Jingyue Ju, Ph.D.

Key Words

  • Erectile Dysfunction

Jingyue Ju, Ph.D.

The ability to sequence DNA accurately, rapidly, and economically is revolutionizing biology and medicine. The Human Genome Project has stimulated an exponential growth in the development of high throughput genetic analysis technologies. Methods for resequencing protein coding and gene regulatory regions of the genome are increasingly necessary to plumb the underlying genetic architecture of common heritable disorders. Thus, resequencing and high throughput mutation scanning technologies have become critical to the identification of mutations underlying disease (molecular epidemiology) and the genetic variability underlying differential drug response (pharmacogenomics). Our laboratory is engaged in the development of novel bioanalytical reagents, systems, and processes to facilitate DNA sequencing and resequencing. Advances in chemistry, engineering, biology, and computer science have permitted us to move from studying individual genes to analyzing and comparing entire genomes.

High Fidelity DNA Sequencing Using Solid Phase Capturable Dideoxynucleotides and Mass Spectrometry. The current “state-of-the-art” technology for high throughput DNA sequencing relies on capillary array DNA sequencers using laser-induced fluorescence detection. While this technology addresses the throughput and read length requirements of large scale DNA sequencing projects it does not provide the high accuracy requirements of resequencing projects. Accurate resequencing, in turn, is critical for studies aimed at mutation detection, forensic identification, and serial analysis of gene expression (SAGE). Electrophoresis-based sequencing methods have inherent limitations for detecting heterozygotes and are compromised by GC compressions; mass spectrometry-based methods, on the other hand, overcome these difficulties, but fall short of the throughput and read length requirements. A fundamental precondition for using mass spectrometry for de novo sequencing, and not merely for single base change (SNP) substitution, is that DNA sequencing fragments must be stringently pure and free of salts, false terminations, and other contaminants before being introduced into the detector. We are developing an approach that relies on the high affinity between biotin and streptavidin to remove undesired components in the sequencing reaction: DNA sequencing fragments are generated using biotinylated dideoxynucleotides and then captured at the 3′ end by a streptavidin-coated solid phase. The captured DNA fragments are then chemically or photo-cleaved from the solid phase and loaded on a matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometer. By mass-tagging the dideoxynucleotides with aromatic functionalities, mass separation of the DNA sequencing fragments is dramatically enhanced to increase resolution and read length. We are also developing a porous-channel based, affinity-capture and light directed-release system for continuous online generation of pure DNA sequencing fragments using solid phase capturable dideoxynucleotides for analysis by MALDI-TOF mass spectrometer.

Mass spectrometry DNA sequencing data from solid phase capturable dideoxynucleotides. The nucleotide is identified above each peak. The first peak is the primer extended by one nucleotide, adenine. The mass difference between a peak and the previous peak is indicated (Edwards et al., Nucleic Acids Research, in press).

Combinatorial Fluorescent Energy Transfer Tags for Multiplex Genomics Analyses. Building upon the original discoveries that led to the development of energy transfer dyes as the industry and academic standards for DNA sequencing (Ju et al. Proc. Natl. Acad. Sci. USA. 1995 92(10): 4347-51), we have developed a novel approach for constructing a large number of combinatorial fluorescence energy transfer (CFET) tags by exploiting energy transfer and combinatorial synthesis to tune the fluorescence emission signatures for multiplex genomics analysis (Tong et al. Nature Biotechnology, 2001, 19: 756-9). All of the CFET tags can be excited at a single wavelength and analyzed by a simple optical system. In collaboration with Dr. Russo (CGC), we are currently applying these CFET tags in multiplex DNA sequencing, single nucleotide polymorphism (SNP) detection, and genome-wide chromosome deletion and insertion analysis.

Fluorescence Imaging Chip System for Massive Parallel DNA Sequencing. The use of electrophoresis for DNA sequencing has been a major bottleneck for high-throughput DNA sequencing projects. The need for electrophoresis is eliminated when sequencing DNA by synthesis, that is, when detecting the identity of each nucleotide as it is incorporated into the growing strand of DNA in a polymerase reaction. Such a scheme, if coupled to the chip format, has the potential to markedly increase the throughput of sequencing projects. Our laboratory is developing a chip-based ‘sequencing by synthesis’ platform. This DNA sequencing system includes the construction of a chip with immobilized single stranded DNA templates that can self prime for the generation of the complementary DNA strand in polymerase reaction, and 4 unique fluorescently labeled nucleotide analogues with 3′-OH capped by a small chemical moiety to allow efficient incorporation into the growing strand of DNA as terminators in the polymerase reaction. A 4-color fluorescence imager is then used to identify the sequence of the incorporated nucleotide on each spot of the chip. Upon removing the dye photochemically and the 3′-OH capping group, the polymerase reaction will proceed to incorporate the next nucleotide analogue and detect the next base. It is a routine procedure now to immobilize high density (>10,000 spots per chip) single stranded DNA on a 4cm x 1cm glass chip. Thus, in the chip based DNA sequencing system, more than 10,000 bases will be identified after each cycle and after 100 cycles million of base pairs will be generated from one sequencing chip. Massively parallel DNA sequencing promises to bring genetic analysis to the next level where we can envision, for example, the comparison on individual genome profiles.

Quantification of Rare Messenger RNA. We have designed and implemented an approach to quantify rare mRNA species recruited during short-term and long-term memory storage in transgenic and control mice. In collaboration with Dr. Gilliam (CGC), Nobel laureate Eric Kandel and Dr. Qingqing Fan (Center for Neurobiology at Columbia), representative libraries of RNA from hippocampal regions of “trained” and “untrained mice are prepared using the SAGE protocol and modified high-throughput sequencing methods to allow detection of reliable gene expression patterns relevant to memory and learning.

Genome Analysis of Model Organism. Dr. Kandel and colleagues have pioneered the molecular neurobiological study of learning and memory (for which Dr. Kandel received the 2000 Nobel Prize in Medicine) largely based upon their studies of the sea slug, Aplysia, an organism with unique advantages for neuroscience research. We have forged a unique collaboration with Dr. Kandel and Incyte Genomics to construct and sequence representative cDNA libraries from the Aplysia central nervous system. Our laboratory is sequencing and analyzing the full-length Aplysia cDNAs, and is measuring the gene expression in Aplysia nervous system for the study of learning and memory using microarray and SAGE in collaboration with Drs. Sergei Kalichikov (CGC) and Qingqing Fan (Center for Neurobiology).

Selected Publications

Tong AK, Li Z, Jones GS, Russo JJ, and Ju J (2001). Combinatorial fluorescence energy transfer tags for multiplex biological assays. Nature Biotechnology 19: 756-759.

Edwards JR, Itagaki Y, and Ju J. (2001). DNA Sequencing Using Biotinylated Dideoxynucleotides and Mass Spectrometry. Nucleic Acids Res. (in press).

Tong AK, Li Z, and Ju J (2001). Combinatorial Fluorescence Energy Transfer Tags: New Molecular Tools for Genomics Applications. Journal of Quantum Electronics Special Issue for Biomedical Applications (in press).

Ju J, Glazer AN, and Mathies RA (1996). Cassette Labeling for Facile Construction of Energy Transfer Fluorescent Primers. Nucleic Acids Res. 24: 1144-1148.

Ju J, Glazer AN, and Mathies RA (1996). Energy Transfer Primers: A New Fluorescence Labeling Paradigm for DNA Sequencing and Analysis. Nature Medicine 2: 246-249.

Kheterpal I, Scherer J, Clark SM, Radhakrishnan A, Ju J, Ginther CL, Sensabaugh GF, and Mathies RA (1996). DNA Sequencing Using a Four-Color Confocal Fluorescence Capillary Array Scanner. Electrophoresis 17: 1852-1859.

Wang Y, Wallin JM, Ju J, Sensabaugh GF, and Mathies RA (1996). High-Resolution Capillary Array Electrophoretic Sizing of Multiplexed Short Tandem Repeat Loci Using Energy-Transfer Fluorescent Primers. Electrophoresis 17: 1485-1490.

Hung SC, Ju J, Glazer AN, and Mathies RA (1996). Cyanine Dyes with High Absorption Cross Section as Donor Chromophores in Energy Transfer Primers. Anal. Biochem. 243: 15-27.

Hung SC, Ju J, Glazer AN, and Mathies RA (1996). Energy Transfer Primers with 5- or 6-Carboxyrhodamine-6G as Acceptor Chromophores. Anal. Biochem. 238: 165-170.

Ju J, Ruan C, Fuller CW, Glazer AN, and Mathies RA (1995). Energy Transfer Fluorescent Dye-Labeled Primers for DNA Sequencing and Analysis. Proc. Natl. Acad. Sci. 92: 4347-4351.

Ju J, Kheterpal I, Scherer J, Ruan C, Fuller C, Glazer AN, and Mathies RA (1995). Design and Synthesis of Energy Transfer Fluorescent Dye-Labeled Oligonucleotide Primers and Their Application for DNA Sequencing and Analysis. Anal. Biochem. 231: 131-140.

Wang Y, Ju J, Carpenter BA, Atherton JM, Mathies RA, and Sensabaugh GF (1995). Rapid Sizing of Short Tandem Repeat Alleles Using Energy Transfer Fluorescent Primers and Capillary Array Electrophoresis. Anal. Chem. 67: 1197-1203.