Wosnick M A, Barnett R W, Vicentini A M, Erfle H, Elliott R, Sumner-Smith M, Mantei N, Davies R W
Allelix Incorporated, Mississauga, Ont., Canada.
Gene. 1987;60(1):115-27. doi: 10.1016/0378-1119(87)90219-8.
We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.
我们测试了几种不同的合成设计和组装方法，以开发一种改进的基因合成策略，该策略能够轻松构建更长的核苷酸序列。该策略部分基于我们合成高质量延长寡聚脱氧核苷酸（长度超过100个碱基）的能力，以及化学5'-磷酸化的使用和简化的低熔点琼脂糖凝胶纯化方法，兼具简便性、速度和高整体效率。我们表明，现在常规合成甚至长基因（至少1-2 kb）都是可行的。为了证明这种能力，我们化学合成并组装了编码牛凝乳酶原（前胃蛋白酶）的基因的两个不同版本。一个基因基本上是天然的牛凝乳酶原基因序列。在第二个基因中，密码子已根据高表达酵母基因的密码子偏好进行了优化。每个合成基因都超过1100 bp，但它们仅由13或14对互补寡聚脱氧核苷酸（寡核苷酸）组装而成，其平均长度分别为87和82 bp。“突变”率足够低，可以评估所有此类寡核苷酸对（总共160-170个核苷酸）中超过75%是无错误的。
