Methods, Devices and System for DNA Sequencing and Molecular Diagnostics

This invention describes a system and methods to capture and identify genetic differences in a given DNA sample. The system incorporates a unique three-dimensional DNA chip, that can be used to amplify and sequence the given sample. The invention addresses both the clinical and research DNA sequencing needs and has applications in whole genome sequencing, identification of genetic variations such as SNPs, whole genome RNA profiling, and promoter methylation screening to identify the risk of diseases such as cancer.

Researchers at Cornell have developed a genomic sequencing array, which is based on a new three-dimensional DNA chip design presented below. Traditional DNA chips on silicon or glass are expensive, and the largest current chips have 2 million addresses. In contrast, the Cornell researchers have designed a new DNA chip on plastic with the ability to capture and sequence DNA on 576 million (2 μm polymer pillars) to 2.3 billion (1 μm polymer pillars) addresses. These chips can be manufactured at a fraction of the cost of traditional chips. This microfabricated array is available in 86 mm x 43 mm, 86 mm x 128 mm, or 128 mm x 128 mm formats, with the capacity to sequence DNA captured and cluster amplified on 128 million, 384 million, 576 million, or 2.3 billion addresses, respectively. The design approach is unique in that the pillars add a third-dimension to the array to increase load compared to a two-dimensional surface and also defines the cluster size to that of the size of the pillar.

Cornell’s approach, is based on what we have termed “Sequoia Amplification”, which provides clusters of unique sequences amplified on individual 1 μm polymer pillars at about 2.5 μm to 5 μm height or 2 μm polymer pillars at about 5 μm to 10 μm height and an inter-pillar spacing of 1 to 2 μm. The pillars are arranged in a Cartesian geometry to allow better utilization of pixels on a CCD camera. The approach is based on a novel primer design that enables amplification of uniform clusters independent of length, from 200 to 5,000 bases, and can produce 10 to 100-fold cleaner signal-to-noise when implemented in a sequencing-by-synthesis approach. Other technologies evolving as part of our Rx-Gen platform include, 400 base long, paired-end reads to allow completion of multiple genomes on a single microfabricated sequencing array (generating 200 to 400 billion bases) in a single run. Further, in contrast to current sequencing machines that sequence random fragments, our unique design also allows for sequencing / digital quantification of only those genes required for the validation studies or clinical test used for each patient.