V-gene amplification revisited - An optimised procedure for amplification of 
rearranged human antibody genes of different isotypes.

Lim, T. S.; Mollova, S.; Rubelt, F.; Sievert, V.; Dübel, S.; Lehrach, H.; Konthur, Z.

doi:10.1016/j.nbt.2010.01.001

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V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes.

MPG-Autoren

Lim, T. S.
Max Planck Society;

Mollova, S.
Max Planck Society;

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Rubelt, F.
In vitro Ligand Screening (Zoltán Konthur), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Sievert, V.
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Lehrach, H.
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Konthur, Z.
In vitro Ligand Screening (Zoltán Konthur), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Zitation

Lim, T. S., Mollova, S., Rubelt, F., Sievert, V., Dübel, S., Lehrach, H., et al. (2010). V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes. Nature biotechnology, 27(2), 108-111. doi:10.1016/j.nbt.2010.01.001.

Zitierlink: https://hdl.handle.net/11858/00-001M-0000-0010-7AF3-B

Zusammenfassung

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.