Use of polymerase chain reaction (PCR) in the development of a recombinant organism.
Tryptic mapping of a purified recombinant antibody expressed in a mammalian cell line demonstrated low level heterogeneity in the heavy chain. Amino acid analysis and N-terminal sequencing of these fragments revealed that a conversion of tyrosine (Y) to glutamine (Q) had occurred at residue 376. DNA sequencing of 30 clones of the original expression construct indicated that the chance of the Y376Q variant being present in the original plasmid was low, < 5%. Once the gene had been cloned into a mammalian cell system, sequencing of 30 clones is considered ineffective for screening large numbers of subclones. Consequently, an alternative method was developed to directly probe total mammalian DNA for the presence of cloned variant. The method described here uses a polymerase chain reaction (PCR) based assay optimizing the ability of Taq polymerase to distinguish whether the 3' end of primers have completely annealed with template DNA during PCR cycling. Ten percent of the subclones producing high levels of the variant were identified and experiments indicated that the Y to Q conversion had developed during transfection of the antibody gene into the mammalian cell system. PCR was shown to be useful as a quick screen for verification of a cloned variant. Tryptic mapping is still considered the most sensitive and appropriate analytical tool for assessing genetic heterogeneity of recombinant cell lines.
Helder
J C
JC
Genentech, Department of Quality Control, South San Francisco, CA.
Utter
S L
SL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology / Q000592:standards; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004274:DNA, Recombinant; D011994:Recombinant Proteins