Cytosine specific DNA sequencing with hydrogen peroxide.
Cytosine specific DNA sequencing with hydrogen peroxide.
复制标题
使用过氧化氢进行胞嘧啶特异性 DNA 测序。
DOI：
10.1093/nar/23.23.4922
复制
发表时间：
1995
影响因子：
14.9
通讯作者：
Church,GM
中科院分区：
文献类型：
--
作者：
Richterich,P;Lakey,ND;Lee,HM;Mao,JI;Smith,D;Church,GM
We report a simple protocol for a C-specific chemical DNA sequencing reaction with hydrogen peroxide. Chemical sequencing offers unique advantages over dideoxy sequencing for specific applications, which include the study of DNA-pro-tein interactions ('footprinting'), probing for alterations in DNA structure, and oligonucleotide sequencing. While dideoxy sequencing is often preferred over chemical methods, chemical sequencing can be useful for'problem templates', for example regions with very high GC-content which have artifact bandsin all four lanes due to'polymerase stops'. Compared with the original C-specific chemical sequencing reactions with hydrazine (1), the hydrogen peroxide reaction is attractive because ituses a less toxic, more stable, andmore easily available reagent. When used in combination with other alterna-tives to the original base-specific chemical sequencing reactions that have been described in the literature (2-6), the entire chemical sequencing procedure can be based on chemicals of reduced toxicity, while simultaneously being more efficient and robust than the original Maxam-Gilbert protocol. The base-specific modification of single-stranded DNA with 2-3 M hydrogen peroxide in Tris and carbonate buffers has been described (7). The reaction is specific for C-residues at pH 7-8.5, and T-specific if the pH is> 9. Under these conditions, reaction with double-stranded DNA is much slower (7), and we could not obtain readable sequence patterns from double-stranded DNA. When triethyl ammonium acetate buffer was used instead of Tris, however, we observed C-specific modification of doublestranded DNA which yielded perfectly readable sequence patterns (Fig. 1).Individual colonies from 20 different libraries in multiplex vectors were pooled and grown together in 40 ml SOA medium overnight (4). DNA was prepared using the Qiagen tip 100 columns according to the manufacturers instructions, digested with Notl, precipitated, and resuspended in 60 glH20 to a typical concentration of 0.3 ig/gl. Five microlitres of DNA was transferred to a V-bottom microtiter plate with hydrophilic coating ('AGTC plates'; Advanced Genetic Technologies, Gaithersburg, MD), and 15 p1 ofa freshly prepared H202 solution [245 mM H202 (Sigma), 10 mM EDTA, 133mM triethyl amine acetate pH 7.0 (Perkin-Elmer)] was added. After taping the plate, vortexing for 5 s, brief centrifugation to collect solutions at the
