Determination of gene usage by differential polymerase chain reaction product hybridization.
All swine VH genes belong to a highly homologous family and have identical leader sequences, and the swine VH locus contains a single JH. The small number of VH genes used by the fetus and neonate in the first 6 weeks have unique CDR1 and CDR2 sequences, permitting each to be identified using specific oligonucleotide probes. We have used this system as a model for the development of a rapid method for determining the proportional usage of closely related genes based on differential polymerase chain reaction (PCR) product hybridization (DPPH). The validity of the method is demonstrated using mixtures of PCR product containing known amounts of VH gene DNAs and by comparing data obtained by this method with those obtained by enumeration of individual hybridizing clones from lymphoid tissue and peripheral blood B cells. Since DPPH is at least 100-fold more efficient than the enumeration of individual hybridizing clones, it is especially useful for analyzing large numbers of samples in population studies. The possible extension of this method to the usage of other genes is discussed.
Sun
J
J
Department of Microbiology, University of Iowa, Iowa City 52242-1109, USA.
Shey
M
M
Butler
J E
JE
D000818:Animals; D000831:Animals, Newborn; D001402:B-Lymphocytes / Q000276:immunology; D004247:DNA / Q000032:analysis* / Q000097:blood / Q000276:immunology; D004622:Embryo, Mammalian / Q000276:immunology; D005803:Genes, Immunoglobulin; D007135:Immunoglobulin Variable Region / Q000097:blood / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013154:Spleen / Q000276:immunology; D013552:Swine
D007135:Immunoglobulin Variable Region; D004247:DNA