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Title of Journal: World J Microbiol Biotechnol

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Abbravation: World Journal of Microbiology and Biotechnology

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Springer Netherlands

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DOI

10.1016/0308-0161(77)90005-9

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1573-0972

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Degenerate primer design to clone the human repert

Authors: Ying Sun HongYan Liu Ling Mu EnJie Luo
Publish Date: 2011/07/08
Volume: 28, Issue: 1, Pages: 381-386
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Abstract

Amplifying the variable Fv or V regions of immunoglobulins Ig has become a challenge in cloning antibody genes for phage display a technique used to study protein–protein protein–peptide and protein–DNA interactions using bacteriophages to connect proteins with the genetic information that encodes them Key parameters affecting the amplification of full antibody repertoires includes the availability of primers that can amplify as many V genes as possible however the strategy used to design these primers and programs used to make the necessary alignments have not been well studied and clearly detailed in the literature Here we present a set of primers computationally designed by iCODEHOP based on a database of human germline Ig sequences We used reverse transcription polymerase chain reaction RTPCR protocols that would recognize the VH genes from human peripheral blood mononuclear cells We identified the most highly conserved region in framework 1 and framework 4 of the Ig cDNA and designed a set of degenerated 5′ primers The VH genes were successfully amplified by RTPCR This new primer has facilitated the creation of more diverse VH libraries than has been previously possible Moreover iCODEHOP improved the primer design efficiency and was found useful both for cloning unknown genes in gene families and for building VH gene librariesWe would like to thank HongYan Liu and Ling Mu for providing Peripheralblood from convalescent patients of HFRS This work was supported by the Natural Science Foundation of Liaoning province No 20062101 and the Shenyang Bureau of Science and Technology 1071186900

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