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

Paper ID Volume ID Publish Year Pages File Format Full-Text
34107 45003 2010 10 PDF Available
Title
V-gene amplification revisited – An optimised procedure for amplification of rearranged human antibody genes of different isotypes
Abstract

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.

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V-gene amplification revisited – An optimised procedure for amplification of rearranged human antibody genes of different isotypes
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Publisher
Database: Elsevier - ScienceDirect
Journal: New Biotechnology - Volume 27, Issue 2, 31 May 2010, Pages 108–117
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us