RESEARCH EXPERIECE

My research experience began at the Genetic Institute, Academia Sinica in the People's Republic of China where I studied plant genetics using pollen-cultured albinos as models. Since coming to the United States, I have focused my research to genome analysis. As a Ph.D. student at the University of Houston, I constructed the physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002 and studied the lrtA (light repressed transcript) transcription/translation regulation gene in that organism. As a postdoctoral fellow and research instructor at the Washington University, I have been developing the tools needed for large-scale genetic analysis of the human genome with single nucleotide polymorphism markers.

I. Development of high throughput DNA testing methods.

I have developed three homogeneous DNA diagnostic assays for single nucleotide polymorphisms (SNPs), namely, the template-directed dye-terminator incorporation (TDI) assay with fluorescence resonance energy transfer (FRET) detection, the dye-labeled oligonucleotide ligation (DOL) assay with FRET detection, and the TDI assay with fluorescence polarization (FP) detection.

FRET occurs when two dyes are physically in close proximity and one dye's (the donor) emission overlaps the other dye's (the acceptor) excitation spectrum. When the donor is excited, instead of emitting fluorescence at its usual intensity, some of the energy is transferred to the acceptor, leading to the observation of quenching in the donor fluorescence and increasing of acceptor fluorescence. We took advantage of the fact that FRET is highly sensitive to the distance between the donor and acceptor dyes to design homogeneous assays for genotyping SNP markers. In the TDI assay, a donor dye labeled probe is designed to anneal to the target molecule with its 3'-end immediately upstream from the polymorphic site. Allele-specific acceptor-dye labeled terminators are incorporated when the primer is extended by DNA polymerase. By monitoring FRET for the allele-specific dye-terminators, one can infer the presence or absence of the alleles in the target molecule without purification or separation of the reaction products from the other reagents.

In the DOL assay, a donor dye labeled oligonucleotide is incubated with the two allele-specific, acceptor dyes labeled oligonucleotides in the presence of DNA ligase. Once again, by monitoring FRET for the allele-specific dye-labeled oligonucleotides, one can determine the alleles present in the target molecule. An additional advantage of this method is that by designing PCR primers that anneal at higher temperatures and oligonucleotide probes that anneal at lower temperatures, one can perform both reactions in the same vessel in one experiment.

FP is based on the observation that when a fluorescent molecule excited by plane polarized light will emit its fluorescence at a fixed plane. The direction of the emission plane is correlated with the molecular volume (molecular mass). A small fluorescence molecule has faster motion (tumbling and rotating) in solution as comparing to a larger molecule. When excited by plane polarized light more depolarized emission will be observed for the smaller molecule. In the FP-TDI assay, an allele-specific dye-terminator that is incorporated onto a 30-mer oligonucleotide probe will increase its molecular mass by 10 times. By monitoring the change of FP in the TDI assay, one can infer the presence of reaction products, therefore the alleles in the targeted template.

All 3 methods are being licensed by major biotech companies and are subjects of 2 patent applications. Moreover, I am the co-investigator of 2 NIH-funded projects to develop and perfect these and other homogeneous genotyping assays based on FRET and FP detection.

II. Single nucleotide polymorphism (SNP) discovery.

I have been part of a team that works on devising efficient strategies for SNP discovery in the human genome. Specifically, I have done preliminary studies of identifying candidate SNPs in expressed sequence tag (EST) clusters and the construction of genomic libraries enriched for SNPs. My work on both of these approaches has resulted in projects funded by the NIH and the Merck Genome Research Institute.

In the first approach, we examined the EST sequence data from the Washington University Genome Sequencing Center (GSC) to identify clusters of EST sequences with >90% sequence identity. The primary sequence data of the clones were aligned and the sequence differences were identified. All sequencing mistakes and variations found in repeat sequences were eliminated. Sequence tagged sites (STSs) were designed to amplify the candidate SNPs for confirmation by pooled DNA sequencing. With this approach, our success rate was 30-40%. In a Merck Genome Research Institute funded project, other members of our group are working with Warren Gish at the GSC to extend our approach to EST sequences anchored by highly accurate, long-range genomic sequences and genomic shotgun sequences anchored by long-range genomic sequences.

In the second approach, I have examined the use of MutS, an E. coli mismatch repair protein that binds to heteroduplex DNA, to construct SNP-enriched libraries. I worked out conditions to capture mismatched heteroduplex DNA with MutS-coated magnetic beads for cloning into a MutS^- E. coli strain. The resultant mixed colonies yielded DNA sequences that preserved the polymorphic site which were easy to identify by DNA sequencing. Other members of our group will extend this approach in a NIH funded project to identify SNPs from any contig of large insert clones.

III. Physical genome mapping of a photosynthetic cyanobacterium.

As a graduate student at the University of Houston, I constructed the physical genome map of the photosynthetic cyanobacterium Synechococcus sp. PCC 7002. In this project, I employed numerous physical mapping techniques including the construction and manipulation of lambda phage and cosmid libraries, intact chromosomal DNA preparation and pulsed field gel electrophoresis. With the genome map, I mapped photo system I (PS I) and PS II genes.

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Last revised: January 13, 2004.