杂交测序——DNA测序新策略

杂交测序──ＤＮＡ测序新策略陈尚武,马涧泉（中山医科大学生化教研室,广州５１００８９）关键词ＤＮＡ,测序,杂交人类基因组计划要求改进ＤＮＡ测序方法,促进了测序技术的发展,一种全新的测序方法──杂交测序（ｓｅｑｕｅｎｃｉｎｇｂｙｈｙｂｒｉｄｉｚａｔｉｏ...     

促髓细胞分化的锌指基因单向PCR扩增直接测序的研究

锌指基因是一种造血调节基因,编码锌指结构蛋白,主要在髓细胞中表达,促进髓细胞分化,在急性早幼粒白血病维甲酸治疗中,促使病情缓解。本文报道了我们从基因分子上研究锌指基因作用中,探索并建立了单向聚合酶链反应（ＰＣＲ）扩增特定单链ＤＮＡ,直接测序的新方法。它能产生质和量均佳的单链ＤＮＡ,无需纯化即可直接用于测序,使复杂的测序研究简便易行,可在２,３天内完成。这种单向ＰＣＲ扩增特定单链ＤＮＡ直接测序的方法,经对锌指基因的ｃＤＮＡ测序,得到验证。此法不仅适用于疾病研究中的ＤＮＡ测序,还可制各单链ＤＮＡ探针,更利于基因结构组成的研究。     

人基因组表达图谱分析和疾病相关基因搜寻──现状与展望(续前)

人基因组表达图谱分析和疾病相关基因搜寻──现状与展望（续前）康毅滨,柴建华（复旦大学遗传学研究所上海２００４３３）二、ｃＤＮＡ随机部分ｆ三、测序及ＥＳＴ的建立以上所讨论的方法均是从基因组ＤＮＡ出发寻找表达序列,与之恰好相反的另一种构建表达图的思路是随机挑选ｃＤＮＡ文库中的克隆进行测序并在染色体上定位,即ｃＤＮＡ策略。人类基因组计划的一个重要目标是获得人基因组３０亿个碱基对的顺序,而以目前的测序技术要在近期内完成这一目标还略显力不从心。     

DNA杂交测序法

ＤＮＡ杂交测序法米志勇,柯,王丽霞（中国科学院发育生物学研究所植物发育分子生物学实验室,北京１０００８０）自从Ｓａｎｇｅｒ双脱氧末端终止法和Ｍａｘａｍ－Ｇｉｌｂｅｒ化学法问世以来,迄今已测定的ＤＮＡ核苷酸序列累计达１×１０８ｂｐ以上。但随着各种基因组计划的实施,这两种传统的测序方法,无论在效率或成本上,都不足以满足将来的测序需要。因而客观上急需发展更加快捷经济的新的ＤＮＡ序列测定技术。     

DNA自动测序技术进展

ＤＮＡ测序是遗传工程的重要技术之一,ＤＮＡ测序技术的自动化对遗传工程的研究具有重要意义。七十年代末期,Ｓａｎｇｅｒ和Ｍａｘａｍ,Ｇｉｌｂｅｒｔ分别提出了切实可行的ＤＮＡ序列测定方法。近二十年来,ＤＮＡ测序技术发展很快。人们从不同方面对该技术进行了改进,并将许多先进的光学探测方法应用于ＤＮＡ测序技术中。目前已出现了许多商品化的ＣＮＡ测序仪。     

基因靶位操作的原理与策略

基因靶位操作（ｇｅｎｅｔａｒｇｅｔｉｎｇ）是８０年代发展起来的一项重要分子生物学技术,是通过外源ＤＮＡ与染色体ＤＮＡ间的同源重组,定点修饰、改造基因组特定位点的技术。１同源重组同源重组是基因靶位操作技术的分子生物学基础。ＤＮＡ同源重组在基因转化和遗传...     

概述DNA测序技术的现状及进展

概述ＤＮＡ测序技术的现状及进展邓炜,柴建华（复旦大学遗传研究所,上海２００４３３）关键词ＤＮＡ测序自１９７７年Ｓａｎｇｅｒ建立“ＤＮＡ双脱氧链未端终止测序法”以来,ＤＮＡ序列测定已成为实验室中常规技术。Ｓａｎｇｅｒ因此而第二次荣膺诺贝尔奖。由于有了成...     

甘薯叶片超氧化物歧化酶基因克隆及测序

以甘薯（Ｉｐｏｍｏｅａｂａｔａｔａｓ（Ｌ．）Ｐｏｉｒ）叶为材料提取植物总ＲＮＡ,经反转录后,利用多聚酶链式反应技术,扩增并克隆超氧化物歧化酶基因的ｃＤＮＡ,并进行测序分析。该序列全长４８２ｂｐ,其读码框编码１５２个氨基酸,与国外文献报道的甘薯块根ＳＯＤ基因的ｃＤＮＡ序列相比,具有９９％的同源性。     

文摘

０００００１ＤＮＡ自动测序技术进展［中］／李玉栋…／／生物工程进展．－１９９９,１９（５）．－６７～７１ＤＮＡ测序是遗传工程的重要技术之一,ＤＮＡ测序技术的自动化对遗传工程的研究具有重要意义。七十年代末期,Ｓａｎｇｅｒ和Ｍａｘａｍ、Ｇｉｌｂｅｒｔ分别...     

DNA序列分析技术及其现状

ＤＮＡ序列分析技术一直是分子生物学领域的一个重要研究课题。“人类基因组计划”的实施有力地推动了高速ＤＮＡ测序技术的发展。本文主要介绍国内外目前流行的经典的测序方法———各种改良的双脱氧链末端终止法,及近年来发展起来的一些全新的ＤＮＡ测序方法     

Nonlinear electrophoresis for purification of soil DNA for metagenomics

Purification of microbial DNA from soil is challenging due to the co-extraction of humic acids and associated phenolic compounds that inhibit subsequent cloning, amplification or sequencing. Removal of these contaminants is critical for the success of metagenomic library construction and high-throughput sequencing of extracted DNA. Using three different composite soil samples, we compared a novel DNA purification technique using nonlinear electrophoresis on the synchronous coefficient of drag alteration (SCODA) instrument with alternate purification methods such as direct current (DC) agarose gel electrophoresis followed by gel filtration or anion exchange chromatography, Wizard DNA Clean-Up System, and the PowerSoil DNA Isolation kit. Both nonlinear and DC electrophoresis were effective at retrieving high-molecular weight DNA with high purity, suitable for construction of large-insert libraries. The PowerSoil DNA Isolation kit and the nonlinear electrophoresis had high recovery of high purity DNA suitable for sequencing purposes. All methods demonstrated high consistency in the bacterial community profiles generated from the DNA extracts. Nonlinear electrophoresis using the SCODA instrument was the ideal methodology for the preparation of soil DNA samples suitable for both high-throughput sequencing and large-insert cloning applications.     

New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis.

We describe improve enzymatic methods for sequencing method for sequencing DNA. They are based on partial digestion of duplex DNA with exonuclease III to produce DNA molecules with 3' ends shortened to varying lengths, followed by repair synthesis to extend and label the 3' ends. After asymmetrical cleavage of the DNA with a restriction enzyme, the labeled products are separated by gel electrophoresis and the sequence read from the autoradiogram. The entire procedures, beginning with unrestricted DNA and followed through gel electrophoresis, takes only one day for sequencing both strands of the DNA molecule. These methods are especially suitable for sequencing DNA cloned in plasmid vectors, and they greatly extend the usefulness of the dideoxynucleotide chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463, 1977). Using these methods we have determined the sequence of a 410 base pair fragment which includes the yeast SUP3 tyrosine tRNA gene.     

Mapping structurally perturbed sites in DNA by replication arrest and run-off replication

We describe a technique for rapid fine mapping of sites of torsion-induced perturbations of DNA structure. The technique involves strand scission or chemical base modification at structurally perturbed sites, replication arrest in a double-strand DNA sequencing reaction, and size analysis of replication products by electrophoresis on sequencing gels. Besides being less complicated and faster than site identification by conventional end-labeling methods, the technique assures high sequence specificity through the use of oligomeric sequencing primers. This property should be useful for in vivo mapping of DNA structural perturbations with known sequence within complex genomes.     

Dna Sequencing After the Human Genome Project

Abstract

In future DNA sequencing, gel electrophoresis, which is particularly effective for de novo sequencing, is likely to be replaced by sequencing by hybridization, mass spectrometry, or combinations of these two methods, which are particularly effective for comparative or diagnostic sequencing.     

Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.

A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The application to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This increased sharpness permits shorter autoradiographic exposure times and longer sequence reads.     

Resuspension of DNA sequencing reaction products in agarose increases sequence quality on an automated sequencer

We are investigating approaches to increase DNA sequencing quality. Since a majorfactor in sequence generation is the cost of reagents and sample preparations, we have developed and optimized methods to sequence directly plasmid DNA isolated from alkaline lysis preparations. These methods remove the costly PCR and post-sequencing purification steps but can result in low sequence quality when using standard resuspension protocols on some sequencing platforms. This work outlines a simple, robust, and inexpensive resuspension protocol for DNA sequencing to correct this shortcoming. Resuspending the sequenced products in agarose before electrophoresis results in a substantial and reproducible increase in sequence quality and read length over resuspension in deionized water and has allowed us to use the aforementioned sample preparation methods to cut considerably the overall sequencing costs without sacrificing sequence quality. We demonstrate that resuspension of unpurified sequence products generated from template DNA isolated by a modified alkaline lysis technique in low concentrations of agarose yields a 384% improvement in sequence quality compared to resuspension in deionized water. Utilizing this protocol, we have produced more than 74,000 high-quality, long-read-length sequences from plasmid DNA template on the MegaBACET 1000 platform.     

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.     

Analysis of the DNA Sequencing Quality and Efficiency of the Apollo100 Robotic Microcycler in a Core Facility Setting

Sanger, or dideoxynucleotide sequencing, is an important tool for biomolecular research. An important trend in DNA sequencing is to find new and innovative ways to provide high-quality, reliable sequences in a more efficient manner, using automated capillary electrophoresis. The Apollo100 combines Sanger cycle sequencing and solid-phase reversible immobilization for product purification in a single instrument with robotic liquid handling and microfluidic (Microscale On-chip Valve) chips that have onboard thermal cycling and pneumatic mixing. Experiments were performed to determine how the DNA sequencing results from the Apollo100 compared with conventional, manual methods used in a core facility setting. Through rigorous experimentation of multiple baseline runs and a dilution series of template concentration, the Apollo100 generated sequencing that exceeded 900 bases with a quality score of 20 or above. When comparing actual client samples of amplicons, plasmids, and cosmids, Apollo100 sequencing results did not differ significantly from those reactions prepared manually. In addition, bacterial genomic DNA was sequenced successfully, directly with the Apollo100, although results were of lower quality than the standard manual method. As a result of the microscale capabilities, the Apollo100 offers valuable savings with respect to the quantity of reagents consumed compared with current manual sequencing methods, thereby continuing the demand for smaller template and reagent requirements. In conclusion, the Apollo100 can generate high-quality DNA sequences for common templates equivalent to those produced using manual sequencing methods and increases efficiency through reduced labor and reagents.     

Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.

The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation.     

Oligonucleotide analysis by gel capillary electrophoresis

Several million oligonucleotides are synthesized each year for a broad variety of molecular biology applications. Steady improvements in the synthesis chemistry efficiency and the automated DNA synthesizers have made production of oligonucleotides routine and reliable. Many applications, such as PCR and sequencing, are often successful when the primers have not been rigorously purified. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary for the dozens of oligonucleotides produced each day by a DNA synthesis laboratory. Traditional methods of analysis have been HPLC and polyacrylamide slab tel electrophoresis (PAGE). Gel capillary electrophoresis is a new option, combining the advantages of the HPLC and PAGE, with unprecedented resolution and speed.