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Nucleic Acids Research, 1991, Vol. 19, No. 25 7097-7104
© 1991

Foreword

Subtractive cDNA cloning using oligo(dt)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells

Eiji Hara1, Tomohiro Kato, Susumu Nakada2, Souei Sekiya3 and Kinichiro Oda2,*

Institute of Medical Science, St Marianna University School of Medicine Kawasaki 216 1Departments of Allpied Biological Science Chiba 278 2Departments of Biological Science and Technology, SCience University of Tokyo Chiba 278 3Department of Obstetrics and Gynaecology, Chiba UNiversity School of Medicine Chiba 280, Japan

*To whom correspondence should be addressed

The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10–2M N,N'-hexamethylene-bis-acetamlde (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)3o-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-treated NEC14 cells using an oligo(dT)30 primer covalently linked to latex particles. After removal of the mRNA template by heat-denaturation and centrifugation, the subtractive hybridization was carried out between the cDNA-oligo(dT)30-Latex and mRNA from untreated NEC14 cells. Unhybridized mRNA collected by centrlfugatlon was hybridized repeatedly to the cONAoligo( dT)30-Latex and subtractive mRNA was converted to cDNA. The subtractive cDNA was then amplified by PCR and cloned info pBluescript || KS-. The cDNA library thus constructed consisted of approximately 10,000 independent clones with cDNA nserts of 1.7 Kb on average. Differential hybridization of these transformants indicated that approximately 3% of them contained cDNA inserts specific to the undifferentiated EC cells, some of which were derived from low abundance mRNAs.


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