PCR-generated cDNA libraries from reduced numbers of mouse oocytes.
We describe a rapid and reproducible method for cloning cDNA amplified from 10 mouse oocytes. The procedure consists in priming cDNA synthesis from a crude cellular extract using an oligo d(T) containing primer and submitting the size-limited cDNA first strand to poly(dG) tailing. The whole cDNA population is then polymerase chain reaction (PCR) amplified using two primers complementary to oligo d(A) and oligo d(G) ends of the cDNA. In this procedure no purification steps are required. We obtained about 5 x 10(6) clones from 10 oocytes. Screening of the library showed that the relative abundance of the transcripts was preserved during amplification and cloning and that the procedure allows cloning of low-abundance sequences at least as rare as 0.008% of the mRNA. The repeatable generation of representative cDNA libraries from reduced numbers of oocytes or embryos should open new opportunities for obtaining genetic information from mammalian preimplantation embryos.
Unite de biologie du developpement INRA, Jouy en Josas, France.
F
F
Revel
J P
JP
Renard
V
V
Duranthon
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011061:Poly A