Sasavage N L, Smith M, Gillam S, Astell C, Nilson J H, Rottman F
Biochemistry. 1980 Apr 29;19(9):1737-43. doi: 10.1021/bi00550a003.
Twelve synthetic oligodeoxynucleotide primers of the general sequence d(pT8-N-N') were tested in a reverse transcriptase reaction for specific initiation of complementary deoxyribonucleic acid (cDNA) synthesis at the poly(adenylic acid) junction of a messenger ribonucleic acid (mRNA) template. Only the sequence d(pT8-G-C) functioned as a specific primer of cDNA synthesis with an enriched fraction of bovine growth hormone mRNA from the anterior pituitary gland and produced unique fragments in a dideoxy sequencing reaction. The nucleotide sequence obtained by this method extended into the protein coding region of bovine growth hormone mRNA and was confirmed by chemical sequencing of the cDNA initiated with [5'-32P]d(pT8-G-C). The 3'-untranslated region of bovine growth hormone mRNA is 104 nucleotides in length and contains regions of significant homology with both rat and human growth hormone mRNAs, including the region surrounding the common AAUAAA hexanucleotide. The method presented here for selection of the d(pT8-N-N') primer complementary to the poly(A) junction of mRNA is of general applicability for nucleotide sequence analysis of partially purified mRNAs.
对12条通用序列为d(pT8-N-N')的合成寡聚脱氧核苷酸引物进行了测试，以在逆转录酶反应中于信使核糖核酸（mRNA）模板的聚腺苷酸连接处特异性起始互补脱氧核糖核酸（cDNA）合成。只有序列d(pT8-G-C)能作为cDNA合成的特异性引物，用于从前垂体中富集的牛生长激素mRNA，并在双脱氧测序反应中产生独特的片段。通过这种方法获得的核苷酸序列延伸至牛生长激素mRNA的蛋白质编码区，并通过以[5'-32P]d(pT8-G-C)起始的cDNA化学测序得到证实。牛生长激素mRNA的3'-非翻译区长度为104个核苷酸，并且与大鼠和人类生长激素mRNA都有显著的同源区域，包括围绕常见的AAUAAA六核苷酸的区域。本文介绍的用于选择与mRNA的聚(A)连接处互补的d(pT8-N-N')引物的方法，对于部分纯化的mRNA的核苷酸序列分析具有普遍适用性。
