Chen Xue-Qing, Wang Ya-Dong, Sun Yong, Duan You-Cai, Ma Gao-Feng
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Di Yi Jun Yi Da Xue Xue Bao. 2005 Feb;25(2):195-7.
To explore the method for in vitro gene assembly of apoptin-encoding DNA sequence, for instance, using a large number of oligodeoxyribonucleotides (oligos).
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.
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.
Gene assembly is a rapid and cost-effective approach for synthesis of genes or vectors, which allows simultaneous mutagenesis of several genes in vitro.
探索例如使用大量寡脱氧核糖核苷酸（oligos）进行凋亡素编码DNA序列体外基因组装的方法。
基于凋亡素基因的编码序列（基因库登录号AY171617），设计了多个oligos在pfu混合反应体系中组装凋亡素基因，每个oligo长度为40个核苷酸（nt），其中使用同义密码子替换消除Bgl II（第172位，agatct - agatcc）和Hind III（第306位，aagctt - aatcct）的限制性酶切位点。组装混合物进一步稀释并用两个末端oligos进行扩增。将靶向序列进行凝胶纯化，再次扩增，然后在Taq聚合酶和dATP存在的情况下在3'末端添加T，再克隆到pGEM - T easy载体中。通过限制性酶切和序列分析确认阳性克隆。
合成混合物在首次组装PCR中呈现明显的“拖尾”现象。混合物稀释并用两个末端oligos扩增后，产生了清晰的DNA梯状条带和清晰的靶向条带。PCR后，将靶向基因克隆到pGEM - T easy载体中，通过序列分析确认阳性克隆与设计的凋亡素基因编码序列一致。
基因组装是一种快速且经济高效的基因或载体合成方法，可在体外同时对多个基因进行诱变。
