Utilization of a potentially universal downstream primer in the rapid identification and characterization of V lambda...

Polymerase chain reaction (PCR) has increased dramatically the speed of cloning and characterizing numerous genes. However, its application to identifying and analysing new germline Ig-variable (V) gene families has been hampered by the lack of sequence information in the downstream flanking regions of the concerned V genes, which are deleted during V(D)J rearrangements. To circumvent this problem, the possibility was explored that a degenerate downstream primer may be used in conjunction with a specific upstream primer, to clone members of new V lambda gene families, as much less is known about V lambda genes than Vh and Vk genes in humans. Firstly the feasibility and the specificity of a degenerate primer was examined by comparing it with an established downstream primer in amplifying known V lambda 1 genes. The results were positive. Thus, the degenerate primer was used to clone and characterize germline V lambda genes of the recently defined V lambda 8 and V lambda 9 gene families. This current strategy may help speed up the identification and characterization of all human V lambda genes. Moreover, a similar strategy can be applied to identify and characterize rapidly new V genes of either known or unknown Ig and T-cell receptor V gene families.