Application for PCR technology to subtractive cDNA cloning: identification of genes expressed specifically in murine plasmacytoma cells.
We describe a simple method for preparing a renewable source of subtractive cDNA which can be used as a hybridization probe or as insert which can be cloned into a variety of convenient vectors. This has been done by ligating a double-stranded oligonucleotide to each end of double-stranded subtractive cDNA, and then using this oligonucleotide sequence to amplify the heterogeneous population of cDNA molecules using the polymerase chain reaction and thermostable Taq DNA polymerase. This method improves the chances for identifying cDNA clones representing low abundance mRNAs that are expressed differentially. Using this approach, we have identified cDNA clones which detect three different low abundance mRNAs that are expressed in mouse plasmacytoma cell lines but not in mouse pre-B or B lymphoma cell lines.
Timblin
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C
NCI-Navy Medical Oncology Branch, National Cancer Institute, National Institutes of Health and Naval Hospital, Bethesda, MD 2084-5015.
Battey
J
J
Kuehl
W M
WM
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D014644:Genetic Variation; D051379:Mice; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D010954:Plasmacytoma / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured / Q000276:immunology
D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm