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          Institute: MPI für molekulare Genetik     Collection: Department of Vertebrate Genomics     Display Documents

ID: 556508.0, MPI für molekulare Genetik / Department of Vertebrate Genomics
V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes.
Authors:Lim, T. S.; Mollova, S.; Rubelt, F.; Sievert, V.; Dübel, S.; Lehrach, H.; Konthur, Z.
Research Context:The work was supported by the German Federal Ministry for Education and Research (BMBF) through the National Genome Research Network (NGFN-II) “Antibody Factory” (Grant No. 01GR0427) and the Max Planck Society. ZK and SD acknowledge additional support from EU-FP6 CA “ProteomeBinders” (RICA. 026008). TSL acknowledges financial support from the Ministry of Higher Education Malaysia and Institute for Research in Molecular Medicine, University Science Malaysia.
Date of Publication (YYYY-MM-DD):2010-05-31
Title of Journal:Nature biotechnology
Journal Abbrev.:N Biotechnol
Issue / Number:2
Start Page:108
End Page:111
Copyright:© 2010 Elsevier B.V. All rights reserved.
Review Status:not specified
Audience:Experts Only
Abstract / Description: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.
Comment of the Author/Creator:Email: konthur@molgen.mpg.de
External Publication Status:published
Document Type:Article
Version Comment:Automatic journal name synchronization
Communicated by:Hans Lehrach
Affiliations:MPI für molekulare Genetik
External Affiliations:1.Free University Berlin, Faculty of Biology, Chemistry and Pharmacy, Takustrasse 3, 14195 Berlin, Germany;
2.Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia;
3.Technical University Braunschweig, Institute for Biochemistry and Biotechnology, Department of Biotechnology, Spielmannstrasse 7, 38106 Braunschweig, Germany.
Identifiers:ISSN:1871-6784 [ID No:1]
URL:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=... [ID No:2]
DOI:10.1016/j.nbt.2010.01.001 [ID No:2]
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