Microarray

Bringing microarrays to the undergraduate and K-12 classroom is the main focus of the research branch of the Tufts HHMI grant. A microarray, or DNA chip, consists of a solid surface such as glass, silicon or plastic. Each microarray contains multiple sites on the surface where different segments of DNA, called probes, can be attached. Since multiple strands of DNA can be affixed to one microarray, a researcher can detect thousands of possible DNA segments in one procedure, and deduce the overall sequence of an unknown bit of DNA. Microarrays are used in multiple applications from determining whether individuals have genetically based medical conditions to linking fragments of DNA to crime scenes for criminal investigations.

Although the technology is widely used, the cost of making a commercial microarray can be upwards of $50 per DNA sample, which is too expensive for widespread use in undergraduate or K-12 settings. The goal of the microarray project is to develop a low cost microarray from readily available materials such as silica beads and filter paper. This will allow students not only to learn about microarray technology, but also to develop their own microarrays to answer open ended research questions.

Summer 2009 and Fall 2009

In addition to using the LEGO microarrays, undergraduates have developed biosensors that can also be used to detect SNPs for analysis. These biosensors offer another opportunity for students to create and use technology during a science experiment. During the fall of 2009 senior Alex Huh and junior Shrikar Rajogopal are testing various ways of reliably detecting SNPs using both LEGO arrays (Huh) and biosensors (Rajogopal).

Spring 2009

Varandt Khodaverdian returned to Malden High School to demonstrate how to make V-arrays during his winter break. Varandt helped lead the classes through the process of V-array fabrication as well as testing the V-arrays to determine whether they were effective. Additionally, Varandt shared his impressions of his research experience with the students, some of whom were fellow classmates two years earlier. This experience was valuable for Varandt, the students, and for the project, as we were able to make modifications to the protocols to make the lab run more smoothly in the classroom setting. The experiment was written up in the Malden Observer. The article can be found here: Sharing A Passion For Science

During the spring of 2009, Ryan Lena and Lia Tucker, both chemical engineering majors, reworked the V-array into the PDMS/ LEGO array. Arrays are created by first curing PDMS into a mold made of LEGO brick imprints, and then immobilizing DNA probe functionalized beads into the resulting wells. We tried making LEGO arrays with a group of fifty fifth graders on a field trip to Tufts. The students were able to make the LEGO arrays, and enjoyed learning about their applications. We plan to conduct the full experiment of making and then using the LEGO arrays in the final year of the project.

Summer 2008

During the summer of 2008, Varandt Khodaverdian created a highly effective array called the “V-Array” consisting of a treated glass slide with silica beads on the surface. Varandt spent his summer working to develop an economical and easy-to-fabricate substrate upon which to perform DNA hybridization experiments. The solid support for the array was fabricated by affixing a piece of silicone sheeting to one side of a glass microscope slide. The array could be further partitioned into discrete assay regions by punching holes in a second piece of adhesive silicone sheeting and layering it on top of the first. A suspension of functionalized silica microspheres (1.25% solids in water or PBS) was spotted onto the silicone surface in user-defined locations and the solvent was allowed to evaporate in an oven. Following evaporation, the substrate was washed with deionized water and dried in a stream of air. This fabrication procedure produced a monolayer of probe-functionalized silica microspheres that strongly adhered to the silicone surface and were extremely resistant to removal. The microsphere layer remained largely intact even after the substrate was subjected to vigorous wash cycles in several different buffers. Direct hybridization experiments using the enzyme amplification readout method developed by Patrice and Kaitlin were carried out on this new substrate. Slight procedural modifications were adopted to eliminate non-specific binding of the HRP-streptavidin conjugate to the silicone surface. These modifications enabled a clear and unambiguous colorimetric detection of complement binding. The total time required to perform this assay, including substrate assembly and DNA hybridization, was 2.5 hours; well within the allotted time for a typical undergraduate laboratory. Future work will focus on applying this substrate format to DNA detection assays of greater complexity, such as the sandwich assay.

2007-2008

The project has been underway since the fall of 2006, with a steadily growing team of undergraduate researchers working together to tackle various scientific problems. We are attempting to develop an inexpensive surface, reliable assays to make genotyping calls, and an inexpensive instrument for visualizing the results of those assays. Students have encountered challenges for each of the aspects of the project but have made significant progress in each area.

We have tried several substrates in an effort to reduce the cost and time of producing microarrays for undergraduate and high school chemistry and biology labs. Several students have worked on filter paper (cellulose), silica particles, polymer microspheres, and streptavidin-coated iron microspheres. Results have been inconsistent with filter paper and silica. Polymer microspheres have many desirable attributes for developing microarrays but coupling DNA sequences typically requires several days, making it undesirable for classroom use. Streptavidin-coated iron microspheres are easily manipulated with a magnet and can be coupled to DNA quite rapidly. The sensitivity of the DNA-coupled iron microspheres is currently being tested and optimized.

We are evaluating methods for the collection and genotyping of mitochondrial DNA (mtDNA) from cells isolated from saliva. Patterns in point mutations known as single nucleotide polymorphisms (SNPs) have been correlated to maternal human migratory patterns. Students have developed protocols for the use of Oragene collection kits purchased from DNA Genotek and have tested for the presence of mtDNA using polymerase chain reaction (PCR) and gel electrophoresis. Another challenge inherent in this project is the ability to accurately detect single base mutations in a long fragment of DNA. The assay currently under development first involves hybridizing fragments of mtDNA to the DNA-coupled microspheres. A second DNA probe will then hybridize to a region of interest. Enzymes will extend this strand by a single base only if the last base in the sequence is complementary. This base has a chemical modification to then capture an enzyme that can be used to turn a colorless substrate into a visible or fluorescent product.

Concurrent with the progress on the solid support and assays, undergraduate students have developed an instrument that students can build in high school and undergraduate classrooms. Affectionately called “The Ray,” this instrument is used to view the fluorescence emitted from the beads when the complimentary DNA is bound. The Ray is constructed from simple, readily available materials such as lenses, filters and PVC pipe, and costs only $200.

Summer 2007

In the summer of 2007, Tufts undergraduates Allistair Mallillin, Ian Althouse and Peter Riviello continued to work on the project. First Allistair and Peter used cellulose based filter paper as the substrate, but after mixed results, they refocused on using cellulose beads. They also improved parts of the initial protocol, for example, they noted that a fresh stock of cyanogen bromide produced much better results.

Post doc Chris LaFratta worked with Allistair and Peter to develop a small instrument which could be used to detect and measure flourescence in the sample. They named this alternative imaging system “The Ray”. Consisting of simple optics, an LED excitation source, and an inexpensive CCD camera, this system is capable of low level DNA detection on a microarray. Additionally, “The Ray” is simple enough for students to be able to construct on their own.

2006-2007

During the 2006-2007 year, Tufts University senior Stacey Watkins investigated different methods for developing microarrays as part of her senior thesis. Mentored by Professor Walt and Ryan Hayman, Stacey investigated various solid supports capable of attaching oligonucleotides through organic chemistry reactions. After researching and testing several different methods, Stacey developed a protocol using cellulose powder as the solid substrate and and cyanogen bromide as the linker molecule between the cellulose and the modified DNA strands. Stacey also had promising results with cellulose filter paper as a solid substrate.

  • Varandt Khodaverdian devised a method of affixing a thin layer of silica beads to a glass slide, called a Varray

    Varandt Khodaverdian devised a method of affixing a thin layer of silica beads to a glass slide, called a "Varray".

  • Allistair Mallillin demonstrates the Ray, an easy to construct imaging instrument.

    Allistair Mallillin demonstrates the Ray, an easy to construct imaging instrument.