About: 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 (RT-PCR) protocols that would recognize the V(H) 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 V(H) genes were successfully amplified by RT-PCR. This new primer has facilitated the creation of more diverse V(H) 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 V(H) gene libraries.   Goto Sponge  NotDistinct  Permalink

An Entity of Type : fabio:Abstract, within Data Space : wasabi.inria.fr associated with source document(s)

AttributesValues
type
value
  • 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 (RT-PCR) protocols that would recognize the V(H) 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 V(H) genes were successfully amplified by RT-PCR. This new primer has facilitated the creation of more diverse V(H) 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 V(H) gene libraries.
subject
  • Bacteriophages
  • Asexual reproduction
  • Gaseous signaling molecules
  • Molecular biology
part of
is abstract of
is hasSource of
Faceted Search & Find service v1.13.91 as of Mar 24 2020


Alternative Linked Data Documents: Sponger | ODE     Content Formats:       RDF       ODATA       Microdata      About   
This material is Open Knowledge   W3C Semantic Web Technology [RDF Data]
OpenLink Virtuoso version 07.20.3229 as of Jul 10 2020, on Linux (x86_64-pc-linux-gnu), Single-Server Edition (94 GB total memory)
Data on this page belongs to its respective rights holders.
Virtuoso Faceted Browser Copyright © 2009-2025 OpenLink Software