LIGATION OF HEXAMERS ON HEXAMER TEMPLATES TO PRODUCE PRIMERS FOR CYCLE SEQUENCING OR THE POLYMERASE CHAIN-REACTION
LIGATION OF HEXAMERS ON HEXAMER TEMPLATES TO PRODUCE PRIMERS FOR CYCLE SEQUENCING OR THE POLYMERASE CHAIN-REACTION
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DOI：
10.1006/abio.1995.1319
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发表时间：
1995-06-10
影响因子：
2.9
通讯作者：
STUDIER, FW
中科院分区：
文献类型：
--
作者：
DUNN, JJ;BUTLERLOFFREDO, LL;STUDIER, FW
描述了一种用于在溶液中有序连接六核苷酸（六聚体）以产生独特的较长寡核苷酸的方法。为了形成18聚体，例如，选择六种不同的六聚体，其可以明确地碱基配对以形成不确定长度的双链复合物。在最有效的排列中，每个六聚体与另外两个六聚体形成三个互补碱基对，产生具有由三个碱基交错的链断裂的连续六聚体的互补链。一条链中两个相邻的六聚体含有5'磷酸基团，其他六聚体未磷酸化。T4和T7 DNA连接酶都可以在50-100 mM范围内的六聚体浓度下将磷酸化的六聚体连接到这种复合物中的相邻六聚体，产生18聚体并留下三个未磷酸化的六聚体。满足明确连接要求的34个复合物中的29个产生了所需的18聚体，其可直接用于循环测序，或在去除未反应的六聚体后用于聚合酶链式反应（PCR）。可比较的连接反应也产生12-、24-和30-聚体。利用所有4096种可能的六聚体的文库，明确连接具有产生超过82%的所有可能的18聚体的潜力，并且可以容易地提供通过引物步移进行DNA测序、PCR或基因合成所需的寡核苷酸。(C)出版社：Academic Press
A method is described for the ordered ligation of hexanucleotides (hexamers) in solution to produce unique longer oligonucleotides. To form an 18-mer, for example, six different hexamers are selected that can base pair unambiguously to form a double-stranded complex of indefinite length. In the most efficient arrangement, each hexamer forms three complementary base pairs with two other hexamers, generating complementary chains of contiguous hexamers with strand breaks staggered by three bases. Two adjacent hexamers in one chain contain 5' phosphate groups and the others are unphosphorylated. Both T4 and T7 DNA ligase can ligate the phosphorylated hexamers to their neighbors in such a complex at hexamer concentrations in the 50-100 mM range, producing an 18-mer and leaving three unphosphorylated hexamers. Twenty-nine of 34 complexes that satisfied the requirements for unambiguous ligation generated the desired 18-mers, which could be used directly for cycle sequencing or, after removal of the unreacted hexamers, for polymerase chain reactions (PCR). Comparable ligation reactions also produced 12-, 24-, and 30-mers. With a library of all 4096 possible hexamers, unambiguous ligation has the potential to produce more than 82% of ah possible 18-mers and could readily supply the oligonucleotides needed for DNA sequencing by primer walking, for PCR, or for gene synthesis. (C) 1995 Academic Press, Inc.