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.     

Simple miniaturized gel system for DNA sequence analysis.

A simple miniaturized gel system suitable for DNA sequencing is described. Small ultrathin polyacrylamide gels are cast, eight or more at a time, using standard microscope slides. Gels, ready to use, can be stored for approximately 2 weeks. Gels are run horizontally in a standard mini-agarose gel apparatus. Typical run times are 6-8 min. A novel sample loading system permits volumes of standard sequencing reactions as small as 0.1 microl to be analyzed. Sequencing ladders were visualized using 35S-labeled DNA by autoradiography and by colorimetric detection. Band resolution compares favorably with that of large gels. The methods introduced here serve as a step toward the miniaturization of DNA sequencing and are amenable to automated sample loading and detection.     

Non-radioactive automated sequencing of oligonucleotides by chemical degradation

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.     

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.     

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)     

Fluorescent labelling of sequencing primers for automated oligonucleotide synthesis.

A fluorescein derivative is described which can be used as a normal phosphoramidite in oligonucleotide synthesis, giving high yields of fluorescein labelled sequencing primers. The labelled primers were used in automated DNA-sequence analysis without modification of existing protocols, the computer-processed sequences being reproducibly readable up to 400 bases. The procedure described makes the fluorescent labelling of oligonucleotides much easier, and the time of labelling can be significantly reduced. It speeds up the "primer walking" approach of automated DNA sequencing.     

Solid phase DNA sequencing using the biotin-avidin system.

A novel method for solid-phase DNA sequencing is described. A plasmid vector, pRIT27, has been designed to allow directional immobilization of double stranded plasmid to avidin agarose. The strategy involves enzymatic incorporation of 11-bio-dUTP into the plasmid and strand specific elution using alkali. The immobilized single stranded DNA is used as template for sequencing reactions and the resulting labelled oligonucleotides are eluted by alkali. The affinity gel containing the immobilized template is consecutively used for the four different dideoxy-nucleotide reactions. The solid-phase technique can be used for both primer specific or extension specific labelling. The possibility to use the system in automated DNA sequencing is discussed.     

Automated fragment analysis method for determining androgen receptor CAG repeat length

Several studies have associated polymorphisms in the androgen receptor gene with the risk of developing hormone-dependent cancers. A highly polymorphic (CAG)n repeat in exon 1 encodes a polyglutamine tract of varying length. The determination of the number of CAG repeats in the androgen receptor has typically been performed on denaturing polyacrylamide gels with autoradiographic orfluorescent detection of differently sized alleles. Samples run on a capillary electrophoresis-based ABI Prism 310 Genetic Analyzer gave anomalous results when internal standards supplied by the manufacturer were used Here we report a modified procedure for androgen receptor allele size determination that can be used on an automated capillary electrophoresis-based DNA sequencer equipped with the appropriate software. The assay is very precise, comparable to DNA sequencing, and is compatible with the latest generation of automated DNA sequencers.     

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.     

Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequencing instrument. Agreement of measured values for velocity, resolution and separation efficiency with theory, predicts further improvements will result from increased electric field strengths (higher voltages and shorter capillaries). Advantages of capillary gel electrophoresis for automatic DNA sequencing instruments and for genomic sequencing are discussed.     

Gamma-irradiation of homodeoxyoligonucleotides 32P-labelled at one end: computer simulation of the chain length distribution of the radioactive fragments

Electrophoresis on polyacrylamide gels of the fragments resulting from gamma-irradiation of single-stranded oligodeoxyribonucleotides labelled at their 5'- or 3'-end proved to be a potent tool for the analysis of the radiation-induced chain breakage of DNA. Owing to the fact that the oligonucleotide may be ruptured at more than one site, the counting of the electrophoresis bands must be corrected and it is necessary to assess the influence of the cleavage position on the band intensities. A complicating factor is the inhomogeneity of the system due to the presence of the four bases A, T, C and G. To circumvent this problem, the homooligodeoxyribonucleotides (dA)15, (dC)15, (dT)15 were used as experimental probes. They were gamma-irradiated in solution, heated in alkali and the resulting fragments separated by gel electrophoresis. A computer simulation of the band intensities was compiled based on the general assumption that the chain breakage is homogeneous. The experimental results obtained from the homooligodeoxyribonucleotides labelled at either the 5' or the 3'-end are in excellent agreement with theoretical calculations. Abacus giving the gel band intensities (percentage) against the nucleotide positions and the remaining intensity of the original oligonucleotide have been obtained.     

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.     

A modified AFLP with fluorescence-labelled primers and automated DNA sequencer detection for efficient fingerprinting analysis in plants

A modified AFLP (amplified fragment length polymorphism) technique is described. Fluorescence-labelled primers were used in the selective amplifications. The amplified fragments were detected on denaturing polyacrylamide gels using an automated ALF DNA sequencer with the fragment option. The modified AFLP technique avoids the use of isotopes or silver staining, but gives a much higher resolution than other AFLP detection systems.     

Use of exonuclease III to determine the site of stable lesions in defined sequences of DNA: the cyclobutane pyrimidine dimer and cis and trans dichlorodiammine platinum II examples

A method to detect chemically stable lesions in DNA has been developed using Exonuclease III, a double strand specific nuclease, to digest 5'-end labeled DNA. The products, when analyzed on high resolution DNA sequencing gels, reveal the sites of DNA modification. Cyclobutane pyrimidine dimers induced by UV irradiation can be localized by comparison of the fragments produced by Exonuclease III digestion with fragments obtained after digestion of the DNA with UV specific endonuclease. The experiments demonstrate the Exonuclease III stops one base away from the cyclobutane pyrimidine dimers. Similar experiments with cis- and trans-dichlorodiammine-platinum (II) showed that modification of DNA by these agents also impede Exonuclease III digestion. In general the same stop sites were found for cis-and trans-platinum adducts. They occur at sites of guanine bases. Additional stop sites were found for cis-platinum at sites of adjacent guanine bases. These results are in agreement with the model that cis-platinum forms intrastrand guanine-guanine dimers, whereas trans-platinum does not.     

The synthesis of protected 5''-mercapto-2'',5''-dideoxyribonucleoside-3''-O-phosphoramidites; uses of 5''-mercapto-oligodeoxyribonucleotides.

The syntheses of the four novel, base protected 5'-(S-triphenylmethyl)mercapto-2',5'-dideoxyribonucleoside-3 '-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) are described. These compounds have been used to prepare 5'-(S-triphenylmethyl) mercapto-oligodeoxyribonucleotides, which are readily purified by reversed phase h.p.l.c., owing to the highly lipophilic trityl group. After cleavage of the S-trityl group by silver or mercuric ions, the free thiol moiety can be coupled to a wide variety of reagents, generating very useful probes. Fluorescent labelled 5'-mercapto-oligodeoxyribonucleotides are being used for automated DNA sequencing without radioactivity, and heavy metal labelled 5'-mercapto-oligonucleotides will be used in X-ray crystallography.     

Increased stability of nucleic acids containing 7-deaza-guanosine and 7-deaza-adenosine may enable rapid DNA sequencing by matrix-assisted laser desorption mass spectrometry.

The use of matrix-assisted laser desorption mass spectrometry (MALDI-MS) has been suggested as an ultrafast readout of Sanger DNA sequencing ladders in a manner analogous to that used with sequencing gels. Currently, a serious limitation of MALDI-MS for the analysis of DNA results from the tendency for oligonucleotides to undergo facile fragmentation in the gas phase. The present study was undertaken to gain an understanding of the influence of various chemical structural features of purine bases on the stability of oligodeoxynucleotide ions produced by MALDI. The study focused on the stability of model compounds of the type d(TTTTTTTTTTXTTTTTTTTT TTTT), where T designates deoxythymidine and X a purine-containing 2'-deoxynucleotide. A variety of different purine derivatives were chosen as the base in the nucleotide X. The mass spectra of the model compounds containing 7-deaza analogues of guanine and adenine reveal a significantly increased stability compared to the 7-aza analogues under the conditions of MALDI-MS. The previously reported incorporation of the 7-deaza-2'-deoxy-adenosine triphosphate and the 7-deaza-2'-deoxy-guanosine triphosphate into DNA by polymerases suggests their use in a Sanger dideoxy sequencing experiment. The dideoxy termination products with the 7-deaza-purines instead of the 7-aza-purines might be sufficiently stable to allow separation and detection of the sequencing ladder by MALDI-MS. Thus, an ultrafast (seconds) read-out of DNA sequence may become feasible.     

纳米孔测序技术应用于食品微生物检测的可行性分析

近些年来DNA测序技术发展迅速,已经从第一代生化测序发展到第三代单分子测序。作为第三代测序技术中的一种不同于当前流行的其他测序技术,纳米孔测序技术是基于电信号的一种物理方法测序。许多研究者通常将高通量测序技术应用于食品微生物的研究,但是将纳米孔测序技术应用于食品中微生物的检测却鲜有报道。Oxford Nanopore Technologies(牛津纳米孔科技公司)研发的DNA测序仪MinION,是世界首例用于商业测序的纳米孔测序仪,经过不断完善,近年来MinION在DNA测序中被广泛应用。MinION 测序一次需要的DNA量约1μg,其标准识别速度为一秒钟识别250个碱基,平均读长可至13kb~20kb,测序准确率可以达到98%。纳米孔测序的高识别速度和高准确率,完全满足快速检测的要求,将其应用于食品中微生物检测是完全可行的。     

Modifications of guanine bases during oligonucleotide synthesis.

Guanine bases are sensitive to modification during automated DNA synthesis and processing reactions. Methods for the detection of two types of guanine modifications are described. The first method uses the higher reactivity of the modified G base to KMn04 oxidation than T bases, and thus allows detection by chemical DNA sequencing. The second method makes use of the Escherichia coli nucleotide excision repair enzyme UvrABC endonuclease which can detect "bulky" base modifications at each nucleotide in the synthetic DNA. Though the chemical structures of the two modifications are not known, they may be related. Both types of G modifications are often found in oligonucleotides synthesized by the methoxy-diisopropyl-phosphoramidite (MEDP) chemistry but non-detectable in the products of the beta-cyanoethyl-diisopropyl-phosphoramidite (CEDP) chemistry. The Rubin and Schmid pyrimidine-specific chemical DNA sequencing procedure (Rubin, C.M., and Schmid, C.W. (1980) Nucleic Acids Res. 8, 4613-4619) was found to be applicable to oligonucleotides synthesized by the CEDP chemistry, and to oligonucleotides synthesized by the MEDP chemistry if precautionary measures are taken to destroy the signals produced by the highly KMnO4 sensitive modified guanine bases. We also show how chemical DNA sequencing might be useful for diagnosing other chemical modifications in synthetic oligonucleotides.