High Throughput Nucleic Acid Purification and Analysis

Nucleic acid extraction and purification represents a significant bottleneck in DNA analysis. Magnetic bead-based extraction methods can rapidly extract DNA from sample solutions; however, these methods suffer from particle aggregation and sedimentation resulting in diminished extraction efficiencies. Circulating tumor DNA (ctDNA) is a promising biomarker for the diagnosis of cancer and monitoring of treatment. However, several challenges prevent ctDNA from being widely used for routine clinical analyses. These include: 1) isolation of ctDNA from plasma or blood and 2) detection and quantification of low levels of ctDNA in the presence of excess circulating DNA. This talk will focus on the design and synthesis of ionic liquids (ILs), magnetic ionic liquids (MILs), and polymeric ionic liquids (PILs) and the use of these materials in a number of applications related to the analysis of nucleic acids. A major challenge faced by DNA and RNA analysis techniques is the selective extraction of particular nucleic acid sequences using rapid and sensitive methodologies. This presentation will discuss how ion-tagged oligonucleotides (ITOs) can be used in conjunction with MILs to efficiently capture DNA sequences from complex samples. This novel liquid-phase approach towards sequence-selective DNA capture provides superior extraction efficiencies to conventional magnetic bead technology as well as a platform for using external fields to manipulate the liquid droplets. The development of isothermal amplification approaches capable of achieving single-nucleotide resolution of nucleic acid sequences will be demonstrated through the use of molecular beacons coupled with 3D printed droplet generators and the use of smartphones to enable field portability. Advances in capturing low copy numbers of nucleic acids coupled to isothermal amplification methods will be demonstrated in the development of diagnostic assays for mycobacteria.