NOVEL METHOD TO MAP TRANSCRIPTS - EVIDENCE FOR HOMOLOGY BETWEEN AN ADENOVIRUS MESSENGER-RNA AND DISCRETE MULTIPLE REGIONS OF VIRAL GENOME
NOVEL METHOD TO MAP TRANSCRIPTS - EVIDENCE FOR HOMOLOGY BETWEEN AN ADENOVIRUS MESSENGER-RNA AND DISCRETE MULTIPLE REGIONS OF VIRAL GENOME
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DOI：
10.1016/0092-8674(77)90182-9
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发表时间：
1977-01-01
期刊：
影响因子：
64.5
通讯作者：
HASSELL, JA
中科院分区：
文献类型：
--
作者：
DUNN, AR;HASSELL, JA
设计了一种方法，该方法允许通过2步杂交程序（夹心杂交）绘制转录本。从用腺病毒-SV-40杂交体（Ad 2 + ND 1）感染的[非洲绿色猴肾CV-1和人宫颈癌HeLa]细胞中提取的RNA与固定在硝酸纤维素滤膜上的2型腺病毒（Ad 2）DNA的[EcoRI、BgI II、Hind II和Hind III]限制性内切酶片段杂交。含有Ad 2和SV-40序列的RNA通过它们的Ad 2序列形成双链体，留下它们的SV-40序列作为突出的尾部。用32 P标记的SV-40 DNA退火导致这些尾部被标记，从而允许放射自显影鉴定与RNA同源的Ad 2 DNA序列。该技术的高灵敏度，通过使用32 P-标记的RNA的高比活性，表明杂交的Ad 2 + ND 1 RNA发生在几个位置上的Ad 2基因组，除了预期的杂交位点附近的SV-40插入。
A method was devised which permits mapping of transcripts by a 2-step hybridization procedure (sandwich hybridization). RNA extracted from [African green monkey kidney CV-1 and human cervical carcinoma HeLa] cells infected with an adenovirus-SV-40 hybrid (Ad2+ND1) was hybridized to [Eco RI, Bgl II, Hind II and Hind III] restriction endonuclease fragments of adenovirus type 2 (Ad2) DNA immobilized on nitrocellulose filters. RNA containing Ad2 and SV-40 sequences formed duplexes through their Ad2 sequences, leaving their SV-40 sequences as protruding tails. Annealing with 32P-labeled SV-40 DNA caused these tails to become labeled, permitting autoradiographic identification of the sequences of Ad2 DNA which are homologous to the RNA. The high sensitivity of this technique, achieved through the use of 32P-labeled RNA of high specific activity, showed that hybridization of Ad2+ND1 RNA occurs at several locations on the Ad2 genome, besides the expected sites of hybridization proximal to the SV-40 insertion.