{"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}
引用次数: 39
Abstract
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.
京公网安备 11010802042870号
京ICP备2023020795号-1
分享
求助内容：
应助结果提醒方式：
扫码关注我们
