Mo-Huang Li

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. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real‐time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.

A self-contained all-in-one cartridge for sample preparation and real-time PCR in rapid influenza diagnosis

Herein we present a fully automated system with pseudo-multiplexing capability for rapid infectious disease diagnosis. The all-in-one system was comprised of a polymer cartridge, a miniaturized thermal cycler, 1-color, 3-chamber fluorescence detectors for real-time reverse transcription polymerase chain reaction (RRT-PCR), and a pneumatic fluidic delivery unit consisting of two pinch-valve manifolds and two pneumatic pumps. The disposable, self-contained cartridge held all the necessary reagents for viral RNA purification and reverse transcription polymerase chain reaction (RT-PCR) detection, which took place all within the completely sealed cartridge. The operator only needed to pipette the patients sample with lysis buffer into the cartridge, and the system would automatically perform the entire sample preparation and diagnosis within 2.5 h. We have successfully employed this system for seasonal influenza A H1N1 typing and sub-typing, obtaining comparable sensitivity as the experiments conducted using manual RNA extraction and commercial thermal cycler. A minimum detectable virus loading of 100 copies per μl has been determined by serial dilution experiments. This all-in-one desktop system would be suitable for decentralized disease diagnosis at immigration check points and outpatient clinics, and would not require highly skilled operators.

Sampling circulating tumor cells for clinical benefits: how frequent?

Circulating tumor cells (CTCs) are cells shed from tumors or metastatic sites and are a potential biomarker for cancer diagnosis, management, and prognostication. The majority of current studies use single or infrequent CTC sampling points. This strategy assumes that changes in CTC number, as well as phenotypic and molecular characteristics, are gradual with time. In reality, little is known today about the actual kinetics of CTC dissemination and phenotypic and molecular changes in the blood of cancer patients. Herein, we show, using clinical case studies and hypothetical simulation models, how sub-optimal CTC sampling may result in misleading observations with clinical consequences, by missing out on significant CTC spikes that occur in between sampling times. Initial studies using highly frequent CTC sampling are necessary to understand the dynamics of CTC dissemination and phenotypic and molecular changes in the blood of cancer patients. Such an improved understanding will enable an optimal, study-specific sampling frequency to be assigned to individual research studies and clinical trials and better inform practical clinical decisions on cancer management strategies for patient benefits.

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°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. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for 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. In addition, we describe an analytical model of PCR gene synthesis based on the thermodynamics and kinetics of DNA hybridization. The kinetics difference between standard PCR amplification and one-step PCR gene synthesis is analyzed using this model and is validated using real-time gene synthesis with eight gene segments (318-1656bp). The effects of oligonucleotide concentration, stringency of annealing temperature, annealing time, extension time, and PCR buffer conditions are examined systematically. Analysis of the experimental results leads to new insights into the gene synthesis process and aids in optimizing gene synthesis conditions. We further extend this method for multiplexing gene assembly with a total DNA length up to 5.74kbp from 1nM oligonucleotide.

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. The program also provides functions on sequence pooling to separate long genes into smaller pieces for multipool assembly and codon optimization for expression based on the highest organism-specific codon frequency. This software has been successfully used in the design and synthesis of various genes with total length >20 kbp. This program is freely available at http://prime.ibn.a-star.edu.sg.

A self-contained polymeric cartridge for automated biological sample preparation

Sample preparation is one of the most crucial processes for nucleic acids based disease diagnosis. Several steps are required for nucleic acids extraction, impurity washes, and DNA/RNA elution. Careful sample preparation is vital to the obtaining of reliable diagnosis, especially with low copies of pathogens and cells. This paper describes a low-cost, disposable lab cartridge for automatic sample preparation, which is capable of handling flexible sample volumes of 10 μl to 1 ml. This plastic cartridge contains all the necessary reagents for pathogen and cell lysis, DNA/RNA extraction, impurity washes, DNA/RNA elution and waste processing in a completely sealed cartridge. The entire sample preparation processes are automatically conducted within the cartridge on a desktop unit using a pneumatic fluid manipulation approach. Reagents transportation is achieved with a combination of push and pull forces (with compressed air and vacuum, respectively), which are connected to the pneumatic inlets at the bottom of the cartridge. These pneumatic forces are regulated by pinch valve manifold and two pneumatic syringe pumps within the desktop unit. The performance of this pneumatic reagent delivery method was examined. We have demonstrated the capability of the on-cartridge RNA extraction and cancer-specific gene amplification from 10 copies of MCF-7 breast cancer cells. The on-cartridge DNA recovery efficiency was 54-63%, which was comparable to or better than the conventional manual approach using silica spin column. The lab cartridge would be suitable for integration with lab-chip real-time polymerase chain reaction devices in providing a portable system for decentralized disease diagnosis.

Solid-phase purification of gene synthesis products using magnetic beads

Herein we report three novel methods which utilize chemical conjugated magnetic beads to purify synthetic gene from its synthesis solution and prepare the synthesized gene in suitable buffer for downstream applications. Silica-coated magnetic beads are applied for non-specific DNA purification to remove short oligonucleotides and monomers. Streptavidin conjugated magnetic beads and (dT)25 Oligo immobilized magnetic beads are introduced for specific DNA extraction. The performances of these methods are investigated and compared using gel electrophoresis. The optimal conditions for enhancing the extraction efficiency are discussed. In addition, the approach to integrate these solid-phase purification methods into microfluidic devices is presented.

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. Additionally, the TopDown gene synthesis has been combined with the LCGreen I DNA fluorescence dye in a real-time gene synthesis approach for investigating the stepwise efficiency and kinetics of PCR-based gene synthesis. The obtained real-time fluorescence signals are compared with gel electrophoresis results to optimize gene synthesis conditions.

A Software for Designing Oligonucleotides for PCR-Based Long DNA Synthesis

Herein we present a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). The program divides the long input DNA sequence based on the input number of oligonucleotides, 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. The software has been successfully used in the design and synthesis of green fluorescent protein fragment (GFPuv) (760 bp), human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp).