PCR of the V Region
To engineer or manipulate antibody or Fv-based molecules, isolation of the V region of the antibodies is necessary. A number of strategies can be adopted for amplification; one such approach is to use subgroup-specific oligonucleotides for the amplification of the V-region genes. Small differences in the conserved regions of variable genes have allowed the division of murine VH and VL genes into subgroups and families, with the members of one family having more homology with each other than members of another family. Therefore, it is possible to categorize the variable genes of particular hybridoma, and there are fewer chances of amplifying VL genes of myeloma partners of the hybridoma with the family-specific primers as compared to a set of highly degenerate primers. Therefore, many (up to six) different 5’ primers can be designed from the FR1 of the kappa chain using the Kabat database (1). Similarly, four different 5’ primers are usually designed for the heavy chain. The 3’ primers are designed from constant segments of the chains (2).
KeywordsGuanidinium Thiocyanate Kappa Chain Reverse Transcriptase Buffer Constant Segment Versus Region Gene
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