Integrated microfluidic devices for DNA sequencing and single molecule/cell genetic analysis.

The genetic analysis of a single DNA molecule is at the heart of a number of new sequencing technologies.

The analysis of the genetic content of individual cells is also critical for infectious disease detection and for understanding genetic variations that lead to diseases such as metastatic cancer.

To address these challenges, a high throughput single copy genetic amplification (SCGA) method has been developed in this dissertation that efficiently and quantitatively amplifies DNA targets for long-range sequencing as well as genetic analysis.

SCGA utilizes a microfabricated droplet generator (muDG) to rapidly encapsulate individual DNA molecules or cells together with reverse primer functionalized microbeads and dye-labelled forward primer in monodisperse nanoliter volume PCR mix-in-oil droplets.

Following bulk PCR amplification of this emulsion, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry.

Optimized conditions show that thousands of DNA targets ranging from 380 to 1139 bp are efficiently and quantitatively amplified at single molecule concentrations, producing up to 100 attomoles of bead-bound DNA product.

These bead-based PCR colonies will enable M icrobead INtegrated DNA Sequencing (MINDS) and single cell genetic analysis.

A MINDS sequencing bioprocessor has been developed in this dissertation that efficiently integrates the steps of Sanger template extension, purification-concentration, injection and separation.

Sanger sequencing from 1 femtomole of PCR product using this bioprocessor produced read lengths of over 550 bases.

An improved inline injection version of this microdevice produced similar results from only 100 attomoles of input template.

Long-range sequencing results from beads, each carrying ∼100 attomoles of a 624 bp product, demonstrate that the amplicons generated by SCGA are competent for achieving attomole-scale Sanger sequencing from a single bead using this integrated bioprocessor.

Genetic analyses of the GAPDH gene in individual human lymphocyte cells and of the gyrB gene in bacterial E. coli K12 cells establish that SCGA is capable of performing high throughput single cell genetic analysis as well.

The methods developed in this dissertation enable the acquisition of digital genetic information from individual biological entities that will reveal the importance of stochastic variations in biological function.