SEQUENCING HEAVY-CHAIN AND LIGHT-CHAIN VARIABLE GENES OF SINGLE B-HYBRIDOMA CELLS BY TOTAL ENZYMATIC AMPLIFICATION

We have devised a protocol to obtain accurate and complete sequences of the immunoglobulin heavy- and light-chain variable-region (V(H) and V(L)) genes of single B-hybridoma cells that express defined V genes. The amplification achieved ranges from 2 x 10(13)- to 1 X 10(14)-fold. Only one potential Taq DNA polymerase error was observed in 7590 nucleotides of sequence, thus permitting the identification of naturally occurring somatic mutations. The two-step nature of the amplification protocol provides sufficient DNA for a minimum of 160 sets of sequencing reactions of both the V(H) and V(L) genes from one cell without cloning. The amplification of relatively long segments of DNA in the first step of the protocol permits second-step amplification and sequencing of regions that flank V(H) and V(L) codons. Fractionating cellular lysates prior to the first step of amplification permits the separate amplification of V genes on opposite sister chromatids and possibly on opposite strands of the same DNA duplex. Accurate sequencing of V(H) and V(L) genes of defined germ-line origin that are expressed by single B cells taken directly from the animal is thus made feasible by this approach.