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Design and analysis of PCR primers for the amplification and cloning of human immunoglobulin Fab fragments

Design and analysis of PCR primers for the amplification and cloning of human immunoglobulin Fab... We have developed PCR primers for the amplification and cloning of the genes encoding human antibody fragments. Two sets of primers were designed to amplify the coding sequence of the complete variable region and the first constant domain of immunoglobulin heavy chains. One set of primers was designed to amplify the coding sequence of complete kappa light chains. These three sets of primers were used in PCR amplifications with cDNA prepared from EBV transformed B cells as the template. The amplified fragments were cloned and their nucleotide sequences were determined. Analysis of the DNA sequences of such PCR generated antibody fragments suggested that our primer sets were able to amplify the major subsets of the heavy and light chain variable region genes. Comparison between the estimated frequency of the different subsets of heavy chain variable region genes and the distribution of our subclones further indicated that one set of our primers was able to proportionally amplify the subsets of the heavy chain family. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Antibodies IOS Press

Design and analysis of PCR primers for the amplification and cloning of human immunoglobulin Fab fragments

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Publisher
IOS Press
Copyright
Copyright © 1994 by IOS Press, Inc
ISSN
1093-2607
eISSN
1875-869X
DOI
10.3233/HAB-1994-51-208
Publisher site
See Article on Publisher Site

Abstract

We have developed PCR primers for the amplification and cloning of the genes encoding human antibody fragments. Two sets of primers were designed to amplify the coding sequence of the complete variable region and the first constant domain of immunoglobulin heavy chains. One set of primers was designed to amplify the coding sequence of complete kappa light chains. These three sets of primers were used in PCR amplifications with cDNA prepared from EBV transformed B cells as the template. The amplified fragments were cloned and their nucleotide sequences were determined. Analysis of the DNA sequences of such PCR generated antibody fragments suggested that our primer sets were able to amplify the major subsets of the heavy and light chain variable region genes. Comparison between the estimated frequency of the different subsets of heavy chain variable region genes and the distribution of our subclones further indicated that one set of our primers was able to proportionally amplify the subsets of the heavy chain family.

Journal

Human AntibodiesIOS Press

Published: Jan 1, 1994

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