Use of reverse-phase chromatography in the Maxam-Gilbert method of DNA sequencing A step toward automation

Reverse-phase chromatography has been used for carrying out Maxam-Gilbert sequencing reactions. The DNA loss has been minimized. Time-consuming ethanol precipitation and lyophilization of piperidine have been eliminated. The method has been used successfully in sequencing oligonucleotides as well as large DNA fragments. Based upon these modifications, we have developed a semiautomatic device for carrying out the Maxam-Gilber sequencing reactions. It is also possible to fully automate the Maxam-Gilbert method of DNA sequencing, and we present the diagram of a fully automated set up. The development of such a machine should expedite the sequencing of the large number of genes being cloned.     

DNA sequencing: an overview of solid-state and biological nanopore-based methods

The field of sequencing is a topic of significant interest since its emergence and has become increasingly important over time. Impressive achievements have been obtained in this field, especially in relations to DNA and RNA sequencing. Since the first achievements by Sanger and colleagues in the 1950s, many sequencing techniques have been developed, while others have disappeared. DNA sequencing has undergone three generations of major evolution. Each generation has its own specifications that are mentioned briefly. Among these generations, nanopore sequencing has its own exciting characteristics that have been given more attention here. Among pioneer technologies being used by the third-generation techniques, nanopores, either biological or solid-state, have been experimentally or theoretically extensively studied. All sequencing technologies have their own advantages and disadvantages, so nanopores are not free from this general rule. It is also generally pointed out what research has been done to overcome the obstacles. In this review, biological and solid-state nanopores are elaborated on, and applications of them are also discussed briefly.     

Swine Genome Sequencing Consortium (SGSC): a strategic roadmap for sequencing the pig genome

The Swine Genome Sequencing Consortium (SGSC) was formed in September 2003 by academic, government and industry representatives to provide international coordination for sequencing the pig genome. The SGSC's mission is to advance biomedical research for animal production and health by the development of DNAbased tools and products resulting from the sequencing of the swine genome. During the past 2 years, the SGSC has met bi-annually to develop a strategic roadmap for creating the required scientific resources, to integrate existing physical maps, and to create a sequencing strategy that captured international participation and a broad funding base. During the past year, SGSC members have integrated their respective physical mapping data with the goal of creating a minimal tiling path (MTP) that will be used as the sequencing template. During the recent Plant and Animal Genome meeting (January 16, 2005 San Diego, CA), presentations demonstrated that a human-pig comparative map has been completed, BAC fingerprint contigs (FPC) for each of the autosomes and X chromosome have been constructed and that BAC end-sequencing has permitted, through BLAST analysis and RH-mapping, anchoring of the contigs. Thus, significant progress has been made towards the creation of a MTP. In addition, whole-genome (WG) shotgun libraries have been constructed and are currently being sequenced in various laboratories around the globe. Thus, a hybrid sequencing approach in which 3x coverage of BACs comprising the MTP and 3x of the WG-shotgun libraries will be used to develop a draft 6x coverage of the pig genome.     

Noninvasive Prenatal Molecular Karyotyping from Maternal Plasma

Fetal DNA is present in the plasma of pregnant women. Massively parallel sequencing of maternal plasma DNA has been used to detect fetal trisomies 21, 18, 13 and selected sex chromosomal aneuploidies noninvasively. Case reports describing the detection of fetal microdeletions from maternal plasma using massively parallel sequencing have been reported. However, these previous reports were either polymorphism-dependent or used statistical analyses which were confined to one or a small number of selected parts of the genome. In this report, we reported a procedure for performing noninvasive prenatal karyotyping at 3 Mb resolution across the whole genome through the massively parallel sequencing of maternal plasma DNA. This method has been used to analyze the plasma obtained from 6 cases. In three cases, fetal microdeletions have been detected successfully from maternal plasma. In two cases, fetal microduplications have been detected successfully from maternal plasma. In the remaining case, the plasma DNA sequencing result was consistent with the pregnant mother being a carrier of a microduplication. Simulation analyses were performed for determining the number of plasma DNA molecules that would need to be sequenced and aligned for enhancing the diagnostic resolution of noninvasive prenatal karyotyping to 2 Mb and 1 Mb. In conclusion, noninvasive prenatal molecular karyotyping from maternal plasma by massively parallel sequencing is feasible and would enhance the diagnostic spectrum of noninvasive prenatal testing.     

Nucleotide sequence changes between Streptococcus pneumoniae R6 and D39 strains determined by an oligonucleotide hybridization DNA sequencing technology

The Gram-positive pathogen Streptococcus pneumoniae, which can be responsible for serious cases of pneumonia and meningitis, has been intensely studied for almost 100 years. Many of the key experiments have been performed in two strains; the non-pathogenic S. pneumoniae R6 and its pathogenic progenitor, S. pneumoniae D39. Whereas the genomic sequence of the R6 strain has been published, there is relatively little genomic information available on D39. Since R6 was derived from D39, we wished to explore the utility of a new technology, Comparative Genome Sequencing, which uses a set of custom oligonucleotide arrays to compare DNA sequences between similar strains. We report here the nucleotide polymorphisms identified between the R6 strain and D39 based on an R6 sequencing array. During the process, we were also able to confirm all of the high confidence changes reported by the oligonucleotide array chip by sequencing the region in the genome around the changes identified with the genome hybridization chip. We also discuss the potential impact of some of the amino acid changes found between these two widely used strains of pneumococci.     

Stepping stones in DNA sequencing

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid technological advances have enormously expanded sequencing opportunities and applications, but also imposed strains and challenges on steps prior to sequencing and in the downstream process of handling and analysis of these massive amounts of sequence data. Traditionally, sequencing has been limited to small DNA fragments of approximately one thousand bases (derived from the organism's genome) due to issues in maintaining a high sequence quality and accuracy for longer read lengths. Although many technological breakthroughs have been made, currently the commercially available massively parallel sequencing methods have not been able to resolve this issue. However, recent announcements in nanopore sequencing hold the promise of removing this read-length limitation, enabling sequencing of larger intact DNA fragments. The ability to sequence longer intact DNA with high accuracy is a major stepping stone towards greatly simplifying the downstream analysis and increasing the power of sequencing compared to today. This review covers some of the technical advances in sequencing that have opened up new frontiers in genomics.     

Commercial high-throughput sequencing and its applications in DNA analysis

DNA sequences can be used for the analysis of genetic variation and gene function. The high-throughput sequencing techniques that have been developed over the past three years can read as many as one billion bases per run, and are far less expensive than the traditional Sanger sequencing method. Therefore, the high-throughput sequencing has been applied extensively to genomic analyses, such as screening for mutations, construction of genomic methylation maps, and the study of DNA-protein interactions. Although they have only been available for a short period, high-throughput sequencing techniques are profoundly affecting many of the life sciences, and are opening out new potential avenues of research. With the highly-developed commercial high-throughput sequencing platforms, each laboratory has the opportunity to explore this research field. Therefore, in this paper, we have focused on commercially-popular high-throughput sequencing techniques and the ways in which they have been applied over the past three years.     

Nanopore sequencing technology: research trends and applications

Nanopore sequencing is one of the most promising technologies being developed as a cheap and fast alternative to the conventional Sanger sequencing method. Protein or synthetic nanopores have been used to detect DNA or RNA molecules. Although none of the technologies to date has shown single-base resolution for de novo DNA sequencing, there have been several reports of alpha-hemolysin protein nanopores being used for basic DNA analyses, and various synthetic nanopores have been fabricated. This review will examine current nanopore sequencing technologies, including recent developments of new applications.     

Sequence analysis of genes and genomes

A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in its genome. The development of powerful techniques for DNA sequencing has enabled sequencing of large amounts of gene fragments and even complete genomes. Important new techniques for physical mapping, DNA sequencing and sequence analysis have been developed. To increase the throughput, automated procedures for sample preparation and new software for sequence analysis have been applied. This review describes the development of new sequencing methods and the optimisation of sequencing strategies for whole genome and cDNA analysis, as well as discusses issues regarding sequence analysis and annotation.     

A non-radioactive automated method for DNA sequence determination

A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.     

Next-generation sequencing of experimental mouse strains

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5?years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.     

第三代测序技术的研究进展与临床应用

Sanger测序法,又称第一代测序技术,作为测序金标准推动了人类基因组“工作框架图”的绘制,但通量低、成本高的缺点限制了其进一步大规模应用。第二代测序技术,又称下一代测序技术,因其通量高、成本低等优点使得基因测序在基础研究与临床诊疗中得到广泛应用,但短读长一直是其不可回避的技术短板。第三代测序技术的出现,因其具有长读长优势,为基因序列上复杂重复区域解析与高质量基因组组装提供了新的技术手段。近年来,第三代测序技术进一步发展与完善,同时在肿瘤、免疫、生殖等相关领域逐步体现出临床应用价值。本文将综述第三代测序技术的研究进展与临床应用。     

三种四膜虫全基因组N6-甲基腺嘌呤比较研究

为了揭示N6-甲基腺嘌呤(6mA)在近缘物种间的分布规律和物种进化过程中的变化, 研究在分离2种四膜虫(Tetrahymena malaccensis和Tetrahymena pyriformi)大核的基础上, 利用三代测序技术绘制了其全基因组单碱基分辨率6mA图谱, 结合公共数据库中已有的模式种Tetrahymena thermophila数据, 开展了3种四膜虫比较6mA甲基化组分析, 发现: (1)3种四膜虫6mA甲基化位点分布特征类似, 包括呈约200 bp周期性分布和具有保守AT基序; (2)6mA主要分布于基因的5′端, 在种间直系同源基因上的分布模式具有区域近似性, 但在单碱基水平不完全保守; (3)在种内近期复制的并系同源基因中, 6mA分布在单碱基水平具有较高的保守性; (4)结合3种四膜虫的分化时间, 估算出了四膜虫中6mA动态变化过程, 6mA位点建立的速度大约为每Mb每百万年69.9—226个位点。     

In vitro production of large single-stranded templates for DNA sequencing.

A method has been developed for the preparation of large single-stranded DNA sequencing templates from primary cloning plasmids or cosmids. The method utilizes the separate action of T7 Gene 6 exonuclease and exonuclease III to generate large quantities of single-stranded template for each strand of a large-cloned fragment. Since the procedure is intended for use on primary clones, it avoids the time-consuming subcloning steps associated with most sequencing programs. The procedure also has the advantage of avoiding clone instability problems associated with subcloning in M13.     

Cooperative oligonucleotides in purification of cycle sequencing products

Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid co-purification of nonextended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.     

Exome Sequencing Is an Efficient Tool for Variant Late-Infantile Neuronal Ceroid Lipofuscinosis Molecular Diagnosis

The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease.     

基于第二代测序技术的基因资源挖掘

转录组研究一直是生命科学研究的一个重要方向,在第二代测序技术问世以前,已经产生了一些行之有效的转录组研究方法,但这些方法存在一定的局限性。第二代测序技术的出现不仅使转录组研究很快进入了高速发展期,同时也为遗传资源的挖掘提供了一套全新的技术平台。本文简要介绍了第二代测序技术的化学原理和特性,重点阐述了利用第二代测序技术进行转录组测序,从而在此基础上挖掘遗传资源的研究。     

Mass-spectrometry DNA sequencing

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been explored widely for DNA sequencing. Compared to gel electrophoresis based sequencing systems, mass spectrometry produces very high resolution of sequencing fragments, rapid separation on microsecond time scales, and completely eliminates compressions associated with gel-based systems. While most of the research efforts have focused on using mass spectrometers to analyze the DNA products from Sanger sequencing or enzymatic digestion reactions, the read lengths attainable are currently insufficient for large-scale de novo sequencing. The advantage of mass-spectrometry sequencing is that one can unambiguously identify frameshift mutations and heterozygous mutations making it an ideal choice for resequencing projects. In these applications, DNA sequencing fragments that are the same length but with different base compositions are generated, which are challenging to consistently distinguish in gel-based sequencing systems. In contrast, MALDI-TOF MS produces mass spectra of these DNA sequencing fragments with nearly digital resolution, allowing accurate determination of the mixed bases. For these reasons mass spectrometry based sequencing has mainly been focused on the detection of frameshift mutations and single nucleotide polymorphisms (SNPs). More recently, assays have been developed to indirectly sequence DNA by first converting it into RNA. These assays take advantage of the increased resolution and detection ability of MALDI-TOF MS for RNA.     

Structure, function, regulation and clinical significance of the 52K pro-cathepsin D secreted by breast cancer cells

In estrogen-receptor-positive human breast cancer cell lines (MCF7, ZR75-1), estrogens specifically increase the secretion into the culture medium of a 52,000 Da (52K) glycoprotein and stimulate cell proliferation. The 52K protein has been purified to homogeneity using monoclonal antibodies and identified as the secreted precursor of a cathepsin D bearing mannose-6-phosphate signals. The secreted precursor 52K protein is mitogenic in vitro in estrogen-deprived MCF7 cells, can be taken up by these cells via mannose-6-phosphate receptors, and can degrade extracellular matrix and proteoglycans following its auto-activation. The protease is also produced constitutively by ER-negative cell lines, and is inducible by tamoxifen in some antiestrogen-resistant variants. The corresponding cDNA has been cloned using N-terminal sequencing of the protein and monoclonal antibodies. Its complete sequencing indicates a strong homology with pro-cathepsin D of normal tissues. Using a cDNA probe, the regulation of 52K cathepsin D mRNA by estrogens and antiestrogens has been studied and chromosome localization determined by in situ hybridization. Clinical studies using both immunohistochemistry and immunoenzymatic assay of breast cancer cytosol have shown that the concentration of total cellular cathepsin D (52K + 48K + 34K) is related to the proliferation of mammary ducts and to the prognosis of breast cancer. Its cytosolic concentration in primary tumors of postmenopausal patients is correlated slightly with lymph node invasion and significantly with shorter disease-free intervals in a 6-year retrospective study with the Danish Breast Cancer Groups and Finsen Institute (S. Thorpe et al.).(ABSTRACT TRUNCATED AT 250 WORDS)     

New technologies for large-genome sequencing

Interest in DNA sequencing has increased in recent years and research into new instrument systems to optimize and automate the process has been intensified. A number of commercial devices, from Europe, Japan, and the United States, which instrument aspects of (mainly) the Sanger method, are now available and several new DNA sequencing techniques have been reported. This review briefly describes current methods of DNA sequencing and provides an account of systems presently available or under development for instrumenting the technology. The problems of automation for large-scale DNA sequencing are discussed.