunto: Ásia Idioma: Chinês Revista: Chinese Journal of Medical Genetics Ano de publicação: 2009 Tipo de documento: Artigo