Paper-Based MicroRNA Expression Profiling from Plasma and Circulating Tumor Cells.

Background: Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging.

Methods: Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancer patient blood.

Microsieve lab-chip device for rapid enumeration and fluorescence in situ hybridization of circulating tumor cells.

Herein we present a lab-chip device for highly efficient and rapid detection of circulating tumor cells (CTCs) from whole blood samples. The device utilizes a microfabricated silicon microsieve with a densely packed pore array (10(5) pores per device) to rapidly separate tumor cells from whole blood, utilizing the size and deformability differences between the CTCs and normal blood cells. The whole process, including tumor cell capture, antibody staining, removal of unwanted contaminants and immunofluorescence imaging, was performed directly on the microsieve within an integrated microfluidic unit, interconnected to a peristaltic pump for fluid regulation and a fluorescence microscope for cell counting.

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis.

Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity.

De novo gene synthesis design using TmPrime software.

This chapter presents TmPrime, a computer program to design oligonucleotide for both ligase chain reaction (LCR)- and polymerase chain reaction (PCR)-based de novo gene synthesis. The program divides a long input DNA sequence based on user-specified melting temperatures and assembly conditions, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences, and potential formation of secondary structures in a PDF file, which will be sent to the user via e-mail.

TopDown real-time gene synthesis.

This chapter introduces a simple, cost-effective TopDown one-step gene synthesis method, which is suitable for the sequence assembly of fairly long DNA. This method can be distinguished from conventional gene synthesis methods by two key features: (1) the melting temperature of the outer primers is designed to be ∼8°C lower than that of the assembly oligonucleotides, and (2) different annealing temperatures are utilized to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. This method eliminates the interference between polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis.

New insights into the de novo gene synthesis using the automatic kinetics switch approach.

Here we present a simple, highly efficient, universal automatic kinetics switch (AKS) gene synthesis method that enables synthesis of DNA up to 1.6kbp from 1nM oligonucleotide with just one polymerase chain reaction (PCR) process. This method eliminates the interference between the PCR assembly and amplification in one-step gene synthesis and simultaneously maximizes the amplification of emerged desired DNA by using a pair of flanked primers.

Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis.

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10 degrees C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process.

Integrated two-step gene synthesis in a microfluidic device.

Herein we present an integrated microfluidic device capable of performing two-step gene synthesis to assemble a pool of oligonucleotides into genes with the desired coding sequence. The device comprised of two polymerase chain reactions (PCRs), temperature-controlled hydrogel valves, electromagnetic micromixer, shuttle micromixer, volume meters, and magnetic beads based solid-phase PCR purification, fabricated using a fast prototyping method without lithography process. The fabricated device is combined with a miniaturized thermal cycler to perform gene synthesis.