Blockerette-ligated capture T7-amplified RT-PCR, a new method for determining flanking sequences.
We have developed a highly sensitive PCR-based technique termed blockerette-ligated capture T7-amplified reverse-transcription PCR (BCT-RT-PCR), which can be used to characterize unknown proviral flanking sequences from a broad range of samples and depends only on knowing the retroviral sequence. This method incorporates several essential elements to make it both sensitive and specific, including a "blockerette" linker, magnetic capture of target sequences, and exponential replication of potentially rare sequences using a nested promoter for T7 RNA polymerase, followed by nested RT-PCR. This linkage of methods was designed to increase sensitivity by decreasing DNA complexity in favor of specific amplification. The resulting PCR products can be directly sequenced to determine integration sequences. We have successfully determined the integration sequences from as little as 30 pg of provirus-containing DNA in the background of 30 ng of untransduced DNA, representing a 0.1% transduction rate. We also show this technique to have single-cell resolution even in the background of 5000 cells. We describe here for the first time the combined use of BCT-RT-PCR and laser-capture microscopy (LCM) for precise isolation of retrovirally transduced cells followed by determination of the 3' retroviral flanking sequence at the single-cell level. This method will aid significantly in determining clonality both in transplant experiments and in the generation of clonal cell populations.
Lenvik
Todd
T
Stem Cell Institute and Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Lund
Troy C
TC
Verfaillie
Catherine M
CM
D024509:3' Flanking Region; D024506:5' Flanking Region; D000818:Animals; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005822:Genetic Vectors; D051379:Mice; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013234:Stem Cells
C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase