The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Abstract

Insertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.

Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites

“…The PCR program for the method with this kind of DNA polymerase decreased the time requirement to 3 h compared with approximately 7 h for other genome walking methods (Liu and Chen 2007 ; Li et al 2015 ). Though significantly less time was spent using this DNA polymerase and PCR program, the obtained PCR products were more than 2 kb, which are much longer than those in many previous reports (Singer and Burk 2003 ; Schmidt et al 2007 ; Zhang et al 2011 ; Ma et al 2014 ; Reddy et al 2008 ; Xu et al 2013 ).…”

Cellular Intrinsic Mechanism Affecting the Outcome of AML Treated with Ara-C in a Syngeneic Mouse Model

“…The abundance of B117P cells present in peripheral blood was also monitored by measuring the B117P-specific proviral insertion tag that has previously been identified by APE-PCR [17] . As shown in Fig.…”

“…Genomic DNA was extracted from B117P and mouse white blood cells using the TIANamp Genomic DNA Kit (TianGen Co., China), according to the manufacturer’s instructions. B117P specific proviral insertion tags were isolated using APE-PCR, as recently reported [17] . One of the tags was chosen as a molecular marker for B117P cells whose abundance can be reflected by measuring this tag using quantitative real-time polymerase chain reaction (See the following description of qPCR for details).…”