Here and there: the double-side transgene localization

Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identification of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact filtration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s flanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two flanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the field of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to efficiently localize genomic transposon integrations, which was confirmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

Real Time PCR; A new, fast and accurate technique in diagnosing the severe type of hemophilia A patients

Introduction: Hemophilia A (HA) is one of the most deleterious X-linked bleeding disorders in male patients. Early and rapid detection could provide management strategies for this disease. This study aims to improve the classic method for detection of important intron 22 inversion mutations (INV22). Materials and Methods: Whole blood samples were taken from 21 male children with a history of severe HA, referred to the Iranian comprehensive hemophilia care center. Two groups were involved for detection of INV22 and two methods were examined in a double-blind manner. The first method used a classical method, inverse PCR, and the second method was Real-time inverse PCR. Results: Results showed that both methods could separately detect the INV22 in 11 out of 21 patients with severe HA (52%) in the same accuracy, but with this difference that only one day was needed for detecting of INV22 by Real-time inverse PCR and on other hand 3 days were needed for the classical methods. Concusion: Because of the medical importance of early detection or prenatal/postnatal diagnosis of this disease, this study recommends the Real-time inverse PCR technique for the rapid detection of this mutation in well-equipped genetic laboratories everywhere.

Targeted MinION sequencing of transgenes

The presence of genetically modified organisms (GMO) is commonly assessed using real-time PCR methods targeting the most common transgenic elements found in GMOs. Once the presence of GM material has been established using these screening methods, GMOs are further identified using a battery of real-time PCR methods, each being specific of one GM event and usually targeting the junction of the plant genome and of the transgenic DNA insert. If, using these specific methods, no GMO could be identified, the presence of an unauthorized GMO is suspected. In this context, the aim of this work was to develop a fast and simple method to obtain the sequence of the transgene and of its junction with plant DNA, with the presence of a screening sequence as only prior knowledge. An unauthorized GM petunia, recently found on the French market, was used as template during the development of this new molecular tool. The innovative proposed protocol is based on the circularization of fragmented DNA followed by the amplification of the transgene and of its flanking regions using long-range inverse PCR. Sequencing was performed using the Oxford Nanopore MinION technology and a bioinformatic pipeline was developed.

Application of Chromosome Conformation Capture Method for Detection MYC/TRD Chromosomal Translocation in Leukemia Cell Line

Background: Chromosomal breakpoints are the most common cause of hereditary diseases and cancers. Today, many standard clinical methods such as cytogenetic and PCR based techniques are used which have limitation regarding detection resolution. Chromosome conformation capture is a method for detecting gene proximity and chromosomal rearrangements. Materials and Methods: In this study, SKW3 cell line was used for detecting t(8;14)(q24;q11) using a 3Cbased technique. SKW3 cell line was used for 3C library preparation. For Inverse PCR, two regions were selected in upstream and downstream of the viewpoint locus on chromosome 8-MYC gene based on EcoRI restriction sites. The captured sequence with intra-chromosomal interaction between chr8-c-MYC and chr14-TRD was selected for the translocation PCR primer design. Results: The DNA fragment captured in 3C PCR showed a specific TRD sequence translocated downstream of the MYC gene. Translocation PCR demonstrated the existence of (8; 14) (q24; q11) MYC /TRD in both library and genomic DNA. Conclusion: This result demonstrated 3C- based method could be used as a useful low-cost easy operating technique in chromosomal rearrangements detection. In this study, the integration of whole genome library monitoring and PCR method was used as a high- through put method in chromosomal breakpoints detection.

Comparison of two molecular diagnostic methods for identifying Beijing genotype of Mycobacterium tuberculosis

Background and Objectives: The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is import- ant for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. Materials and Methods: This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. Results: In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. Conclusion: Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family.