2011 Theses Doctoral

Novel molecular engineering approaches for genotyping and DNA sequencing

Qiu, Chunmei

The completion of the Human Genome Project has increased the need for investigation of genetic sequences and their biological functions, which will significantly contribute to the advances in biomedical sciences, human genetics and personalized medicine. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an attractive option for DNA analysis due to its high accuracy, sensitivity and speed. In the first part of the thesis, we report the design, synthesis and evaluation of a novel set of mass tagged, cleavable biotinylated dideoxynucleotides (ddNTP-N3-biotins) for DNA polymerase extension reaction and its application in DNA sequencing and single nucleotide polymorphism (SNP) genotyping by mass spectrometry. These nucleotide analogs have a biotin moiety attached to the 5 position of the pyrimidines (C and U) or the 7 position of the purines (A and G) via a chemically cleavable azido-based linker, with different length linker arms serving as mass tags that contribute large mass differences among the nucleotides to increase resolution in MS analysis. It has been demonstrated that these modified nucleotides can be efficiently incorporated by DNA polymerase, and the DNA strand bearing biotinylated nucleotides can be captured by streptavidin coated beads and efficiently released using tris(2-carboxyethyl) phosphine in aqueous solution which is compatible with DNA and downstream procedures. Reversible solid phase capture (SPC) mass spectrometry sequencing using ddNTP-N3-biotins was performed, and various DNA templates, including biological samples, were accurately sequenced achieving a read-length of 37 bases. In mass spectrometric SNP genotyping, we have successfully exploited our reversible solid phase capture (SPC)-single base extension (SBE) assay and been able to detect as low as 2.5% heteroplasmy in mitochondrial DNA samples, with interrogation of human mitochondrial genome position 8344 which is associated with an important mitochondrial disease (myoclonic epilepsy with ragged red fibers, MERRF); we have also quantified the heteroplasmy level of a real MERRF patient and determined several mitochondrial MERRF mutations in a multiplex approach. These results demonstrated that our improved mass spectrometry genotyping technologies have great potential in DNA analysis, with particular applications in sequencing short-length targets or detecting SNPs with high accuracy and sensitivity requirements, such as DNA fragments with small indels, or SNPs in pooled samples. To truly implement this mass spectrometry-based genotyping method, we further explored the use of a lab-on-a-chip microfluidic device with the potential for high throughput, miniaturization, and automation. The microdevice primarily consists of a micro-reaction chamber for single base extension and cleavage reactions with an integrated micro heater and temperature sensor for on-chip temperature control, a microchannel loaded with streptavidin magnetic beads for solid phase capture, and a microchannel packed with C18-modified reversed-phase silica particles as a stationary phase for desalting before MALDI-TOF analysis. By performing each functional step, we have demonstrated 100% on-chip single base incorporation, sufficient capture and release of the biotin-ddNTP terminated single base extension products, and high sample recovery from the C18 reverse-phase microchannel with as little as 0.5 pmol DNA molecules. The feasibility of the microdevice has shown its promise to improve mass spectrometric DNA sequencing and SNP genotyping to a new paradigm. DNA sequencing by synthesis (SBS) appears to be a very promising molecular tool for genome analysis with the potential to achieve the $1000 Genome goal. However, the current short read-length is still a challenge. Therefore, the second part of the thesis focuses on strategies to overcome the short read-length of SBS. We developed a novel primer walking strategy to increase the read-length of SBS with cleavable fluorescent nucleotide reversible terminators (CF-NRTs) and nucleotide reversible terminators (NRTs) or hybrid-SBS with cleavable fluorescent nucleotide permanent terminators and NRTs. The idea of the walking strategy is to recover the initial template after one round of sequencing and re-initiate a second round of sequencing at a downstream base to cover more bases overall. The combination of three natural nucleotides and one NRT effectively regulated the primer walking: the primer extension temporarily paused when the NRT was incorporated, and resumed after removing the 3' capping group to restore the 3'-OH group. We have successfully demonstrated the integration of this primer walking strategy into the sequencing by synthesis approach, and were able to obtain a total read-length of 53 bases, nearly doubling the read-length of the previous sequencing. On the other hand, we explored the sequencing bead-on-chip approach to increase the throughput of SBS and hence the total genome coverage per run. The various prerequisite conditions have been optimized, allowing the accurate sequencing of several bases on the bead surface, which demonstrated the feasibility of this approach. Both of these approaches could be integrated into current SBS platforms, allowing increased overall coverage and lowering overall costs. As a step beyond genotyping, the in vivo visualization of biomolecules, like DNA and its encoded RNA and proteins, provides further information about their biological functions and mechanisms. The third part of the thesis focuses on the development of a novel quantum dot (QD)-based binary molecular probe, which takes advantage of fluorescent resonance energy transfer (FRET), for detection of nucleic acids, aiming at their eventual use for detection of mRNAs involved in long term memory studies in the model organism Aplysia californica. We reported the design, synthesis, and characterization of a binary probe (BP) that consists of carboxylic quantum dot (CdSe/ZnS core shell)-DNA (QD-DNA) conjugated donor and a cyanine-5 (Cy5)-DNA acceptor for the detection of a sensorin mRNA-based synthetic DNA molecule. We have demonstrated that in the absence of target DNA, the QD fluorescence is the main signal observed (605 nm); in the presence of the complementary target DNA sequence, a decrease of QD emission and an increase of Cy5 emission at 667 nm was observed. We have demonstrated the distance dependence of FRET, with the finding that the target with 16 base separation between the QD and Cy5 after probe hybridization gave the most efficient FRET. Further studies are in progress to evaluate the effectiveness of this QD-based probe inside a cell extract and in living cells.