Creation of Recombinant Antibodies: Using 5′-RACE to Amplify Immunoglobulin Sequences

Abstract

Modern molecular biology techniques have been applied to the production of therapeutic antibodies. Nonetheless, recombinant antibodies remain the exception rather than the rule for the antibodies used in most research and diagnostic applications. The lack of penetration of recombinant antibodies into the research arena can be attributed largely to the fact that the Ig gene families are large and the variable region gene segments are, indeed, variable, precluding the use of polymerase chain reaction (PCR) with two simple primers to amplify the heavy and light chain gene segments. Because of the complexity of the V gene family and the number of possible sequences for amplification, there may be a distinct advantage to using a PCR method that does not require a specific 5′ primer to amplify the gene segment. 5′-RACE is just such a method. In the original 5′-RACE method, mRNA served as a template for cDNA synthesis using either oligo(dT) priming or a gene-specific primer. Terminal deoxynucleotide transferase (TdT) was used to tail the first strand with a region of known sequence, such as poly(A). Once tailed, the second strand can be amplified using poly(T). This 5′-RACE protocol uses the TdT activity of reverse transcriptase, which allows nontemplated addition of nucleotides to the end of the nascently made cDNA first strand.