A novel method for sequencing members of multi-gene families

摘要

We have adapted a method of amplifying and sequencing genomic DNA (1) for rapidly identifying and partially sequencing genes or transcripts from members of a multi-gene family in which one end is conserved and the other end is highly variable. By utilizing nested, conserved sequence primers in conjunction with primers specific for 6 bp restriction enzyme recognition sequences in a PCR, a family of related sequences can be amplified. Further specificity can be obtained by analyzing known gene family members for restriction enzyme sites, and designing restriction primers which will only amplify the desired subset. We applied this technique to identify Va and V beta usage and obtain CDR3 sequence information for T cell receptor (TCR) a and 3 chains. Asamplification of only a small portion of the Va and Vp genes is required for their identification, due to the absence of somatic hypermutation in V region genes (2), restriction sites found in a majority of Va and VP genes were used as a basis for primer design, with PCR amplification specificity conveyed by the use of nested oligonucleotide primers homologous to the downstream Ca or CP genes. Sequences of murine Va and V(3 regions were obtained from GenBank v. 73 (October 1992) and analyzed for commonrestriction sites. T7 Restriction Primers specific for the seven common sites were constructed by tailing a 20 bp T7 promoter sequence at the 3' end with recognition sequences of the selected restriction enzymes and synthesized in the Molecular BiologyCore Facility at the Mayo Clinic. Tables 1 and 2 lists the sequences of all primers utilized in this study. Target sequence amplified by the PCR was transcribed with T7 polymerase into RNA which was directly sequenced (1). Downstream amplification from these internal V region sites provided not only rapid V and J region identification, but also the complete CDR3 junctional region sequence.