Applications of the polymerase chain reaction in retroviral-mediated gene transfer and the analysis of gene-marked human TIL cells.

TLDR: Quantification of data obtained from PCR dilution experiment indicates that, under appropriate conditions, amplification is linear with respect to the amount of input DNA, permitting estimations of gene dosage in unknown samples.

Abstract: The polymerase chain reaction (PCR), a widely used new technology, was applied to several aspects of retroviral-mediated gene transfer. Ten oligonucleotide primer pairs were analyzed for their ability to amplify specific regions of a retroviral vector, including the long terminal repeat (LTR), and a NeoR selectable marker gene. By using the appropriate oligonucleotide primers, cells transduced by retroviral vectors could readily be detected and analyzed for deletions in proviral sequences by PCR, without Southern blotting. In combination with a simplified RNA isolation/reverse transcription protocol, an approximate titer of vector producer cell lines could be estimated by PCR in a single day, eliminating the need for time-consuming transductions and biological selection. Quantification of data obtained from PCR dilution experiment indicates that, under appropriate conditions, amplification is linear with respect to the amount of input DNA, permitting estimations of gene dosage in unknown samples....