Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA.
Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA.
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DNA 核苷酸序列的直接测定：噬菌体 phiX172 DNA 片段的结构。
DOI：
10.1016/0022-2836(74)90093-x
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发表时间：
1974
影响因子：
5.6
通讯作者：
E. Ziff
中科院分区：
文献类型：
--
作者：
F. Galibert;J. Sedat;E. Ziff
本文建立了直接测定DNA核苷酸序列的方法，并用于测定噬菌体φ X 174 DNA的完整一级结构。在低温和高离子强度下，用T4噬菌体诱导的核酸内切酶IV消化，从φX DNA中释放出该片段。在剧烈条件下用核酸内切酶IV再消化该片段，并将产物二维分级，提供该片段的特征性核酸内切酶IV“指纹”。Burton（Burton & Petersen，1960）脱嘌呤反应用于表征再消化产物并鉴定其5′和3′末端的嘧啶残基。然后，这些寡核苷酸产物通过用脾和蛇毒磷酸二酯酶进行部分外切核酸酶消化进行全测序，并通过碱基组成、脱嘌呤和用外切核酸酶I进行5′端基团分析来分析分级的寡核苷酸。还推导出了部分核酸外切酶消化物二维指纹的解释规则，通过简单的检查即可快速提供序列信息。为了推导出片段的完整结构，通过脱嘌呤反应表征大的、重叠的、部分核酸内切酶IV消化产物，对完全测序的寡核苷酸进行排序。所描述的测序方法是通用的，并且可以用于直接确定其他DNA片段的一级结构。
Methods for the direct determination of nucleotide sequences in DNA have been developed and used to determine the complete primary structure of a fragment of bacteriophage φX174 DNA which is 48 residues in length. This fragment was liberated from φX DNA by digestion at low temperature and high ionic strength with the T4 phage-induced endonuclease IV. The fragment was redigested with endonuclease IV under vigorous conditions and the products fractionated two-dimensionally providing a characteristic endonuclease IV “fingerprint” of the fragment. The Burton (Burton & Petersen, 1960) depurination reaction was used to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini. These oligonucleotide products were then fully sequenced by partial exonuclease digestion with spleen and snake venom phosphodiesterase and analysis of the fractionated digests by base composition, depurination, and 5′ end-group analysis using exonuclease I. Rules for the interpretation of two-dimensional fingerprints of partial exonuclease digests, which rapidly provide sequence information by simple inspection, were also deduced. To derive the complete structure of the fragment, the fully sequenced oligonucleotides were ordered by characterizing large, overlapping, partial endonuclease IV digestion products by means of the depurination reaction. The sequencing methods described are general and may be used for the direct determination of the primary structures of other fragments of DNA.