Application of linker-ligation-PCR for construction of phage display epitope libraries.
An efficient method for construction of random epitope libraries using filamentous phage is described. Random DNA fragments generated by DNase I digestion were blunt ended by T4 DNA polymerase and ligated with a 12-mer linker, followed by PCR amplification using the same oligonucleotide linker as primers. The results showed that only the ligated product containing linker sequences on both ends was specifically amplified. When the linker ligated-PCR (LL-PCR) product was used for the construction of phage display epitope libraries, the total number of independent clones in the libraries was increased by 100 to 1000 fold in comparison to that obtained for libraries constructed using other methods. In addition, the LL-PCR strategy also increased the probability of isolating recombined DNA fragments which are derived by random in-frame ligation of two or more discontinuous peptide-coding sequences before being inserted into the display vector. Such randomly recombined DNA fragments might be useful in defining conformational epitopes.
Nagesha
H S
HS
CSIRO Australian Animal Health Laboratory, Geelong, Victoria, Australia.
Yu
M
M
Wang
L F
LF
D000358:African Swine Fever Virus / Q000235:genetics / Q000276:immunology*; D000956:Antigens, Viral / Q000235:genetics; D001435:Bacteriophages; D002213:Capsid / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D000939:Epitopes / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*
D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D019151:Peptide Library; C063745:capsid protein p72, African swine fever virus; D015245:Deoxyribonuclease BamHI; D003850:Deoxyribonuclease I