D Sblattero1, A Bradbury. 1. International School for Advanced Studies, Trieste, Italy. bradbury@icgeb.trieste.it
Abstract
BACKGROUND: The creation of large diverse phage antibody libraries from natural sources relies on primers which are able to amplify as many V genes as possible. All functional germline V genes have recently been catalogued in a database, V BASE [1]. Previously published primer sets are unable to recognise all these V genes. OBJECTIVES: The design of a human primer set able to recognise all functional human V genes which can be used to create diverse phage antibody libraries. STUDY DESIGN: A new set of primers able to recognise all functional V genes were designed using the following criteria: at least 16 bp homology of the 3' end of the primer to the V gene, no more than 8-fold total degeneracy and minimum primer dimer formation. These primers were tested in all possible combinations in PCR using cDNA from human peripheral blood lymphocytes or from human cord blood lymphocytes. RESULTS: By computer analysis, all V genes in V BASE could be amplified using this primer set. This theoretical result was tested practically by PCR and all primer pairs were shown to be functional, producing PCR products of the expected size. The intensity of the PCR products reflected information available on the expression of the different V gene families recognised and their expression in these two different V gene sources. CONCLUSIONS: This new primer set will facilitate the creation of more diverse phage antibody libraries than has been hitherto possible using presently available primer sets.
BACKGROUND: The creation of large diverse phage antibody libraries from natural sources relies on primers which are able to amplify as many V genes as possible. All functional germline V genes have recently been catalogued in a database, V BASE [1]. Previously published primer sets are unable to recognise all these V genes. OBJECTIVES: The design of a human primer set able to recognise all functional human V genes which can be used to create diverse phage antibody libraries. STUDY DESIGN: A new set of primers able to recognise all functional V genes were designed using the following criteria: at least 16 bp homology of the 3' end of the primer to the V gene, no more than 8-fold total degeneracy and minimum primer dimer formation. These primers were tested in all possible combinations in PCR using cDNA from human peripheral blood lymphocytes or from human cord blood lymphocytes. RESULTS: By computer analysis, all V genes in V BASE could be amplified using this primer set. This theoretical result was tested practically by PCR and all primer pairs were shown to be functional, producing PCR products of the expected size. The intensity of the PCR products reflected information available on the expression of the different V gene families recognised and their expression in these two different V gene sources. CONCLUSIONS: This new primer set will facilitate the creation of more diverse phage antibody libraries than has been hitherto possible using presently available primer sets.
Authors: M B Zwick; A F Labrijn; M Wang; C Spenlehauer; E O Saphire; J M Binley; J P Moore; G Stiegler; H Katinger; D R Burton; P W Parren Journal: J Virol Date: 2001-11 Impact factor: 5.103
Authors: Daniel Rodriguez-Pinto; Jason Sparkowski; Martin P Keough; Kathryn N Phoenix; Frank Vumbaca; David K Han; Eckart D Gundelfinger; Philip Beesley; Kevin P Claffey Journal: Cancer Immunol Immunother Date: 2008-06-21 Impact factor: 6.968
Authors: J Glanville; S D'Angelo; T A Khan; S T Reddy; L Naranjo; F Ferrara; A R M Bradbury Journal: Curr Opin Struct Biol Date: 2015-08 Impact factor: 6.809