UC San Diego

Recent advances in DNA sequencing technologies have allowed researchers to decrease the cost and time requirements for genomic sequencing by orders of magnitude. Investments in novel sequencing methods and improvements to existing next-generation sequencing platforms have resulted in much higher accuracy and lower cost-per-base for genomic sequencing using a variety of chemistries. However, one of the bottlenecks for all next- generation sequencing methods is the amount of time and resources required for template and library preparation. This process typically results in considerable sample loss and low throughput. Two of the steps that are in need of improvement are the fragmentation of long DNA strands and the amplification of fragmented genomes. To enable the creation of high quality genomic libraries, I have developed an automated device, based on a syringe pump, for the random fragmentation of genomic DNA. The length of the resulting fragments is tunable using a single parameter and the ends are easily repaired for the efficient ligation of adapters. I have also developed a method for the unbiased linear amplification of long DNA fragments using the concerted activities of a nicking endonuclease and a polymerase. The optimization of reaction conditions resulted in markedly better performance than existing similar protocols and I have demonstrated the utility of this method in amplifying a fragmented phage genome. I explored methods of using a highly specific homing endonuclease for use with this technique, including the incorporation of non-native nucleotides and the engineering of the enzyme's catalytic site. Lastly, I have made significant progress in efforts towards the engineering of the nuclease's DNA recognition residues in order to create a highly specific nicking enzyme for use in the modification or amplification of large genomes