V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes.
For studying human antibody variable (V)-gene usage in any group of individuals or for the generation of recombinant human antibody libraries for phage display, quality and yield of the amplified V-gene repertoire is of utmost importance. Key parameters affecting the amplification of full antibody repertoires are V-gene specific primer design, complementary DNA (cDNA) synthesis from total RNA extracts of peripheral blood mononuclear cells (PBMCs) and ultimately the polymerase chain reaction (PCR). In this work we analysed all these factors; we performed a detailed bioinformatic analysis of V-gene specific primers based on VBASE2 and evaluated the influence of different commercially available reverse transcriptases on cDNA synthesis and polymerases on PCR efficiency. The primers presented cover near to 100% of all functional and putatively functional V-genes in VBASE2 and the final protocol presents an optimised combination of commercial enzymes and reaction additives for cDNA synthesis and PCR conditions for V-gene amplification. Finally, applying this protocol in combination with different immunoglobulin (Ig) chain specific reverse primers we were able to amplify rearranged antibody genes of different isotypes under investigation.
Lim
Theam Soon
TS
Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Berlin, Germany.
Mollova
Svetlana
S
Rubelt
Florian
F
Sievert
Volker
V
Dübel
Stefan
S
Lehrach
Hans
H
Konthur
Zoltán
Z
D000906:Antibodies / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D014644:Genetic Variation; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D057127:Single-Chain Antibodies / Q000235:genetics*
D000906:Antibodies; D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies