One main goal of the HHMI grant is to provide students with access to realistic research experiences. These experiences will include laboratory research opportunities for undergraduate students in the Walt Laboratory, as well as restructuring undergraduate laboratories to include research oriented projects. There are two different parts of this project – using microarrays to determine maternal ancestry and developing cost effective microarray methods.

Using Microarrays in the Classroom

Nuclear DNA in cells is a combination of both parents’ genetic code; mitochondrial DNA (mtDNA), however, is maternally inherited. As a result, mutation patterns in the mitochondrial DNA have given insights into human migratory routes over time. These mutations often involve individual bases, and are thus called Single Nucleotide Polymorphisms, or SNPs. By genotyping, or determining the identity of these SNPs, maternal ancestry can be traced back to certain geographic locations. This framework provides an excellent context for students to ask meaningful questions about their own origins and to employ microarrays, a cutting edge scientific technique, to find the answer. During the 2006-2007 academic year, Jennifer Torpey worked to identify SNPs which would be useful in determining human ancestry and migration. After a thorough literature search, she compiled these SNPs and designed a genotyping assay based on a commercially available microarray platform manufactured by Illumina. The assay was designed to take advantage of the highly multiplexed Illumina platform for screening sequences; the arrays would be downsized after this proof of concept. This challenging undertaking was complicated by the highly repetitive nature of mtDNA. In the summer of 2007, Ian Althouse and Brett Lieblich continued work on the project. They tested the first generation assay but results were inconclusive, likely due to an incompatibility of Jennifer’s sequences and the assay chemistry. After reviewing the sequences and discussing the issue with the Illumina technical support group, it was concluded that the SNPs initially chosen were too close and thus competed for binding of the assay’s oligonucleotide probes. Ian and Brett reworked the list of possible SNPs, choosing longer segments of mtDNA between SNPs to decrease the competition between the different sites. Tjani Warren joined the project at the end of the summer, and will continue working on the project during the fall of 2007.

Determining Maternal Ancestry

Bringing microarrays to the undergraduate and K-12 classroom is the main focus of the research aspect of the Tufts HHMI grant. A DNA microarray consists of a solid surface made of glass, silicon or plastic, to which short segments of DNA, called probes, are attached. Using state of the art fabrication techniques, these arrays are pattered with high precision and feature density. Since multiple sequences of DNA can be affixed to one microarray, a researcher can simultaneously detect thousands of nucleic acid targets. Microarrays are used in multiple applications from high throughput screening for genetically-based medical conditions to painstaking forensic investigations. Although the technology is widely used, the cost of utilizing a commercial microarray can be upwards of $50 per DNA sample, too expensive for widespread use in undergraduate or K-12 settings. The primary goal of the microarray project is to leverage organic chemistry to develop a low cost solid support from readily available materials such as silica beads and filter paper and couple DNA probes in a cost- and time-effective manner. These adaptations will expose progressively younger students to learn about a rapidly developing field in biotechnology and to develop their own microarrays to probe new and interesting research questions. During the 2006- 2007 academic year, Tufts University Senior Stacey Watkins investigated different methods for making microarrays as part of her honors thesis. She tested several different methods to couple amine-modified DNA probes to a variety of solid substrates. Stacey developed a protocol using cellulose powder as the solid substrate and cyanogen bromide as the linker molecule and also had promising results with cellulose filter paper. In the summer of 2007, Tufts undergraduates Allistair Mullilin and Peter Riviello continued to work on the project. Efforts first focused on using cellulose-based filter paper, but after mixed results they refocused on using cellulose beads as the substrate. They noted that a fresh stock of cyanogen bromide produced much better results. With the help of Dr. Chris Lafratta, they also developed a small instrument which could be used to detect fluorescence in the sample, and named this alternative imaging system “The Ray”.

Developing Cost Effective Microarray Methods

Although microarrays are used extensively in laboratory research, the cost of microarrays prohibits their widespread use in K-12 classrooms. For example, the cost of the microarrays mentioned above is $50 per initial run of arrays, and $20 for each subsequent manufacturing of the array. We are working on a more cost effective array using silica gel to achieve similar results, with the goal of reducing the cost to pennies per sample.

In the summer of 2007, the Walt Laboratory will host undergraduate students from Tufts and other universities and a high school teacher in the Malden school district to continue working on the projects started by the undergraduates. A specific emphasis will be on continuing the work that has been started on the maternal ancestry project and the silica gel array, as well as developing materials which can be used in high school classrooms.