DNA sequencing and genotyping in miniaturized electrophoresis systems

Advances in microchannel electrophoretic separation systems for DNA analyses have had important impacts on biological and biomedical sciences, as exemplified by the successes of the Human Genome Project (HGP). As we enter a new era in genomic science, further technological innovations promise to provide other far-reaching benefits, many of which will require continual increases in sequencing and genotyping efficiency and throughput, as well as major decreases in the cost per analysis. Since the high-resolution size- and/or conformation-based electrophoretic separation of DNA is the most critical step in many genetic analyses, continual advances in the development of materials and methods for microchannel electrophoretic separations will be needed to meet the massive demand for high-quality, low-cost genomic data. In particular, the development (and commercialization) of miniaturized genotyping platforms is needed to support and enable the future breakthroughs of biomedical science. In this review, we briefly discuss the major sequencing and genotyping techniques in which high-throughput and high-resolution electrophoretic separations of DNA play a significant role. We review recent advances in the development of technology for capillary electrophoresis (CE), including capillary array electrophoresis (CAE) systems. Most of these CE/CAE innovations are equally applicable to implementation on microfabricated electrophoresis chips. Major effort is devoted to discussing various key elements needed for the development of integrated and practical microfluidic sequencing and genotyping platforms, including chip substrate selection, microchannel design and fabrication, microchannel surface modification, sample preparation, analyte detection, DNA sieving matrices, and device integration. Finally, we identify some of the remaining challenges, and some of the possible routes to further advances in high-throughput DNA sequencing and genotyping technologies.     

Recent developments in DNA sequencing by capillary and microdevice electrophoresis

The present review covers papers published in the years 1997 and 1998 on DNA sequencing by capillary and microdevice electrophoresis. The article does not include other electrophoretic DNA applications such as analysis of oligonucleotides, genotyping, and mutational analysis. Capillary gel electrophoresis (CGE) is starting to become a viable competitor to slab gel electrophoresis for DNA sequencing. Commercially available multicapillary array sequencers are now entering sequencing facilities which to date have totally relied on traditional slab gel technology. CGE research on DNA sequencing therefore becomes increasingly concerned with the critical task of fine-tuning the operational parameters to create robust sequencing systems. Electrophoretic microdevices are being considered the next technological step in DNA sequencing by electrophoresis.     

DNA荧光标示研究进展

DNA(脱氧核糖核酸)荧光标示技术是分子生物学中的一项重要技术,普遍应用于DNA测序、寡核苷酸杂交、荧光PCR等领域。本文综述了二十世纪八十年代以来DNA荧光标示方法(手动标示、自动标示)、标示试剂及其研究进展,介绍了DNA荧光标示的一些新的应用,并结合我国实际提出了有益的建议。     

An integrated method for mutation detection using on-chip sample preparation, single-stranded conformation polymorphism, and heteroduplex analysis

This work integrates rapid techniques for mutation detection by producing single-stranded DNA and (renatured) double-stranded DNA on-chip, labeling these with fluorescent DNA stains and then performing two complementary methods of mutation detection-single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis (HA). This involves the denaturation of double-stranded polymerase chain reaction (PCR) product into single-stranded DNA, the mutation analysis of the single-stranded DNA by SSCP and the rehybridized double-stranded DNA by HA. These steps were performed entirely on-chip within several minutes of operation. The combination of these two mutation detection methods on-chip provides a highly sensitive method of mutation detection for either genotyping or screening. Many mutation analysis methods rely upon fluorescently labeled samples from a PCR with fluorescently labeled primers. By labeling on-chip we not only attain improved signal strength, but the method is considerably more versatile. Although we used PCR products in this work, this method could be used to analyze DNA from any source. We believe that this combination of several procedures on a single chip represents a significant step in the development of higher levels of integration upon microfluidic devices.     

Numerical model for DNA loading in microdevices: stacking and autogating effects

Many electrophoresis-based DNA sequencing and genotyping microdevices rely on field-driven effects to load and preconcentrate the sample. A quantitative model is developed for a broad class of electrophoresis-based microfabricated sample injectors. Quantitative predictions of DNA preconcentration are compared with experimental data and are shown to qualitatively reproduce the detailed time-evolving sample distribution in the injector. The model provides practical guidance on device and protocol design, in order to optimize this critical aspect of microfluidic devices.     

Multiple-primer DNA sequencing method.

A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.     

Genotyping with microfluidic devices

In the past few years, electrophoresis microchips have been increasingly utilized to interrogate genetic variations in the human and other genomes. Microfluidic devices can be readily applied to speed up existing genotyping protocols, in particular the ones that require electric field-mediated separations in conjunction with restriction fragment analysis, DNA sequencing, hybridization-based techniques, allele-specific amplification, heteroduplex analysis, just to list the most important ones. As a result of recent developments, microfabricated electrophoresis devices offer several advantages over conventional slab-gel electrophoresis, such as small sample volume requirement, low reagent consumption, the option of system integration and easy multiplexing. The analysis speed of microchip electrophoresis is significantly higher than that of any other electric field-mediated separation techniques. State-of-the-art microfluidic bioanalytical devices already claim their place in most molecular biology laboratories. This review summarizes the recent developments in microchip electrophoresis methods of nucleic acids, particularly for rapid genotyping, that will most likely play a significant role in the future of clinical diagnostics.     

Applications of capillary electrophoresis on microchip

CE on microchip is an emerging separation technique that has attracted wide attention and gained considerable popularity. Because of miniaturization of the separation format, CE on chip typically offers shorter analysis time and lower reagent consumption with potential development of portable analytical instrumentation. This review with 143 references is focused on proteins and peptides analysis, DNA separation including fragment sizing, genotyping, mutation detection and sequencing, and also the analysis of low-molecular-weight compounds, namely explosive residues and warfare agents, pharmaceuticals and drugs of abuse, and various small molecules in body fluids.     

焦测序技术及其在遗传分析中的应用

焦测序技术是一种新的实时DNA测序技术。它在DNA聚合酶、三磷酸腺苷硫酸化酶、荧光素酶和三磷酸腺苷双磷酸酶4种酶的协同作用下,使引物延伸聚合脱氧核糖核酸(dNTP)释放焦磷酸盐(PP i)、PP i转换三磷酸腺苷(ATP)、ATP产生荧光信号与dNTP和ATP的降解等化学反应偶联起来,检测结果准确可靠。本文综述了焦测序技术的基本原理、操作步骤和它在单核苷酸多态性(SNP)研究、微生物的分型鉴定和基因甲基化检测等遗传分析中的应用,并对焦测序技术的发展作了展望。     

Copy number variants calling for single cell sequencing data by multi-constrained optimization

Variations in DNA copy number carry important information on genome evolution and regulation of DNA replication in cancer cells. The rapid development of single-cell sequencing technology allows one to explore gene expression heterogeneity among single-cells, thus providing important cancer cell evolution information. Single-cell DNA/RNA sequencing data usually have low genome coverage, which requires an extra step of amplification to accumulate enough samples. However, such amplification will introduce large bias and makes bioinformatics analysis challenging. Accurately modeling the distribution of sequencing data and effectively suppressing the bias influence is the key to success variations analysis.Recent advances demonstrate the technical noises by amplification are more likely to follow negative binomial distribution, a special case of Poisson distribution. Thus, we tackle the problem CNV detection by formulating it into a quadratic optimization problem involving two constraints, in which the underling signals are corrupted by Poisson distributed noises. By imposing the constraints of sparsity and smoothness, the reconstructed read depth signals from single-cell sequencing data are anticipated to fit the CNVs patterns more accurately. An efficient numerical solution based on the classical alternating direction minimization method (ADMM) is tailored to solve the proposed model. We demonstrate the advantages of the proposed method using both synthetic and empirical single-cell sequencing data. Our experimental results demonstrate that the proposed method achieves excellent performance and high promise of success with single-cell sequencing data.     

Multiple-channel microchips for high-throughput DNA analysis by capillary electrophoresis

Capillary electrophoresis on microfabricated multiple-channel chips has great potential for high-throughput analysis. This review focuses on multiple-channel chips used for high-throughput DNA analysis. It covers progress in the design and fabrication of multiple-channel chips and detection schemes used on these chips. Applications are concentrated on DNA fragment sizing, genotyping, and sequencing.     

High-throughput biopolymer desalting by solid-phase extraction prior to mass spectrometric analysis.

In the last 10 years mass spectrometry (MS) has become an important method for analysis of peptides, proteins and DNA. It was recently utilized for accurate high-throughput protein identification, sequencing and DNA genotyping. The presence of non-volatile buffers compromises sensitivity and accuracy of MS biopolymer analysis; it is essential to remove sample contaminants prior to analysis. We have developed a fast and efficient method for desalting of DNA oligonucleotides and peptides using 96-well solid-phase extraction plates packed with 5 mg of Waters Oasis HLB sorbent (Waters, Milford, MA, USA). This reversed-phase sorbent retains the biopolymer analytes, while non-retained inorganic ions are washed out with pure deionized water. DNA oligonucleotides or peptides are eluted using a small amount (20-100 microl) of acetonitrile-water (70:30, v/v) solution. The SPE desalting performance meets the requirements for MS applications such as protein digest analysis and DNA genotyping.     

DNA sequencing

Determination of the sequence of DNA is one of the most important aspects of modern molecular biology. New sequencing methods currently being developed enable DNA sequence to be determined increasingly faster and more efficiently. One of the major advances in sequencing technology is the development of automated DNA sequencers. These utilize fluorescent rather than radioactive labels. A laser beam excites the fluorescent dyes, the emitted fluorescence is collected by detectors, and the information analyzed by computer. Robotic work stations are being developed to perform template preparation and purification, and the sequencing reactions themselves. Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing. Success in this endeavor would mean that the gel electrophoresis step in DNA sequencing could be eliminated. A major innovation has been the application of polymerase chain reaction (PCR) technology to DNA sequence determination, which has led to the development of linear amplification sequencing (cycle sequencing). This very powerful yet technically simple method of sequencing has many advantages over conventional techniques, and may be used in manual or automated methods. Other recent innovations proposed recently to increase speed and efficiency include multiplex sequencing. This consists of pooling a number of samples and processing them as pools. After electrophoresis, the DNA is transferred to a membrane, and sequence images of the individual samples are obtained by sequential hybridizations with specific labeled oligonucleotides. Multiplex DNA sequencing has been used in conjunction with direct blotting electrophoresis to facilitate transfer of the DNA to a membrane. Chemiluminescent detection can also be used in conjunction with multiplex DNA sequencing to visualize the image on the membrane.     

脱氧核糖核酸电化学研究进展

本文首先介绍了DNA与电极的相互作用、DNA的电化学反应、DNA与过渡金属配合物相互作用的电化学研究及技术， 然后重点对过渡金属配合物在DNA的长程电子转移、DNA的电致化学发光标记分析、DNA电化学传感器、DNA损伤与修复等方面应用的研究现状作了归纳和评述。提出了今后研究工作的方向。     

QSNPlite, a software system for quantitative analysis of SNPs based on capillary array SSCP analysis

We present a newly developed software called "QSNPlite" that comprehensively interprets the data of SSCP and sequencing analyses obtained from capillary array electrophoresis systems used in the quantitative characterization of SNPs. QSNPlite assists in the genotyping of individuals with SNPs and in estimating the allele frequencies of SNPs using pooled DNA. We show that this estimation is accurate (mean absolute error, 1.4%) by comparing the results of the pooled analysis using QSNPlite with the true frequencies based on the allele counting after performing individual genotypings. The QSNPlite program runs on Windows XP and can be used to determine the allele frequencies of SNPs among a large number of individuals, such as in association studies of disease-responsible genes using the candidate gene approach.     

High resolution DNA separations using microchip electrophoresis

Planar microfluidic devices have emerged as effective tools for the electrophoretic separation of a variety of different DNA inputs. The advancement of this miniaturized platform was inspired initially by demands placed on electrophoretic performance metrics by the human genome project and has provided a viable alternative to slab gel and even capillary formats due to its ability to offer high resolution separations of nucleic acid materials in a fraction of the time associated with its predecessors, consumption of substantially less sample and reagents while maintaining the ability to perform many separations in parallel for realizing ultra-high throughputs. Another compelling advantage of this separation platform is that it offers the potential for integrating front-end sample preprocessing steps onto the separation device eliminating the need for manual sample handling. This review aims to compile a recent survey of various electrophoretic separations using either glass or polymer-based microchips in the areas of genotyping and DNA sequencing as well as those involving the growing field of DNA-based forensics.     

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.     

Recent advances in DNA sequencing by capillary and microdevice electrophoresis

A number of significant improvements in the electrophoretic performance and design of DNA sequencing devices have culminated in the introduction of truly industrial grade production scale instruments. These instruments have been the workhorses behind the massive increase in genomic sequencing data available in public and private databases. We highlight the recent progress in aspects of capillary electrophoresis (CE) that has enabled these achievements. In addition, we summarize recent developments in the use of microfabricated devices for DNA sequencing that promise to bring the next leap in productivity.     

Polymeric microfluidic system for DNA analysis

The application of micro total analysis system (μTAS) has grown exponentially in the past decade. DNA analysis is one of the primary applications of μTAS technology. This review mainly focuses on the recent development of the polymeric microfluidic devices for DNA analysis. After a brief introduction of material characteristics of polymers, the various microfabrication methods are presented. The most recent developments and trends in the area of DNA analysis are then explored. We focus on the rapidly developing fields of cell sorting, cell lysis, DNA extraction and purification, polymerase chain reaction (PCR), DNA separation and detection. Lastly, commercially available polymer-based microdevices are included.     

Single-Base Extension and ELISA-Based Approach for Single-Nucleotide Polymorphisms Genotyping

Single-nucleotide polymorphisms (SNPs) emerge as a fundamental tool in personalized medicine due to their association with drug responses or disease predisposition. Single-base extension (SBE) is a common method for characterizing known SNPs, but involves complicated procedures or requires costly analytical instruments. Here, we describe a novel SNP genotyping based on SBE and enzyme-linked immunosorbent assay (ELISA). During the SBE, the 5′ end fluorescein isothiocyanate-labeled allele-specific primer will extend with biotinylated dideoxynucleotides which are complementary to the SNP sites. The extension product will then be captured by streptavidin-coated nanoparticle and develop blue color in the ELISA assay. We validated this method by detecting SNPs for TP53 gene codon 273 from 68 individuals and the data were 100% in concordant with DNA sequencing. Thus, SBE and ELISA-based SNPs assay is a simple and accurate method for SNP genotyping.