Establishment of an improved high-efficiency thermal asymmetric interlaced PCR for identification of genomic integration sites mediated by phiC31 integrase.

Abstract

Streptomyces phage phiC31 integrase is widely used to mediate the integration of exogenous genes into host genomes for gene therapy and genomic modification, as it autonomously performs efficient, unidirectional, site-specific integration into pseudo attP sites of the host genome. Although pseudo attP sites are rarely found within exons, it is necessary to map their precise locations to avoid the risk of insertion mutagenesis. High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) is a technique that has been developed to recover genomic sequences that flank insertion tags. We have found, however, that this technique is poorly efficient, as it amplifies many non-specific targets and frequently does not generate sufficient product for downstream analysis. Therefore, we have modified the hiTAIL-PCR procedure and re-designed the random primers. As a result, both the amount and specificity of the reaction product were enhanced for each integration site. Restriction analysis of known sequences within the integrated vector, which co-amplified with the flanking genomic sequences, validated 90% of these bands for sequencing. In contrast, only 30% of the bands produced by previous hiTAIL-PCR could be validated. Compared with the original hiTAIL-PCR, our improved hiTAIL-PCR procedure identified phiC31 integration sites more accurately and efficiently.