Construction of representative immunoglobulin variable region cDNA libraries from human peripheral blood lymphocytes without in vitro stimulation.
We have developed a sensitive and specific method for preparation of representative IgM and IgG cDNA libraries of peripheral blood lymphocytes without in vitro stimulation of B cells and without primer-based bias. The procedure involves preparation of double-stranded cDNA from initial C mu and C gamma constant region primers. Linkers are attached to the cDNA and nonselective polymerase chain reaction (PCR) amplification is performed with the linkers as primers. This product is ligated to M13 RF DNA, and selective PCR amplification is achieved with a nested constant region primer and a vector primer. The product is cloned into M13 phage. More than 85% of the white plaques are positive with a JH probe. Diversity of the library is confirmed by hybridization with VH gene probes and by DNA sequencing. A sample of 7-30 ml of blood from a normal human adult can yield both mu and gamma cDNA libraries. Analysis of the libraries yields a picture of Ig variable gene usage at the time of sampling.
Huang
C
C
Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111.
Stollar
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BD
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008214:Lymphocytes / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis
D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D004247:DNA