This Investigator-Initiated Small Grant (R03) application proposes a novel physical approach to achieve desired random fragmentation of genomic DNA for preparation of a DNA fragment library suitable for next-generation sequencing. In contrast to existing apparatuses which mechanically break DNA, the proposed method uses an essentially different principle. Electromagnetic energy is applied on the molecules to fragment the DNA. The amount of energy delivered can be precisely tuned, is uniformly distributed within a large compartment enabling multiple samples to be processed simultaneously, and readily penetrates through the container into the enclosed solution.
The aim i s to prove the suitability of the method for preparation of libraries for multiple next-generation sequencing platforms, and develop procedures to achieve improvement in efficiency and quality of DNA fragment library generation. To achieve this, human genomic DNA will be fragmented by the proposed method, repaired, blunt-ended, ligated with sequencing adaptors, amplified and sequenced on next-gen sequencing platforms. Library preparation processes will be adapted as needed to yield blunt ended, ligatable DNA. The output will be analyzed to determine accuracy (absence of introduced or systematic errors), reproducibility and applicability across different platforms. .
The revolutionary new DNA sequencing technology, massively parallel DNA sequencing or next-generation sequencing, has been greatly accelerating our understanding of genes and genetic variations and alterations related to the progress of human diseases and aging. The technology has promising perspectives on medicine, health care, preventive and clinical medication. Nevertheless, technical developments are urgently needed to improve upstream DNA sample preparation for better efficiency and consistency, and for processing multiple DNA samples in parallel. One common technical hurdle is fragmenting the enormous 3-billion- basepair human genomic DNA to short fragments of DNA, typically from 100 - 500 bases in size, for next- generation DNA sequencing. Current apparatuses mechanically break DNA by violent shearing. A novel approach based on a different principle is proposed that involves the application of electromagnetic energy to achieve the desired random fragmentation of DNA. The method is simple, fast, precise and safe to use in research and future medical molecular diagnosis.
| Yang, Yu; Hang, Jun (2013) Fragmentation of genomic DNA using microwave irradiation. J Biomol Tech 24:98-103 |
