Randerath K, Randerath E, Chia L S, Gupta R C, Sivarajan M
Nucleic Acids Res. 1974 Sep;1(9):1121-41. doi: 10.1093/nar/1.9.1121.
A tritium derivative method for sequence analysis of polyribonucleotides is detailed, which is based on borotritide reduction of oligonucleotide dialdehydes generated by treatment of polyribonucleotides with alkaline phosphatase and excess periodate at pH 8 (borate buffer; no primary amine present in the reaction mixture). While neither phosphatase nor periodate possess any intrinsic exonuclease activity their combination mimics an RNA-specific exonuclease ("pseudo-exonuclease"). Procedures are described for separation and characterization of tritiumlabeled oligonucleotide derivatives. The sequence is deduced by identification of labeled 3'-termini following separation of the reduced nucleotide intermediates according to chain length. The sensitivity of the method is indicated by the fact that as little as 0.01 O.D.260 unit of a nonradioactive decanucleotide is sufficient for sequence determination.
详细介绍了一种用于多聚核糖核苷酸序列分析的氚衍生物方法，该方法基于在pH 8（硼酸盐缓冲液；反应混合物中不存在伯胺）条件下，用碱性磷酸酶和过量高碘酸盐处理多聚核糖核苷酸所产生的寡核苷酸二醛的硼氚化物还原反应。虽然磷酸酶和高碘酸盐都不具有任何内在的核酸外切酶活性，但它们的组合模拟了一种RNA特异性核酸外切酶（“假核酸外切酶”）。描述了分离和表征氚标记寡核苷酸衍生物的程序。通过根据链长分离还原的核苷酸中间体后鉴定标记的3'-末端来推导序列。该方法的灵敏度体现在，低至0.01 O.D.260单位的非放射性十聚核苷酸就足以进行序列测定。
