Fluorescence-based mutation detection

Conventional SSCP analysis of DNA amplified by polymerase chain reaction (PCR-SSCP) is one of the simplest and most reliable tools for identifying point mutations, and small insertions or deletions. The sensitivity of the technique is increased by using the Applied Biosystems (ABI) semiautomated DNA sequencer equipped with GENESCAN 672 software for F-SSCP. The four-dye ABI system permits a red dye-labeled internal lane standard to be run in the same lanes as the DNA being examined, leaving three dye colors for labeling DNA of interest. The internal lane standard is used to normalize gels or correct for minor differences in apparent electrophoretic mobility between lanes. Correction for these lane-dependent differences in migration and the capability to stack data from two different lanes on the computer screen makes it possible to detect sequence variants that produce very small mobility shifts. Coelectrophoresis of control and unknown DNA in the same lane, using different dye labels for each, is also helpful for detecting sequence variants that produce small mobility changes. Multiplexing multiple F-SSCP targets in the same lane increases sample throughput.     

Automated Sanger DNA sequencing with one label in less than four lanes on gel

Novel Sanger dideoxy sequencing with only one fluorescent dye label for the four bases of one clone and sequence determination in two lanes on polyacrylamide gel is presented, loading A greater than G in one lane and T greater than C in the other. Sequencing reactions for the two bases in each lane are carried out in one tube. At present the ratio of ddATP:ddGTP and ddTTP:ddCPT is set to 5:1 in the two tubes. Distinction between the two bases in one lane is done by comparing the different magnitudes of the peaks. This method increases the capacity since more clones may be run simultaneously on one gel, while keeping the reliability and simplicity that comes with the use of only one fluorescent dye for the four bases of one clone. At present about 200 bases are determined with the one-dye two-lane method on the EMBL's automated fluorescent DNA sequencer, using T7 DNA polymerase. The error rate in the deduced sequence is about 1%. The technique is used for the determination of overlaps in mapping projects. In principle, it is possible to determine the sequence with one dye in only one lane on the gel by choosing the proper ddNTP ratios for all four bases, carrying out reactions in one tube and applying the product in one lane, but the error rate for this one-lane method seems too high at present and further improvements in the uniformity of peaks obtainable with the T7 DNA polymerase or other enzymes are required.     

The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner

A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Practical applications in molecular biology of sensitive fluorescence detection by a laser-excited fluorescence image analyzer.

A new kind of fluorescence image analyzer was developed for a variety of uses, especially in molecular biology. Compounds labeled with fluorescent groups on a gel or nitrocellulose membrane are excited with 532 nm of light from a green laser. The fluorescence emitted passes through light-collecting fibers to a photomultiplier. Imaging data converted from the emitted light are analyzed by a microcomputer and stored on a magnetic optical disk. Dideoxy DNA sequencing was done with the same amount of DNA used for autoradiography, and the sequencing ladders obtained from gel scanning were automatically converted to sequence data by the analyzer. When an agarose gel was analyzed after electrophoresis, DNA stained with ethidium bromide was detected by the analyzer with higher sensitivity rather than by the conventional photographic method. Nylon and nitrocellulose membranes could be read by the analyzer, so blot hybridization experiments can be done without radioisotopes. High-quality computer storage of the imaging data from gel electrophoresis and hybridized membranes, including pulsed-field gels, make it possible to quantify image intensity and to construct many kinds of databases.     

Automated sequencing of fluorescently labelled DNA by chemical degradation

A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.     

Hierarchical analysis of large-scale two-dimensional gel electrophoresis experiments

Large-scale two-dimensional gel experiments have the potential to identify proteins that play an important role in elucidating cell mechanisms and in various stages of drug discovery. Such experiments, typically including hundreds or even thousands of related gels, are notoriously difficult to perform, and analysis of the gel images has until recently been virtually impossible. In this paper we describe a scalable computational model that permits the organization and analysis of a large gel collection. The model is implemented in Compugen's Z4000 system. Gels are organized in a hierarchical, multidimensional data structure that allow the user to view a large-scale experiment as a tree of numerous simpler experiments, and carry out the analysis one step at a time. Analyzed sets of gels form processing units that can be combined into higher level units in an iterative framework. The different conditions at the core of the experiment design, termed the dimensions of the experiment, are transformed from a multidimensional structure to a single hierarchy. The higher level comparison is performed with the aid of a synthetic "adaptor" gel image, called a Raw Master Gel (RMG). The RMG allows the inclusion of data from an entire set of gels to be presented as a gel image, thereby enabling the iterative process. Our model includes a flexible experimental design approach that allows the researcher to choose the condition to be analyzed a posteriori. It also enables data reuse, the performing of several different analysis designs on the same experimental data. The stability and reproducibility of a protein can be analyzed by tracking it up or down the hierarchical dimensions of the experiment.     

基于PC/Linux的核酸序列分析系统的构建及其应用

基于PC机和Linux操作系统, 利用Phred/Phrap/Consed软件和Blast软件, 构建了核酸序列大规模自动分析系统. 该套系统可自动完成从测序峰图向核酸序列的转化、载体序列去除、序列自动拼接、重复序列鉴定以及序列的相似性分析, 可加速对大规模测序数据的分析和利用.     

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.     

AFLP fingerprinting as a tool to study the genetic diversity of Rhizobium galegae isolated from Galega orientalis and Galega officinalis

AFLP fingerprints of Rhizobium galegae strains that infect Galega orientalis and Galega officinalis obtained from different geographical sources, and of taxonomically diverse rhizobia representing the recognized species, were generated. Comparisons of the fingerprints from fluorescent labeled AFLP products using capillary electrophoresis on ABI prism 310, slab gel electrophoresis on ABI prism 377 genetic analyzers and silver staining were in good agreement. All methods delineated the G. orientalis strains from G. officinalis strains, the G. orientalis strains formed a tight cluster whereas the G. officinalis strains seem to show a greater level of genetic diversity. Comparison of fluorescent AFLP with other detection methods revealed that fluorescent labeling is more sensitive and practical, in addition, the deleterious effect of radioactivity associated with 32P-labeling, the delicate process of blotting polyacrylamide gels or the tedious procedure of silver staining can be avoided. The automated system facilitated a large number of runs at a time and the subsequent analysis of the data by generating exportable raw data. The congruency of the experiments was analyzed using the Bionumerics software.     

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.     

System for DNA sequencing with resolution of up to 600 base pairs

A system capable of resolving about 500 bases is of interest for sequencing of longer DNA molecules. Studies on further optimization of resolution on DNA sequencing gels were carried out. The effect of physico-chemical properties of gels and buffers on resolution were tested, e.g. ionic strength and pH of buffers, different buffer systems, acrylamide concentration, crosslinker concentration, type of crosslinker, temperature of polymerization, denaturing conditions, gel length and thickness. Tested were as well different running conditions like electric field, gel temperature, dimension of sample slots. Gels 0.1-0.2 mm thick and up to 1.2 m long were cast and tested routinely. Gel lengths of 60-70 cm (for sequencing up to 350-400 bases) to about 100 cm (above 400 bases) are practicable. Little is gained in resolution by increasing the gel length from 1 to 1.2 m. Resolution was improved using 0.1 mm thick gels, at a higher pH value of 8.6-8.8, and molarity increased to 0.2 M. The sequencing pattern in the region of higher bases could be better resolved on a twice-magnified picture of that region on the autoradiogram. With the long gels (70-120 cm), it is advantageous to obtain the sequence overlap by running in parallel gels of different concentrations, without re-application of samples, all loaded at the same time. Buffer chamber for running of two of three gels and thermostating plates up to 1.2 m long were designed. In this way four to six thermostated gels can be run from a power supply with two inputs. Three 1 m long gels (concentrations: 4%, 6%, 12-16%) are loaded with several samples of DNA to be sequenced and run in parallel without re-application of the samples. With good samples, the sequence overlap from the gels could be counted up to 500 base pairs, with exceptionally good samples closer to 600 bases. At present this number seems to be near the limit of the resolving power of the polyacrylamide gels.     

Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules.

We have developed a technique to detect, recognize, and track each individual low density lipoprotein receptor (LDL-R) molecule and small receptor clusters on the surface of human skin fibroblasts. Molecular recognition and high precision (30 nm) simultaneous automatic tracking of all of the individual receptors in the cell surface population utilize quantitative time-lapse low light level digital video fluorescence microscopy analyzed by purpose-designed algorithms executed on an image processing work station. The LDL-Rs are labeled with the biologically active, fluorescent LDL derivative dil-LDL. Individual LDL-Rs and unresolved small clusters are identified by measuring the fluorescence power radiated by the sub-resolution fluorescent spots in the image; identification of single particles is ascertained by four independent techniques. An automated tracking routine was developed to track simultaneously, and without user intervention, a multitude of fluorescent particles through a sequence of hundreds of time-lapse image frames. The limitations on tracking precision were found to depend on the signal-to-noise ratio of the tracked particle image and mechanical drift of the microscope system. We describe the methods involved in (i) time-lapse acquisition of the low-light level images, (ii) simultaneous automated tracking of the fluorescent diffraction limited punctate images, (iii) localizing particles with high precision and limitations, and (iv) detecting and identifying single and clustered LDL-Rs. These methods are generally applicable and provide a powerful tool to visualize and measure dynamics and interactions of individual integral membrane proteins on living cell surfaces.     

preAssemble: a tool for automatic sequencer trace data processing

Background  

Trace or chromatogram files (raw data) are produced by automatic nucleic acid sequencing equipment or sequencers. Each file contains information which can be interpreted by specialised software to reveal the sequence (base calling). This is done by the sequencer proprietary software or publicly available programs. Depending on the size of a sequencing project the number of trace files can vary from just a few to thousands of files. Sequencing quality assessment on various criteria is important at the stage preceding clustering and contig assembly. Two major publicly available packages – Phred and Staden are used by preAssemble to perform sequence quality processing.     

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.     

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.     

Loader Lite: a new software tool for the ABI PRISM 3700 DNA sequencer

Here we describe the development of a novel software tool entitled Loader Lite that generates plate records or sample sheetsfor the ABI PRISMs 3700 DNA sequencer. The major advantage of this program is that it enables the ongoing operation of sequencing instruments without reference to external network(s). The autonomous operation of sequencing instruments is critical if sample throughput is to be maintained during periods of network outage. Loader Lite employs a deliberate strategy of inputting anonymous tray barcodes at run time. After sequencing, the barcodes are reconciled with relevant project details by reference to a database. This software takes advantage of barcode scanning technology by creating plate records directly on the local computer, serving an individual sequencer, immediately before importing and linking. This real-time synthesis of the plate records at the point of loading all but eliminates loading errors. Loader Lite is user-friendly, fully configurable, and permits the running of partial or full 384-well sample trays, using any standard combinations of run modules, dye sets, mobility files, analysis modules, etc. The 96-well format is not supported; however, this capability will appear in subsequent versions that are currently under development. This application is designed as an added value, adjunct program to the regular ABI PRISM 3700 Data Collection software. We have successfully used Loader Lite over the past six months to load approximately 7 million sequencing reactions and believe its utility and functionality will prove to be attractive to the wider sequencing community.     

The 4-6-8 method of sequence analysis

A new method to obtain more sequence data from a single gel run is described. This method allows the reading of over 500 bases of sequence data from a single gel by taking advantage of the differential migration of specific sized dideoxy terminated chain lengths in sequencing gels containing variable percentages of acrylamide. The method is easy to use, requires no special equipment and requires no special technical abilities. We feel the methodology described will be useful for the average laboratory doing sequencing work.