A rapid method for purifying PCR products for direct sequence analysis

An HPLC approach for purification and sequencing of double-stranded DNA obtained directly from a PCR is described. This simple and reliable procedure has several advantages; the DNA fragment is rapidly eluted (less than 7 minutes), requires no organic cleanup, produces several hundred bases of sequence and is sensitive enough to obtain DNA sequence from a single 100-microliters PCR. This method is demonstrated by sequencing tumor necrosis factor alpha (TNF alpha) gene amplified from mouse tail DNA.     

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.     

Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template

A simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded DNA. This approach employs the thermostable DNA polymerase from Bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. The procedure requires few pipetting steps, no preannealing step and very short reaction time. This method can significantly reduce the cost associated with DNA polymerase and the amount of template and time required to perform the enzymatic sequencing reactions. As little as a 10-ng aliquot of such sequencing reactions can be analyzed on a fluorescence-based capillary gel electrophoresis instrument recently developed in our laboratory. This highly sensitive detection, in conjunction with the ability to efficiently sequence nanogram amounts of template, strongly suggests the feasibility of direct DNA sequencing of single bacteriophage M13 plaques without prior amplification.     

Parallel tagged sequencing on the 454 platform

Parallel tagged sequencing (PTS) is a molecular barcoding method designed to adapt the recently developed high-throughput 454 parallel sequencing technology for use with multiple samples. Unlike other barcoding methods, PTS can be applied to any type of double-stranded DNA (dsDNA) sample, including shotgun DNA libraries and pools of PCR products, and requires no amplification or gel purification steps. The method relies on attaching sample-specific barcoding adapters, which include sequence tags and a restriction site, to blunt-end repaired DNA samples by ligation and strand-displacement. After pooling multiple barcoded samples, molecules without sequence tags are effectively excluded from sequencing by dephosphorylation and restriction digestion, and using the tag sequences, the source of each DNA sequence can be traced. This protocol allows for sequencing 300 or more complete mitochondrial genomes on a single 454 GS FLX run, or twenty-five 6-kb plasmid sequences on only one 16th plate region. Most of the reactions can be performed in a multichannel setup on 96-well reaction plates, allowing for processing up to several hundreds of samples in a few days.     

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.     

Assignment of position-specific error probability to primary DNA sequence data.

DNA sequence predicted from polyacrylamide gel-based technologies is inaccurate because of variations in the quality of the primary data due to limitations of the technology, and to sequence-specific variations due to nucleotide interactions within the DNA molecule and with the gel. The ability to recognize the probability of error in the primary data will be useful in reconstructing the target sequence of a DNA sequencing project, and in estimating the accuracy of the final sequence. This paper describes the use of linear discriminant analysis to assign position-specific probabilities of incorrect, over- and under-prediction of nucleotides for each predicted nucleotide position in primary sequence data generated by a gel-based DNA sequencing technology. Using this method, most of the error potential in primary sequence data can be assigned to a limited number of discrete positions. The use of probability values in the sequence reconstruction process, and in estimating the accuracy of consensus sequence determination is described.     

Selection of chemical spacers to improve isoelectric focusing resolving power: implications for use in two-dimensional electrophoresis

Presented here is a straightforward and inexpensive method for expanding isoelectric focusing pH gradients relative to the gradients that are formed by commercially available narrow range ampholytes. This method requires no special equipment or techniques and is applicable to isoelectric focusing in acrylamide gels, in Sephadex, and in agarose. The utility of separators in improving the resolving power of two-dimensional polyacrylamide gel electrophoresis is demonstrated using proteins from the exocytotic trichocyst organelle of Paramecium tetraurelia. The mode of action of separators is briefly described.     

Double-stranded dideoxy sequencing from "dirty" DNA--done in a day.

A rapid method for preparing and directly sequencing plasmid and phagemid miniprep DNA is described. This protocol is a novel combination of two fairly standard procedures, resulting in quick and easy generation of sequence data. The lack of extensive manipulations in the purification process allows the production of DNA sequence data in a single day.     

Extraction of proteins and peptides from Coomassie Blue-stained sodium dodecyl sulfate--polyacrylamide gels.

A method is described for extracting proteins and peptides from stained sodecyl sulfate-polyacrylamide gels. Coomassie blue and sodium dodecyl sulfate present in stained gel sections are removed to allow subsequent analysis of the peptides (e.g., amino acid analysis or tryptic digestion and fingerprinting). The method is simple, requires no radioisotopes or special equipment, and can be carried out with a minimum of handling of the sample. The process can be used for samples at the nanomole level with recoveries of 90%.     

Large scale sequencing projects using rapidly prepared double-stranded plasmid DNA.

We have developed a simple rapid plasmid DNA mini-preparation method which yields DNA of sufficient quality to be used in large scale sequencing projects. The method, which is a modification of the alkaline method of Birnboim and Doly (1979), requires less than two hours. We have eliminated the use of organic extractions, RNase digestion and alkaline denaturation of the DNA for annealing of the primer. The proportion of supercoiled plasmid DNA obtained is close to 100%. Greater than 80% of the clones yield at least 500 bp of sequence information per primer. The sequencing reactions from these double-stranded templates can be done on both strands using the universal and reverse sequence primers with the usual two reactions per primer, one to read close to the primer and one to read far from it. Thus, each clone yields at least 1 kb of sequence information. The preparation of the templates and the sequencing reactions can be done in less than three hours so that the sequencing gel can be run the same day.     

Analysis of glycopeptides as borate complexes by polyacrylamide gel electrophoresis

A new method using polyacrylamide gel electrophoresis in a Tris-borate buffer to analyze Pronase-derived glycopeptides is described. Examination of immunoglobulin, Sindbis virus, and ovalbumin-radiolabeled glycopeptides by this system demonstrates a pattern similar to that seen after Bio-Gel-P-6 chromatography and, in addition, exposes a heterogeneity in the immunoglobulin and Sindbis virus glycopeptides not apparent after gel filtration. The resolution of glycopeptides by gel electrophoresis depends on the inclusion of borate ions in the sample, the gel, and the electrophoresis buffer. The borate ions react with neutral sugars, converting them to charged complexes which migrate during electrophoresis. The number of borate ions bound to a glycopeptide is a function of the composition, sequence, and linkages of the carbohydrates. Gel electrophoresis of glycopeptides in a borate buffer has several advantages: (1) The method requires no new equipment or special skills beyond those necessary for conventional polyacrylamide gel electrophoresis; (2) when performed on a slab gel, up to 24 samples can be analyzed simultaneously; and (3) since detection is by radio-autography, small amounts of radiolabeled glycopeptides can be visualized by prolonging the exposure time. These characteristics are advantageous for studies of glycopeptides based on digestion products resulting from incubations with specific exo- and endo-glycosidases. Untreated glycopeptides have been compared on the same gel with glycopeptides sequentially treated with different glycosidases to gain structural information.     

A strategy of DNA sequencing employing computer programs.

With modern fast sequencing techniques and suitable computer programs it is now possible to sequence whole genomes without the need of restriction maps. This paper describes computer programs that can be used to order both sequence gel readings and clones. A method of coding for uncertainties in gel readings is described. These programs are available on request.     

The current status and portability of our sequence handling software.

I describe the current status of our sequence analysis software. The package contains a comprehensive suite of programs for managing large shotgun sequencing projects, a program containing 61 functions for analysing single sequences and a program for comparing pairs of sequences for similarity. The programs that have been described before have been improved by the addition of new functions and by being made very much easier to use. The major interactive programs have 125 pages of online help available from within them. Several new programs are described including screen editing of aligned gel readings for shotgun sequencing projects; a method to highlight errors in aligned gel readings, new methods for searching for putative signals in sequences. We use the programs on a VAX computer but the whole package has been rewritten to make it easy to transport it to other machines. I believe the programs will now run on any machine with a FORTRAN77 compiler and sufficient memory. We are currently putting the programs onto an IBM PC XT/AT and another micro running under UNIX.     

A simple and rapid method for the isolation of peptides from sodium dodecyl sulfate-containing polyacrylamide gels

A rapid and simple method is described for the recovery of peptides from sodium dodecyl sulfate-containing polyacrylamide gels. It involves the electrophoretic concentration of a peptide in the stacking gel followed by elution into glycerol. The method requires no special equipment or chemicals, and the elution can be made using the same electrophoretic systems used in the separation step. The method is more rapid than normal extraction procedures, and simpler than most electrophoretic elution methods described. The method can be used for isolation of microgram as well as milligram quantities of an individual peptide with yields of approximately 100%.     

Rapid and reliable fluorescent cycle sequencing of double-stranded templates.

Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.     

用普通琼脂糖代替低熔点胶回收DNA片段

为了建立一种直接从普通琼脂糖凝胶中回收DNA片段的简便实用的方法,采用聚合酶链式反应扩增人P53基因外显子7、8和其间的内含子7序列,用普通琼脂糖凝胶电泳,直接从凝胶中切下产物带,用加热熔化法回收DNA;紫外比色法测定回收率;用测序法鉴定回收产物质量。并用QIAquick Spin纯化柱对照。结果表明,本法回收的产物质量明显优于用QIAquick Spin柱回收,本法回收的产物用于测序效果极佳,回收率达80％,用QIAquick Spin柱回收率不到20％,差异非常显著（P<0.01）。证明这种方法回收PCR产物质量可靠,能代替低熔点胶回收DNA,有较大的实用价值。 Abstract： In order to find a simple and efficient method to isolate single or double?strand DNA fragment amplified by polymerase chain reaction (PCR),we used PCR method to amplify exon 7,exon 8 and intron 7 of human P53 gene, electrophoresis to identify products,fusion and phenol-chlorofom extraction (FPC) to isolate specific DNA from agarose gel,ultraviolet colorimetry to deteminate collected rate,and direct sequencing to identify the quality of recollected DNA. A control test was also made by using QIAquick Spin Colum．The results showed that the quality of PCR products recollected by using FPC method was very good．When the recollected DNA was used in sequencing,no matter what was single or double-strand DNA,the sequence data was clear and even,with low noise．The recollected rate of using FPC,which was over 80 per cent, was higher than that of using colum (lessthan 20 per cent), there were statistical significances (P<0.01).In the control test, it had a little non-specific DNA in the collected products,and the sequencing experiment of using double-strand products was failure．All above mentioned suggested that general agarose gelis efficient in place of low melting-temperature for isolating DNA fragment．     

Genomic sequencing of single microbial cells from environmental samples

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.     

Solid-phase sequence analysis of polypeptides eluted from polyacrylamide gels. An aid to interpretation of DNA sequences exemplified by the Escherichia coli unc operon and bacteriophage lambda

An approach to sequencing proteins by the solid-phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described. Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels. They are detected with Coomassie blue, eluted, selectively reacted with porous glass derivatives and sequenced in their amino-terminal regions with the aid of a new microsequencer. Alternatively they can be analysed or digested with enzymes and fingerprinted. It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences. Nine genes, four from the unc operon of Escherichia coli encoding the alpha, beta, gamma and epsilon subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.     

A gel electrophoresis system for resolving over 500 nucleotides with a single sample loading

A procedure for the resolution of over 500 nucleotides in a single loading on a sequencing gel is described. The method uses exponential wedge spacers to achieve compression of the band spacing in the lower portion of the gel. The method is simple and reliable and reduces the cost of electrophoresis and autoradiography supplies by at least a factor of two.     

Short template amplicon and multiplex megaprimer-enabled relay (STAMMER) sequencing, a simultaneous approach to higher throughput sequence-based typing of polymorphic genes

Sequence-based typing (SBT) is a powerful method of genotyping in highly polymorphic gene systems. In standard SBT methods, both strands of a double-stranded template amplicon are sequenced in separate reactions in order to achieve high quality data across the region of interest. The amount of informative data that is obtained from the second strand sequence is often low, whilst the impact of performing second strand sequencing on costs and throughput are significant. Here we present short template amplicon and multiplex megaprimer-enabled relay (STAMMER) sequencing, a novel simultaneous sequence-based typing methodology that allows the detection of any practical amount of useful sequence from a plurality of distinct polymerase chain reaction products in a single sequencing reaction. In addition to simultaneous bidirectional sequencing, we show how the STAMMER approach can be used to simultaneously sequence a number of regions of interest that are not physically linked within the range of a single sequencing reaction. The efficiencies of this method could impact significantly on the output of SBT laboratories.