[A novel method of amplifying the heavy-chain variable regions from immunoglobulin cDNAs].
The amplification of variable regions (Fv) of immunoglobulins has become a crucial technique for studying antigen-antibody interactions and cloning the monoclonal antibodies (mAbs). But this work becomes a major challenge in cloning antibody genes either from hybridoma cell lines or B cells. Isolation of the immunoglobulin heavy-chain variable regions (VH) from two mouse hybridoma cell lines based on conventional protocols using both the specific consensus primers and the commercial primer set of VH regions, was unsuccessful. This prompted us to design our own primers for VH regions of immunoglobulins. A novel algorithms and reverse polymerase chain reaction (PCR) protocol--3' RACE and 5' RACE were established in order to recognize the VH genes. The most conserved region in the end of framework region (FR1) of the VH cDNA region was identified with the program and a degenerated primer were designed in the most conserved region followed by 3' RACE and 5' RACE. The new protocol rescued the failed PCR amplifications. Thereby, a improvement in the degenerate primers designing programs and a notable increase in amplification specificity were achieved. This approach can be useful in primer design and amplification of VH and light-chain variable regions (VL) gene libraries or cloning of the unknown genes in the large gene families and extend to other species or members of the immunoglobulin superfamily.
Wang
Ying
Y
Medical Laboratory Center, First Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
Chen
Wei
W
Li
Xu
X
Cheng
Bing
B
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015321:Gene Rearrangement / Q000235:genetics; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region