{"title":"pcr扩增总cDNA的克隆:小鼠卵母细胞cDNA文库的构建","authors":"John Welsh , Jeh-Ping Liu , Argiris Efstratiadis","doi":"10.1016/0735-0651(90)90038-H","DOIUrl":null,"url":null,"abstract":"<div><p>We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)<sup>+</sup> RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)<sup>+</sup> RNA in this mammalian oocyte.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 1","pages":"Pages 5-17"},"PeriodicalIF":0.0000,"publicationDate":"1990-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90038-H","citationCount":"39","resultStr":"{\"title\":\"Cloning of PCR-amplified total cDNA: Construction of a mouse oocyte cDNA library\",\"authors\":\"John Welsh , Jeh-Ping Liu , Argiris Efstratiadis\",\"doi\":\"10.1016/0735-0651(90)90038-H\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)<sup>+</sup> RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)<sup>+</sup> RNA in this mammalian oocyte.</p></div>\",\"PeriodicalId\":77714,\"journal\":{\"name\":\"Gene analysis techniques\",\"volume\":\"7 1\",\"pages\":\"Pages 5-17\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"1990-02-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/0735-0651(90)90038-H\",\"citationCount\":\"39\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Gene analysis techniques\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/073506519090038H\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Gene analysis techniques","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/073506519090038H","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Cloning of PCR-amplified total cDNA: Construction of a mouse oocyte cDNA library
We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)+ RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)+ RNA in this mammalian oocyte.
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