Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA.
BACKGROUND
The amplification of variable regions of immunoglobulins has become a major challenge in the cloning of antibody genes, whether from hybridoma cell lines or splenic B cells. Using conventional protocols, the heavy-chain variable region genes often are not amplified successfully from the hybridoma cell lines.


RESULTS
A novel method was developed to design the degenerated primer of immunoglobulin cDNA and to amplify cDNA ends rapidly. Polymerase chain reaction protocols were performed to recognize the VH gene from the hybridoma cell line. The most highly conserved region in the middle of the VH regions of the Ig cDNA was identified, and a degenerated 5'primer was designed, using our algorithms. The VH gene was amplified by both the 3'RACE and 5'RACE. The VH sequence of CSA cells was 399 bp.


CONCLUSIONS
The new protocol rescued the amplifications of the VH gene that had failed under conventional protocols. In addition, there was a notable increase in amplification specificity. Moreover, the algorithm improved the primer design efficiency and was shown to be useful both for building VH and VL gene libraries and for the cloning of unknown genes in gene families.
Wang
Ying
Y
Center for Laboratory Medicine, First Teaching Hospital of Medical School, Xi'an Jiaotong University, Xi'an 710061, China. ywang00@sohu.com
Chen
Wei
W
Li
Xu
X
Cheng
Bing
B
D002460:Cell Line; D017931:DNA Primers / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D015321:Gene Rearrangement / Q000235:genetics; D006825:Hybridomas / Q000502:physiology; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region