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用于寡脱氧核糖核苷酸合成的改良化学方法显著提高了对一种合成35聚体的限制性内切酶切割效率。

Improved chemistry for oligodeoxyribonucleotide synthesis substantially improves restriction enzyme cleavage of a synthetic 35mer.

作者信息

Farrance I K, Eadie J S, Ivarie R

机构信息

Department of Genetics, University of Georgia, Athens 30602.

出版信息

Nucleic Acids Res. 1989 Feb 11;17(3):1231-45. doi: 10.1093/nar/17.3.1231.

DOI:10.1093/nar/17.3.1231
PMID:2537951
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC331741/
Abstract

Two DNA duplexes of identical sequence and 35 nt in length were synthesized by an original and a highly improved version of phosphoramidite chemistry. By base composition analysis, DNA synthesized by improved chemistry (termed DMTS-imp) contained no detectable modified bases while DNA synthesized by the original chemistry (termed DMTS-std) had a large number of modifications. Under optimal reaction conditions, HhaI and RsaI cleaved the DMTS-std duplex to 76-77% completion and the DMTS-imp duplex to 96-99% completion. Restriction analysis and piperidine treatment yielded estimates of approximately 3.0% modified nucleotides in DMTS-std and approximately 1.0% in DMTS-imp. Overall, the improvements in chemistry increased the restriction efficiency of synthetic DNA up to 10-fold.

摘要

通过亚磷酰胺化学的原始版本和高度改进版本合成了两个序列相同、长度为35个核苷酸的DNA双链体。通过碱基组成分析，改进化学方法合成的DNA（称为DMTS-imp）未检测到修饰碱基，而原始化学方法合成的DNA（称为DMTS-std）有大量修饰。在最佳反应条件下，HhaI和RsaI将DMTS-std双链体切割至76 - 77%的完成度，将DMTS-imp双链体切割至96 - 99%的完成度。限制性分析和哌啶处理得出，DMTS-std中修饰核苷酸的估计比例约为3.0%，DMTS-imp中约为1.0%。总体而言，化学方法的改进使合成DNA的限制性效率提高了多达10倍。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47f4/331741/9e1d3bb9676f/nar00212-0413-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47f4/331741/1ee693fad6f5/nar00212-0407-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47f4/331741/71e623789a54/nar00212-0410-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47f4/331741/9e1d3bb9676f/nar00212-0413-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47f4/331741/1ee693fad6f5/nar00212-0407-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47f4/331741/71e623789a54/nar00212-0410-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47f4/331741/9e1d3bb9676f/nar00212-0413-a.jpg

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本文引用的文献

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Influence of local nucleotide sequence on substitution of 2-aminopurine for adenine during deoxyribonucleic acid synthesis in vitro.体外脱氧核糖核酸合成过程中局部核苷酸序列对2-氨基嘌呤取代腺嘌呤的影响。
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Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter.噬菌体λ PR 启动子的化学合成与生化反应活性
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Interaction of cell-type-specific nuclear proteins with immunoglobulin VH promoter region sequences.细胞类型特异性核蛋白与免疫球蛋白重链可变区启动子区域序列的相互作用。
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