Extended fitness of variable region primers by a novel PCR protocol.
In an attempt to isolate the variable region genes of five monoclonal natural autoantibodies, we failed to amplify all ten variable region cDNAs using a V region primer set and following conventional PCR protocols. A novel PCR procedure was established in which the reaction began with three cycles of low temperature annealing and chain extension using 5'-primer as the only primer present, and continuing with conventional PCR cycles following addition of 3'-primer. Combined with solid-phase cDNA synthesis on oligo(dT)cellulose, this new protocol rescued all failed PCR amplifications based on a conventional protocol, thereby extending the universal utility of the V region primers. A notable increase in amplification specificity was also achieved. Such a protocol could be useful in primer design and the amplification of non-antibody genes whose sequences are not well-understood.
Lan
F
F
Department of Clinical Laboratory Diagnostics, DongFang Hospital, Fuzhou City, China.
Umeda
M
M
Inoue
K
K
D000818:Animals; D017931:DNA Primers; D005803:Genes, Immunoglobulin; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D007135:Immunoglobulin Variable Region