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.
Department of Genetics and Development, Columbia University, New York, NY 10032.
J
J
Welsh
J P
JP
Liu
A
A
Efstratiadis
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis*; D015698:Genomic Library; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D014981:Xenopus
D012313:RNA; D004247:DNA