Bill W. Colston

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Birefringence characterization of biological tissue by use of optical coherence tomography.

An improved polarization-sensitive optical coherence tomography (OCT) system is developed and used to measure birefringence in porcine myocardium tissue and produce two-dimensional birefringence mapping of the tissue. Signal-to-noise issues that cause systematic measurement errors are analyzed to determine the regime in which such measurements are accurate. The advantage of polarization-sensitive OCT systems over standard OCT systems in avoiding image artifacts caused by birefringence is also demonstrated.

On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets.

The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused-silica device to generate a stream of monodisperse picoliter-scale droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment and will be useful in viral discovery and gene-profiling applications.

Evaluation of optical coherence quantitation of analytes in turbid media by use of two wavelengths

We introduce a novel method for determining analyte concentration as a function of depth in a highly scattering media by use of a dual-wavelength optical coherence tomography system. We account for the effect of scattering on the measured attenuation by using a second wavelength that is not absorbed by the sample. We assess the applicability of this technique by measuring the concentration of water in an Intralipid phantom, using a probe wavelength of 1.53 microm and a reference wavelength of 1.31 microm. The results of our study show a strong correlation between the measured absorption and the water content of the sample. The accuracy of the technique, however, was limited by the dominance of scattering over absorption in the turbid media. Thus, although the effects of scattering were minimized, significant errors remained in the calculated absorption values. More-accurate results could be obtained with the use of more powerful superluminescent diodes and a choice of wavelengths at which absorption effects are more significant relative to scattering.

Imaging of the Oral Cavity Using Optical Coherence Tomography

Optical coherence tomography is a new method for noninvasively imaging internal tooth and soft tissue microstructure. The intensity of backscattered light is measured as a function of depth in the tissue. Low coherence interferometry is used to selectively remove the component of backscattered signal that has undergone multiple scattering events, resulting in very high resolution images (< 15 microns). Lateral scanning of the probe beam across the biological tissue is then used to generate a 2-D intensity plot, similar to ultrasound images. This imaging method provides information that is currently unobtainable by any other means, making possible such diverse applications as diagnosis of periodontal disease, caries detection, and evaluation of restoration integrity. This chapter presents an overview of this exciting new imaging technique and its current application to dental diagnosis.

Noninvasive diagnosis of early caries with polarization-sensitive optical coherence tomography (PS-OCT)

There is no diagnostic technology presently available utilizing non-ionizing radiation that can image the state of demineralization of dental enamel in vivo for the detection, characterization and monitoring of early, incipient caries lesions. In this study, a Polarization Sensitive Optical Coherence Tomography (PS-OCT) system was evaluated for its potential for the non-invasive diagnosis of early carious lesions. We demonstrated clear discrimination in PS-OCT imags between regions of normal and demineralized enamel in bovine enamel blocks containing well-characterized artificial lesions. Moreover, high-resolution, cross- sectional images were acquired that clearly discriminate between the normal and carious regions of extracted human teeth. Regions that appeared to be demineralized in the PS- OCT imags were verified using histological thin sections examined under polarized light. The PS-OCT system discriminates between normal and carious regions by measuring the state of polarization of the back-scattered 1310 nm light, which is affected by the state of demineralization of the enamel. The demineralized regions of enamel have a large scattering coefficient, thus depolarizing the incident light. This initial study shows that PS-OCT has great potential for the detection, characterization, and monitoring of incipient caries lesions.

Multiplex primer prediction software for divergent targets

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (∼3700 18-mers or ∼2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus.

Optical coherence tomography for diagnosing periodontal disease

We have, in this preliminary study, investigated the use of optical coherence tomography for diagnosis of periodontal disease. We took in vitro OCT images of the dental and periodontal tissues from a young pig and compared them to histological sections. These images distinguish tooth and soft tissue relationships that are important in diagnosing and assessing periodontal disease. We have imaged the attachment of gingiva to the tooth surface and located the cemento-enamel junction. This junction is an important reference point for defining attachment level in the diagnosis of periodontal disease. the boundary between enamel and dentin is also visible for most of the length of the anatomical crown, allowing quantitation of enamel thickness and character.

Intraoral fiber-optic-based diagnostic for periodontal disease

The purpose of this initial study was to begin development of a new, objective diagnostic instrument that will allow simultaneous quantitation of multiple proteases within a single periodontal pocket using a chemical fiber optic senor. This approach could potentially be adapted to use specific antibodies and chemiluminescence to detect and quantitate virtually any compound and compare concentrations of different compounds within the same periodontal pocket. The device could also be used to assay secretions in salivary ducts or from a variety of wounds. The applicability is, therefore, not solely limited to dentistry and the device would be important both for clinical diagnostics and as a research too.

OCT for diagnosis of periodontal disease

We have developed a hand-held in vivo scanning device for use in the oral cavity. We produced, using this scanning device, in vivo OCT images of dental tissues in human volunteers. All the OCT images were analyzed for the presence of clinically relevant anatomical structures. The gingival margin, periodontal sulcus, and dento-enamel junction were visible in all the images. The cemento-enamel junction was discernible in 64% of the images and the alveolar bone presumptively identified for 71% of the images. These images represent, to our knowledge, the first in vivo OCT images of human dental tissue.