Use of family specific leader region primers for PCR amplification of the human heavy chain variable region gene repertoire.
We have designed a set of six, non-degenerate oligonucleotide primers, corresponding to the 5' leader regions of each of the six human VH gene families. A general strategy for family specific polymerase chain reaction amplification is described using these primers and a conserved 3' primer corresponding to frame work 3, JH, or constant region. This strategy was used to isolate and sequence novel human germline VH genes belonging to the VH2 and VH4 families. Under certain conditions, chimeric VH sequences were created by a "jumping polymerase chain reaction", combining DNA segments from different germline genes, but this could be avoided by limiting the number of amplification cycles. PCR amplification with these family specific primers will facilitate studies of the repertoire of germline VH genes as well as studies on VH gene usage in normal and aberrant (B cell malignancies, autoimmune diseases, etc.) B cell populations.
Campbell
M J
MJ
Stanford University School of Medicine, Department of Medicine, CA 94305-5306.
Zelenetz
A D
AD
Levy
S
S
Levy
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002999:Clone Cells; D004277:DNA, Single-Stranded / Q000235:genetics; D015322:Gene Rearrangement, B-Lymphocyte; D005803:Genes, Immunoglobulin / Q000235:genetics*; D005854:Germ Cells; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid
D004277:DNA, Single-Stranded; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region