Isolation Of Leukemic Genomic Insertion Sites Of Proviruses Using APE-PCR And Analysis Of Targeted Gene(s)

Leukemia is a clonal malignant disease of hematopoietic stem cell, and itspathogenesis is not yet clear. Many factors may affect the onset of the disease. Virusmay be one of the reasons. At present, it is generally believed that proto-oncogene canbe activated by inserted provirus and then cause leukemogenesis. Therefore,examination of the insertion sites of proviruses, detection of leukemia related geneticvariation and identification of the related genes is of great importance. Furthermore,Insertional mutagenesis is a productive strategy for the exploration of genetic regulationof important biological and pathological processes, such as tumorigenesis. Successfulimplementation of this strategy depends heavily on an efficient approach to theidentification of insertion sites present in the host genome.Objective:Here we aim to develop an improved PCR-based method, which is efficient,convenient and fast for cloning of insertion sites of proviruses (PISs) and the insertionsites of other types of DNA elements; and to identify the common insertion sites ofprovirus and their relevant genes in acute myeloid leukemia (AML).Methods:We have introduced a PCR method to clone the genomic insertion sites of DNAelements, called Adenosine-ended Primer Extension Polymerase Chain Reaction(APE-PCR), which was developed from the previous PCR methods in many aspects,such as a combination of enzymes, the efficiency of ligation, the method of purificationand so on; APE-PCR was adopted to map a large amount of PISs present in leukemiacells derived from the AML mouse model which was constructed using syngeneictransplantation followed by the treatment with Ara-C; finally, using the method of highthroughput sequencing analysis to find common insertion sites and related genes. Results:We have demonstrated that APE-PCR is able to amplify more and larger specificproviral insertion site (PIS)-derived fragments, with a lower non-specific backgroundproduced, fewer steps and less DNA samples required, flexibility in choice of restrictionenzymes applied, at a lower cost. Replacement of regular magnetic beads withnano-scale ones in the protocol can further increase its power. Moreover, even withsmall amount of sample DNA, PISs can be recovered and analyzed. In addtion, we haveobtained preliminary screening results indicating that some genes may be related to thedevelopment of leukemia, which also needs further verification.Conclusions:Based on the results provided from this study, we believe that APE-PCR representsan efficient method in mapping of PISs and likely, the insertion sites of other types ofDNA elements as well. In this work, we have obtained some candidate leukemia genesand provided a reliable basis for the study of functional genomics in leukemogenesis.