体外拼装凋亡素基因  

Assembly of apoptin gene using oligodeoxyribonucleotides in vitro

作　　者：陈学清[1] 王亚东[1] 孙勇[1] 段佑才[2] 马高峰[1] 

机构地区：[1]南方医科大学南方医院消化内科,广东广州510515 [2]广东省第二人民医院,广东广州510317

出　　处：《第一军医大学学报》2005年第2期195-197,共3页Journal of First Military Medical University

基　　金：南方医院院长基金~~

摘　　要：目的以凋亡素基因为例,探讨用寡核苷酸片段体外拼装基因的方法。方法按已知的凋亡素基因序列(GeneBank登录号:AY171617),设计40bp的凋亡素基因寡核苷酸片段,并将其中的172位(BglII,agatct→agatcc)和306位碱基(HindIII,aagctt→aatcct)进行同义突变,Taq消除这两个酶切位点。在pfu酶的PCR系统中,对寡核苷酸片段进行拼装和扩增。产物稀释后进一步纯化扩增,在Tag酶存在的条件下对产物两末端加T,然后TA克隆于pGEM-Teasy载体中。阳性克隆经测序鉴定。结果凋亡素基因寡核苷酸片段第1次PCR拼装后,产物呈拖尾状。但产物经进一步稀释,并用两未端引物进行2次扩增后,即可见明确的产物带及其他梯形带状产物。目的产物TA克隆后,筛选得到阳性克隆,其经测序鉴定,与设计的凋亡素基因序列完全一致。结论体外基因拼装法是一种非常有效的基因克隆方法,可用于基因或载体的合成,及一次性对基因进行广泛的突变。Objective To explore the method for in vitro gene assembly of apoptin-encoding DNA sequence, for instance, using a large number of oligodeoxyribonucleotides (oligos). Methods Based on the encoding sequence of apoptin gene (GeneBank accession number AY171617), a number of oligos were designed to assembly apoptin gene in pfu mix reaction system, and each oligo was 40 nucleotides (nt) in length, in which synonymous codon substitution was used to eliminate the restriction enzyme sites of Bgl II ( position 172, agatct→agatcc) and Hind III (position 306, aagctt→aatcct). The assembly mixture was further diluted and amplified with two end oligos. The targeting sequence was gel-purified, amplified for one more time, followed by the addition of T to the 3' end in the presence of Taq polymerase and dATP before cloning into pGEM-T easy vector. The positive clones were confirmed by restriction enzyme digestion and sequence analysis. Results The synthetic mixture presented obvious 'tails' in the first PCR for assembly. After dilution of the mixture and amplification with two end oligos, clear DNA ladder bands with a clear targeted band were yielded. After PCR, the targeted gene was cloned into pGEM-T easy vector, and the positive clones were confirmed by sequence analysis with be identical to the designed coding sequence of apoptin gene. Conclusion Gene assembly is a rapid and cost-effective approach for synthesis of genes or vectors, which allows simultaneous mutagenesis of several genes in vitro.

关 键 词：基因拼装 凋亡素 聚合酶链式反应 

分 类 号：Q785[生物学—分子生物学]