Aric Joneja received a Bachelor's degree in Biological Resources Engineering from the University of Maryland in 2004. That fall he enrolled in the PhD program in Bioengineering at UCSD, and in 2006 began working in Dr. Xiaohua Huang's laboratory. His current project involves the construction of genomic DNA libraries. Genomic library construction is an essential step in the sequencing of the human genome. This requires the fragmentation and amplification of DNA in a way that facilitates sequencing by various chemistries and subsequent reassembly of the genome by algorithm. Improvements in these areas are needed in order to continue progress towards a $1000 accurate human genome.
Hydrodynamic Shearing of Genomic DNA – In order to build an effective genomic library, it is essential to be able to randomly break the genomic DNA into fragments of similar and manageable size. In this project we avoid the bias of enzymes by using shear stresses to tear the DNA into fragments. Our improvements on previous devices have yielded an instrument with superior performance, and we have been contacted by local San Diego biotech companies as well as the National Cancer Institute for assistance in exporting our device.
Amplification of DNA fragments – The next step towards the creation of a genomic library is the amplification of random DNA fragments. We have investigated methods of circularizing linear fragments and amplifying via RCA (Rolling Circle Amplification).
We are also seeking to amplify random linear DNA fragments without the additional step of circularizing. Our work focuses on optimizing Strand Displacement Amplification (SDA) of long DNA fragments. In order to improve specificity and yield, the suitability of select nucleases, polymerases, and various classes of nucleic acid analogues have been characterized.
Further, we are attempting to engineer existing enzymes for use in SDA. Inactivating one cutting site on restriction endonucleases and, when necessary, changing their specificity to impart directionality should yield an active, highly specific nicking enzyme suitable for use with genomic DNA. We plan to select active variants from a large pool of mutants utilizing a water-in-oil microemulsion, biotin-streptavidin binding, and adaptor-specific PCR. Progress has been made towards establishing a platform for this selection strategy.