Patent ID: 7241598

Claim:
A method for preparing single-chain variable fragments encoding an antigen-specific antibody, comprising the steps of: a) extracting genomic DNAs of lymphocytes from a non-immunized mammal or a mammal immunized with a specific antigen; b) recovering variable regions of immunoglobulin heavy and light chains from the genomic DNAs; c) amplifying the variable regions using PCR with a forward primer and a set of different reverse primers designed for the variable regions, wherein the reverse primers are represented by gAcDgTgASHRDRgTBccTKSRccccANNNNNN for V H or YYTKgTSccHBcDccgAAYgTNNNNNN for V L-κ , in which N is A, c, T or g, R is A or g, Y is c or T, M is A or c, K is T or g, S is c or g, H is A, T or c, B is T, c or g, and D is A, T or g; d) introducing both an adaptor and a linker to the variable regions obtained in step c); and e) linking the variable regions obtained in step d) by an overlap-extensive PCR to obtain single-chain variable fragments.