Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50°C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.     

Size‐selective DNA separation: Recovery spectra help determine the sodium chloride (NaCl) and polyethylene glycol (PEG) concentrations required

In the presence of sodium chloride (NaCl), DNA fragments can be size‐selectively separated by varying the final concentration of polyethylene glycol (PEG). This separation strategy in combination with the use of paramagnetic particles provides a valuable platform for achieving the desired DNA size interval, which is important in automated library preparation for high‐throughput DNA sequencing. Here, we report the establishment of recovery spectra of DNA fragments that enable the determination of suitable NaCl and PEG concentrations for size‐selective separation. Firstly, at a given NaCl concentration, the recovery equation was obtained by fitting the DNA recovery ratios versus the PEG concentrations using the logistic function to determine the required parameters. Secondly, the slope function of the recovery equation was achieved by deducing its first derivative. Therefore, the recovery spectrum can be generated using the slope function based on those parameters. According to the recovery spectra of different length DNA fragments, suitable NaCl and PEG concentrations can be determined, respectively, by calculating their resolution values and recovery ratios. The strategy was effectively applied to the size‐selective separation of 532‐, 400‐, and 307‐bp fragments at the selected reagent concentrations with recoveries of 96.9, 64.7, and 85.9%, respectively. Our method enables good predictions of NaCl and PEG concentrations for size‐selective DNA separation.     

DNA sequencing by a subcloning-walking strategy using a specific and semi-random primer in the polymerase chain reaction.

In its basic concept, in vitro DNA amplification by the polymerase chain reaction (PCR) is restricted to those instances in which segments of known sequence flank the fragment to be amplified. Recently, techniques have been developed for amplification of unknown DNA sequences. These techniques, however, are dependent on the presence of suitable restriction endonuclease sites. Here, we describe a strategy for PCR amplification of DNA that lies outside the boundaries of known sequence. It is based on the use of one specific primer, homologous to the known sequence, and one semi-random primer. Restriction sites in the 5' proximal regions of both primers allow for cloning of the amplified DNA in a suitable sequencing vector or any other vector. It was shown by sequence analysis that the cloned DNA fragments represent contiguous DNA fragments that are flanked at one side by the sequence of the specific primer. When omitting the semi-random primer, a single clone was obtained, which originated from PCR amplification of target DNA by the specific primer in both directions.     

一个RNAi载体上反向重复片段的测序策略

相邻的反向重复DNA片段有形成单链内二级结构的倾向,属于一种测序困难的DNA模板。解决RNAi载体插入的反向重复片段的测序问题,为该类载体正确性的测序验证奠定基础。采用常规分子克隆方法构建表达小麦TaATG2串联反向重复片段的RNAi载体,设计2种策略对经菌落PCR初步鉴定的载体进行测序验证:一种是以完整的载体质粒为模板进行测序;另一种是先对载体进行酶切处理,切除反向重复片段中的一个后对保留另一个片段的线性载体进行测序。结果表明,第一种测序策略受到串联反向重复片段形成的单链内部二级结构的影响,测序信号在反向重复片段处出现衰减或乱峰,无法读取序列。第二种测序策略排除了2个反向重复片段之间的干扰,保留在载体上的片段测序信号清晰,序列准确。采用酶切切除一个片段后进行测序的方法,经过2次酶切和2次测序可以有效地对载体上的2个反向重复片段分别进行序列测定,进而确认构建载体的正确性。     

PCR and DNA sequencing

Specific DNA segments defined by the sequence of two oligonucleotides can be enzymatically amplified up to a millionfold using the polymerase chain reaction (PCR). One of the most significant uses of this technique is for generation of sequencing templates, either from cloned inserts or directly from genomic DNA. To avoid the problem of reassociation of the linear DNA strands in the sequencing reaction, ssDNA templates can be produced directly in the PCR or generated directly from dsDNA by enzymatic treatment, electrophoretic separation or affinity purification. By combining PCR with direct sequencing, both the amplification and the sequencing reaction can be performed in the same vial. Finally, use of fluorescently labeled terminators or sequencing primers will allow the whole procedure to be amenable to complete automation.     

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.     

Tetrabutylammonium phosphate-assisted separation of multiplex polymerase chain reaction products in non-gel sieving capillary electrophoresis

A method based on non-gel sieving capillary electrophoresis (NGS–CE) with ultraviolet (UV) detection has been developed for the separation of multiplex polymerase chain reaction (PCR) products of three pathogenic bacteria in which hydroxypropylmethylcellulose was used as the sieving medium and dynamic capillary coating. In the method, an ion pair reagent, tetrabutylammonium phosphate (TBAP), was first used in NGS–CE to improve the detection sensitivities and resolutions of DNA fragments. The interaction of TBAP and DNA was proved using the UV spectra of DNA with and without TBAP. Field-enhanced sample injection was used as an on-line preconcentration method to improve the detection sensitivity. The separation of DNA fragments ranging from 100 to 1000 bp was accomplished in 30 min. Three pairs of primers and three PCR products of bacteria were successfully separated in 25 min using the developed method. The intraday relative standard deviations (RSDs) for the migration time and peak area for each PCR product were less than 2.4% (n = 5), and the interday RSDs were less than 6.1% (n = 15).     

Combination of amplification and post-amplification strategies to improve optical DNA sensing

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.     

桥式PCR，一种简易连接DNA标签序列的方法

MAST方法采用人工文库的DNA标签序列鉴定mRNA的可接近位点。大量的标签序列通过扩增和克隆测序达到阐明mRNA结合位点图。设计了单一引物的PCR,其引物在标签序列两端结合搭桥,在扩增中DNA标签序列在搭桥引物的作用下进行连接,连接的标签序列再克隆和测序。十几条这样的连接产物包含了上千条标签序列。该PCR方法简单、高效以用于高通量的方式对标签序列测序。     

Terminal restriction fragment length polymorphism monitoring of genes amplified directly from bacterial communities in soils and sediments

Terminal Restriction Fragment Length Polymorphism (T-RFLP) or Fluorescent Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (FluRFLP) have made a significant impact on the way in which PCR products amplified from mixed community DNA extracts have been assessed. Technically, these approaches are essentially the same. PCR products are generated that contain at one 5′ end label, typically a fluorescent moiety, that will be detected by a DNA sequencing machine. Upon digestion using a specific restriction endonuclease, labeled and unlabeled fragments are generated. This restriction endonuclease is chosen such that following this digestion, each labeled fragment corresponds to a different sequence variant. During electrophoretic separation, the DNA sequencing machine detects only these labeled fragments and therefore detects only the sequence variants. The aim of this article is to describe the protocois and demonstrate that this profiling can be performed using different DNA sequencing machines. The analysis and applications of this approach are also discussed.     

Efficient identification of point mutations by automated DNA sequencing of artificial heterozygote samples

DNA sequencing templates of individual point mutants of thelacI target gene were amplified by polymerase chain reaction (PCR). By mixing the PCR fragments from two individual mutants in a defined ratio, samples of artificial heterozygous composition were prepared. These samples were then submitted to automated DNA sequencing. The simultaneous, visual comparison of the mixed mutant traces using a graphics program efficiently revealed all heterozygous positions. Based on the individual intensities of the heterozygous base signals the identified point mutations could be assigned to the corresponding mutants. This efficient approach doubles the sample throughput for both the sequencing reactions and the gel electrophoresis using an automated DNA sequencing system.     

Amplification of bacterial genomic DNA by the polymerase chain reaction and direct sequencing after asymmetric amplification: application to the study of periplasmic permeases.

The polymerase chain reaction (PCR) has been used to amplify DNA fragments by using eucaryotic genomic DNA as a template. We show that bacterial genomic DNA can be used as a template for PCR amplification. We demonstrate that DNA fragments at least as large as 4,400 base pairs can be amplified with fidelity and that the amplified DNA can be used as a substrate for most operations involving DNA. We discuss problems inherent in the direct sequencing of the amplified product, one of the important exploitations of this methodology. We have solved the problems by developing an "asymmetric amplification" method in which one of the oligonucleotide primers is used in limiting amounts, thus allowing the accumulation of single-stranded copies of only one of the DNA strands. As an illustration of the use of PCR in bacteria, we have amplified, sequenced, and subcloned several DNA fragments carrying mutations in genes of the histidine permease operon. These mutations are part of a preliminary approach to studying protein-protein interactions in transport, and their nature is discussed.     

DNA sequence analysis by MALDI mass spectrometry.

Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.     

Analysis and purification of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography

Sizing of DNA fragments is a routine analysis traditionally performed on agarose or polyacrylamide gels. Electrophoretic analysis is labor-intensive with only limited potential for automation. Recovery of DNA fragments from gels is cumbersome. We present data on automated, size-based separation of DNA fragments by ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) - DNA chromatography - on the WAVE DNA Fragment Analysis System with the DNASep cartridge. This system is suitable for accurate and rapid sizing of double-stranded (ds) DNA fragments from 50 to ca. 2000 base pairs (bp). Fluorescently labeled DNA fragments are compatible with the technology. Length-dependent separation of dsDNA fragments is sequence independent and retention times are highly reproducible. The resolving capabilities of DNA chromatography are illustrated by the analysis of multiple DNA size markers. Resolved dsDNA fragments are easily collected and are suitable for downstream applications such as sequencing and cloning. DNA chromatography under denaturing conditions with fluorescently labeled DNA fragments offers a means for the separation and purification of individual strands of dsDNA. Analysis of DNA fragments on the WAVE System is highly automated and requires minimal manual intervention. DNA chromatography offers a reliable and automated alternative to gel electrophoresis for the analysis of DNA fragments.     

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.     

Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA.

A modification of the asymmetric PCR method is described, which reliably facilitates sequencing of PCR-amplified DNA. This procedure produces single-stranded DNA fragments as long as two kilobases that are suitable for dideoxy DNA sequencing. First, a PCR for double-stranded DNA is preformed under optimal conditions (double-stranded PCR). Then, a 5-10-microliters fraction of the double-stranded PCR and a single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer is approximately 0.4 microM. Usually 15 to 25 cycles of single-stranded PCR are optimal to produce single-stranded DNA for four to eight sequencing reactions. The single-stranded DNA is purified by centrifugal ultrafiltration and used directly in dideoxy sequencing. This method was employed to produce high-quality single-stranded DNA templates from a variety of organisms for efficient DNA sequencing of PCR-amplified DNA.     

一种高效构建同源重组DNA片段的方法——融合PCR

融合PCR技术（fusion PCR）采用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸,从而将不同来源的任意DNA片段连接起来,此技术在不需要内切酶消化和连接酶处理的条件下实现DNA片段的体外连接,为同源重组片段的构建提供了快速简捷的途径。对原有的融合PCR技术进行改进,以三个同源重组线性DNA片段的构建为例,详细论述了改进的融合PCR技术的反应过程及技术体系。结果表明,改进的融合PCR技术可以同时进行三个片段及四个片段的融合反应,产物长度均在4.5kb以上,各同源重组片段在扩增过程中均无突变发生,获得的片段可以用于后续实验分析。     

A simple method for direct automated sequencing of PCR fragments.

A simple and rapid method for direct sequencing of PCR-generated fragments has been developed for use on Applied Biosystems 373A Automated DNA Sequencer utilizing the DyeDeoxy terminator chemistry. Standard PCR conditions are used to generate a DNA fragment, which is subsequently gel-purified to remove excess primers and unwanted PCR products. The sequencing reactions are carried out in a thermal cycler using the purified product as template DNA and the Dye-Deoxy terminators. The sequence of 500-bp region in the bacteriophage lambda genome and a 320-bp fragment of the human genomic erythropoietin gene were sequenced with greater than 99% accuracy using this method.     

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.