利用Taq DNA聚合酶对PCR产物直接进行顺序分析

Taq DNA聚合酶具有反应速度快、温度作用范围广及良好的续进性等特点,可视为一种理想的DNA顺序分析酶。本文首先对非对称性PCR扩增过程中单、双链DNA产物的积累情况进行了分析,然后采用标记延伸二步法,对Taq DNA聚合酶的性质及影响因素进行分析。为进一步改进Taq DNA聚合酶测序的方法,本反应建立了“Klenow-型”的直接掺入标记同位素测序法,即在反应液中加入与标记核苷酸相应的一定浓度的冷dNTP。此法不但解决了二步法中引物后部分DNA顺序无法读出的缺点,而且简化了反应步骤,亦能得到令人满意的顺序分析结果,每次可读出至少400碱基的序列。     

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.     

A simplified biochemistry for DNA sequencing

A simplified method of DNA sequencing by dideoxy chain termination is developed that approaches a single-step protocol. Utilizing the sequencing advantages contributed by a thermophilic polymerase and a guanine analog, stable sequencing reaction concentrates have been obtained that readily perform the entire sequencing reaction simply by adding prepared DNA to each of the four reaction concentrates required by this method. The mechanics and dynamics of these reactions have been investigated and the capacity of these reactions to withstand normal user variation is demonstrated. This study focuses on one form of this simplified method embodied in the FASTaq DNA sequencing kit.     

Adenine specific DNA chemical sequencing reaction.

Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.     

DNA sequencing using rolling circle amplification and precision glass syringes in a high-throughput liquid handling system

An automated high-throughput method that employs rolling circle amplification (RCA) to generate template for large-scale DNA sequencing has been developed using liquid handling systems equipped with precision glass syringes. A protocol was designed to perform the sequencing analysis from template preparation to thermal cycle sequencing within the same vessel, thus minimizing the amount of liquid handling and transfer. The amplified DNA was directly used for cycle sequencing with no need for any purification procedures. Total RCA reaction volumes as low as 500 nL generated sufficient templates for successful sequencing. Reducing the RCA total reaction volumes by a 40-fold factor, from a total of 20 microL to 500 nL, resulted in a significant reduction in cost, from $1.25/reaction to less than $0.04/reaction. Additionally, the volume of the sequencing reactions was reduced from a total of 20 to 10 microL, thus generating a further cost advantage. This high-throughput DNA sequencing protocol maximizes the speed and precision of processing while significantly reducing the cost of amplification.     

Colorimetric-detected DNA sequencing

A sensitive, colorimetric method for visualizing the band pattern of DNA sequencing reaction is described. The enzymatic incorporation of radioactive nucleotides commonly used for the band detection is replaced by biotin conjugated to the 5'-terminus of a synthetic oligonucleotide by chemical synthesis. The oligonucleotide so labeled is used as a primer for dideoxy DNA sequencing in a primer extension reaction. The products of the sequencing reactions are analyzed on a denaturing polyacrylamide gel using the direct blotting electrophoresis technique. This technique makes it possible to transfer the band pattern during the electrophoresis onto an immobilizing matrix, on which it is made visible by an enzymatic reaction in less than 3 h. This biotin-based detection method is so sensitive that the sequencing reactions can be performed under the same conditions and concentrations as those for the radioactive detection.     

Automated Sanger dideoxy sequencing reaction protocol

The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps. An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions. The system is set up in a short time for routine operation and it is faster and more reliable than a human operator. It is flexible and allows variations and optimization of the standard procedure. Disposable microtiter plates at a controlled temperature are used. In one reaction cycle (about 50 min) up to 48 templates are processed. Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot. The protocol is applicable to fluorescent as well as to radioactive labeling.     

Editor's choice: Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging

The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics.     

三酶焦测序体系的建立及其在唐氏综合征快速诊断中的应用

为了避免四酶焦测序体系中由于三磷酸腺苷双磷酸酶(apyrase)造成的测序结果偏差, 文章建立了一种定量性能好的无三磷酸腺苷双磷酸酶的三酶焦测序体系。方法是将生物素修饰的DNA模板、荧光素酶和ATP硫酸化酶固定在磁性微球表面进行焦测序反应, 当加入一种dNTP进行焦测序反应完后, 采用磁性分离技术, 除去焦测序反应产生的ATP和剩余的dNTP, 然后加入另一种dNTP进行测序, 按同样的方法去除影响下一轮测序反应的成分, 实现循环测序。此体系能准确判读待测DNA的碱基序列, 且可定量测定单核苷酸序列多态性(SNP)中两种等位基因型的相对比值。文章成功检测了16例正常人和8例唐氏综合征患者样本中21号染色体上两个杂合率较高位点(rs1042917和 rs4818219)的等位基因型比值, 所得结果能够明确说明待测样本中来自于父方和母方的21号染色体数目是否相等。该法具有良好的定量性能, 适合于SNP等位基因型的定量分析, 可以用于唐氏综合征的快速检测。     

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing Was developed by a synthesis method(DP-SBS)for high-throughput DNA sequencing,in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays.In this method,adifferent set of degenerate primers containing a give nnumber(n)of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface.The nucleotides(n 1)on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides.The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately.The main advanmge of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension.From the present study,it is found that the DP-SBS method is reliable,simple,and cost-effective for laboratory-sequencing a large amount of short DNA fragments.     

In vitro double transposition for DNA identification

We present a double transposition technique that inserts two different transposons into target DNA to act as priming sites for amplifying the region between the two transposons for sequencing applications. Unlike some current sequencing approaches, the genome of the unknown target remains intact in this method. The transposition reaction, DNA repair, and subsequent sequencing were performed entirely in vitro, without the need for transformation into bacteria, and resulted in sequence homology with the plasmid DNA target. This approach can reduce the time required for the assay by more than a day compared with standard techniques and reduces the number of required enzymatic steps. In addition, the in vitro method enables transposition to be carried out in automated microfluidic platforms without the need for significant sample manipulation. As a demonstration of incorporating transposition techniques into high-throughput technologies, single transposition reactions were carried out in picoliter-sized droplets generated on a microfluidic platform.     

Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues

The increasing use of high‐throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low‐salt CTAB (MoLSC) extraction protocol to accommodate contaminant‐rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in‐house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuitable for some applications of massively parallel sequencing. Our open‐source MoLSC protocol provides a cost‐effective, scalable, alternative DNA extraction method that can be easily optimized and adapted for sequencing applications in other contaminant‐rich samples.     

Use of a chemically modified T7 DNA polymerase for manual and automated sequencing of supercoiled DNA

Procedures are presented for reliable and accurate nucleotide sequence analysis using as template supercoiled DNA prepared by a modified rapid boiling minipreparation protocol. This method yields DNA templates suitable for sequencing within 1 h of bacterial harvest. We describe optimal reaction conditions for supercoiled miniprep DNA sequencing using a modified T7 DNA polymerase (Sequenase) in dideoxynucleotide chain termination reactions. We demonstrate that under these conditions, the sequencing data obtained with miniprep DNA is indistinguishable from that obtained with CsCl purified supercoiled DNA or from that obtained using single stranded DNA templates. We further show that the supercoiled DNA sequencing reactions can be analyzed on a commercially available automated DNA sequencing system that detects 32P labeled DNA during its electrophoretic separation. Taken together, these developments represent a significant improvement in the process of nucleotide sequence analysis.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.     

A facile method for the construction of synthetic genes

A simple protocol for rapid assembly of chemically synthesized deoxyoligonucleotides into double stranded DNA is described. Several parameters of a ligation-free method were investigated to allow efficient assembly of a large number of oligonucleotides into double stranded DNA by polymerase chain reaction. Synthesis of a 701 bp DNA was carried out in a single reaction by assembling 28 oligonucleotides designed with partial overlaps at complementary ends. An estimate of error rate was made by sequencing several independent clones of the synthesized DNA     

DNA sequencing with solid-phase-capturable dideoxynucleotides and energy transfer primers

False terminations occurring in fluorescent dye-primer DNA sequencing, and nonsequencing primer extension DNA fragments generated in dye-terminator sequencing cause background noise in fluorescent electropherograms, leading to errors in sequence determination. We describe here a DNA sequencing chemistry that produces accurate and clean sequencing data on a fluorescent DNA sequencer, eliminating the false terminations and background noise. The procedure involves coupling fluorescence energy transfer (ET) primers that produce high fluorescent signals with solid-phase-capturable biotinylated dideoxynucleotides to generate Sanger DNA sequencing fragments. After the sequencing reaction,the DNA extension fragments that carry a biotin at the 3' end are captured with streptavidin-coated magnetic beads, while the other components in the sequencing reaction are washed away. Only pure DNA extension products terminated by the biotinylated dideoxynucleotides are released from the magnetic beads and are loaded onto a sequencing gel to produce accurate sequencing data.     

Simple and efficient synthesis of 5' pre-adenylated DNA using thermostable RNA ligase

We report a simple method of enzymatic synthesis of pre-adenylated DNA linkers/adapters for next-generation sequencing using thermostable RNA ligase from Methanobacterium thermoautotrophicum (MthRnl). Using RNA ligase for the reaction instead of the existing chemical or T4 DNA ligase-based methods allows quantitative conversion of 5'-phosphorylated single-stranded DNA (ssDNA) to the adenylated form. The MthRnl adenylation reaction is specific for ATP and either ssDNA or RNA. In the presence of Mg(+2), the reaction has a pH optimum of 6.0-6.5. Unlike reactions that use T4 DNA ligase, this protocol does not require synthesis of a template strand for adenylation. The high yield of the reaction simplifies isolation and purification of the adenylated product. Conducting the adenylation reaction at the elevated temperature (65°C) reduces structural constraints, while increased ATP concentrations allow quantitative adenylation of DNA with a 3'-unprotected end.     

Rapid determination of short DNA sequences by the use of MALDI-MS

We have developed a protocol for rapid sequencing of short DNA stretches (15–20 nt) using MALDI-TOF-MS. The protocol is based on the Sanger concept with the modification that double-stranded template DNA is used and all four sequencing reactions are performed in one reaction vial. The sequencing products are separated and detected by MALDI-TOF-MS and the sequence is determined by comparing measured molecular mass differences to expected values. The protocol is optimized for low costs and broad applicability. One reaction typically includes 300 fmol template, 10 pmol primer and 200 pmol each nucleotide monomer. Neither the primer nor any of the nucleotide monomers are labeled. Solid phase purification, concentration and mass spectrometric sample preparation of the sequencing products are accomplished in a few minutes and parallel processing of 96 samples is possible. The mass spectrometric analyses and subsequent sequence read-out require only a few seconds per template.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry protocol for monitoring the progress of enzymatic (13)C/(15)N-labeled DNA syntheses

We demonstrate that a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry protocol provides a rapid and accurate method for monitoring the stage and completeness of enzymatic DNA syntheses. A crucial step of these syntheses is to quench the reaction at the desired nucleotide length. This is especially important when expensive, e.g., (13)C/(15)N-labeled DNA segments, are synthesized for multinuclear magnetic resonance purposes to reveal detailed structural information. The analyses of three templates for a human telomeric 22-mer, a wild type, and a mutant human c-MYC promoter (18- and 22-mer) DNA and their reactions with the 3'-5' exo(-) Klenow fragment of DNA polymerase I demonstrate the usefulness of our protocol. Small amounts of samples (ca. 1-2 microl each) were taken from the reaction mixtures at different times and analyzed promptly by MALDI-TOF, applying our successive on-plate desalting method that eliminates the insensitivity of the MALDI technique at high salt content. The progress of the reaction was detected by monitoring the relative intensity ratios of ions corresponding to the desired products and the primer-template complexes. The effectiveness of NH(3) cleavage leading to final products was also followed by MALDI-TOF in successful enzymatic reactions.