Multiple displacement amplification enables large-scale clonal analysis following retroviral gene therapy |
| |
Authors: | Bleier S Maier P Allgayer H Wenz F Zeller W J Fruehauf S Laufs S |
| |
Affiliation: | Molecular Oncology of Solid Tumors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. |
| |
Abstract: | Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|