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Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

10.7K Views

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12:53 min

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June 14th, 2017

DOI :

10.3791/55812-v

June 14th, 2017

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Gajendra W. Suryawanshi*¹^,², Song Xu*³, Yiming Xie¹, Tom Chou³, Namshin Kim⁴, Irvin S. Y. Chen¹^,⁵, Sanggu Kim⁶

¹UCLA AIDS Institute, University of California at Los Angeles (UCLA), ²Department of Microbiology, Immunology, & Molecular Genetics, University of California at Los Angeles (UCLA), ³Departments of Biomathematics and Mathematics, University of California at Los Angeles (UCLA), ⁴Personalized Genomic Medicine Research Center, Division of Strategic Research Groups, Korea Research Institute of Bioscience and Biotechnology, ⁵Department of Medicine, University of California at Los Angeles (UCLA), ⁶Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University (OSU)

Summary

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

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Keywords Bidirectional Retroviral Integration Site PCR

Quantitative Data Analysis Workflow

Retroviral Vector Integration Sites

Vector Host DNA Junctions

DNA Purification

Chapters in this video

0:05

Title

1:11

LTR-specific Biotin-primer Extension

2:53

Rsal and CviQI Digestion and Blunt-ending

4:12

Straptavidin Bead Binding

5:31

Linker Ligation

6:39

Pre-amplification of Both Left and Right Junction DNA

8:15

Left- and Right-junction-specific Amplifications

10:23

Results: The IS Bidirectional Assay Improves Detection Rates and Sequence Quantification Accuracy

12:09

Conclusion

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