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Denaturing Electrophoresis in DNA Sequencing: A Brief History and Current Protocols
Abstract
Two common DNA sequencing methods rely on the principle of measuring relative distances of individual nucleotides from a fixed point in a DNA chain (Sanger and Coulson 1975; Maxam and Gilbert 1977). Base-specific sequencing reactions produce nested sets of fragments, sharing one end and terminating at all positions of a given base, for example guanine, in a DNA chain. The ability to determine a sequence is thus dependent on a separation system which is capable of resolving two DNA fragments that differ in length by a single nucleotide. Until very recently, the only system with adequate resolution, suggested in the 1960s, was based on electrophoresis in a flat polyacrylamide gel with a Tris-borate buffer system (Peacock and Dingman 1967). Electrophoresis was conducted between two glass plates in a simple apparatus, based on the design of Studier (1973). After a certain time, controlled by the mobility of marker dyes, the electrophoresis was terminated and the gel was exposed to an X-ray film or to a phosphor-imaging screen to detect DNA bands labelled with 32P, 35S or 33P (batch technique). The nucleotide sequence was then read from the image using band order and specificity of the sequencing reaction (track identity) (Fig. 1A).
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The effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800-1000 bases. The use of one-dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size-mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequences is discussed.
The long-term goal of this project is the elucidation of the complete sequence of the Caenorhabditis elegans genome. During the first year methods have been developed and a strategy implemented that is amenable to large-scale sequencing. The three cosmids sequenced in this initial phase are surprisingly rich in genes, many of which have mammalian homologues.
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Ansorge W, Sproat B, Stegemann J, Schwager C, Zenke M. Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis. Nucleic Acids Res. 1987 Jun 11;15(11):4593–4602. [PMC free article] [PubMed]
We describe optimized procedures for colorimetrically-detected DNA sequencing with direct blotting electrophoresis. One-step protocols for Sequenase and Klenow enzyme are given. The clapping technique has been adapted to allow convenient casting of very thin gels with an optimal lower gel (transfer) surface. This gives very sharp band patterns, enabling more than 350 bases from a single loading to be read with confidence. The crucial points for direct blotting electrophoresis are discussed. Background problems resulting from unspecific binding of streptavidin to the nylon membranes have been eliminated by the use of high concentrations of SDS in the incubation buffer; and using a single large glass tube for all incubation and washing steps is a very convenient and effective development protocol. Automation of the colorimetric development process is described.
A mismatch-free hybridization of oligonucleotides containing from 11 to 20 monomers to unknown DNA represents, in essence, a sequencing of a complementary target. Realizing this, we have used probability calculations and, in part, computer simulations to estimate the types and numbers of oligonucleotides that would have to be synthesized in order to sequence a megabase plus segment of DNA. We estimate that 95,000 specific mixes of 11-mers, mainly of the 5'(A,T,C,G)(A,T,C,G)N8(A,T,C,G)3' type, hybridized consecutively to dot blots of cloned genomic DNA fragments would provide primary data for the sequence assembly. An optimal mixture of representative libraries in M13 vector, having inserts of (i) 7 kb, (ii) 0.5 kb genomic fragments randomly ligated in up to 10-kb inserts, and (iii) tandem "jumping" fragments 100 kb apart in the genome, will be needed. To sequence each million base pairs of DNA, one would need hybridization data from about 2100 separate hybridization sample dots. Inevitable gaps and uncertainties in alignment of sequenced fragments arising from nonrandom or repetitive sequence organization of complex genomes and difficulties in cloning "poisonous" sequences in Escherichia coli, inherent to large sequencing by any method, have been considered and minimized by choice of libraries and number of subclones used for hybridization. Because it is based on simpler biochemical procedures, our method is inherently easier to automate than existing sequencing methods. The sequence can be derived from simple primary data only by extensive computing. Phased experimental tests and computer simulations increasing in complexity are needed before accurate estimates can be made in terms of cost and speed of sequencing by the new approach. Nevertheless, sequencing by hybridization should show advantages over existing methods because of the inherent redundancy and parallelism in its data gathering.
The increasing demand for DNA sequences can be met by replacement of each DNA sample in a device with a mixture of N samples so that the normal throughput is increased by a factor of N. Such a method is described. In order to separate the sequence information at the end of the processing, the DNA molecules of interest are ligated to a set of oligonucleotide "tags" at the beginning. The tagged DNA molecules are pooled, amplified, and chemically fragmented in 96-well plates. The resulting reaction products are fractionated by size on sequencing gels and transferred to nylon membranes. These membranes are then probed as many times as there are types of tags in the original pools, producing, in each cycle of probing, autoradiographs similar to those from standard DNA sequencing methods. Thus, each reaction and gel yields a quantity of data equivalent to that obtained from conventional reactions and gels multiplied by the number of probes used. To date, even after 50 successive probings, the original signal strength and the image quality are retained, an indication that the upper limit for the number of reprobings may be considerably higher.
Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.
A method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during electrophoresis has been developed. A blotting membrane moves with constant speed across the end of a very short, denaturing gel and collects the molecules according to size. A constant distance between bands for molecules differing in length by one nucleotide is obtained over a large range (approximately 600 nucleotides with a 5% gel), simplifying the determination of DNA sequences considerably. Reliable sequences of 500 nucleotides can be read and sequence features up to greater than 1000 nucleotides are revealed in a single experiment. The sequencing of a potential Z-DNA-forming fragment from Escherichia coli DNA is given as an example and possible further developments are discussed.
This chapter discusses the large-scale complementary DNA sequencing methods. Sequence analysis of cDNA clones is a useful means for characterizing genes in Caenorhabditis elegans and investigating gene expression at specific stages of development. To provide high efficiency when cloning cDNAs, bacteriophage lambda vectors are typically used. This chapter describes methods for PCR amplification and single-pass fluorescent sequencing of C. elegans cDNAs. To produce the highest-quality data while maintaining throughput at a maximum, the PCR templates are purified by polyethylene glycol (PEG) precipitation. Cycle sequencing with dye-labeled primers is the method of choice for sequencing. The chapter also describes the methods for complete sequencing of C. elegans cDNAs. The exonuclease III deletion method of Henikoff is especially useful for cDNAs in the size range 2 to 6 kb, and the multiple cloning sites of some of the newer lambda vectors typically provide an adequate choice of restriction enzymes for unidirectional deletion. A second method for complete sequencing of cloned cDNAs is the random of “shotgun” strategy. Although this may be generally preferred for cDNAs larger than 5 kb, it is possible to stitch several small cDNAs together prior to shotgun subcloning.
This chapter discusses the genomic DNA sequencing methods. The method currently in use for large-scale DNA sequence analysis of the Caenorhabditis elegans (C. elegans) genome employs a random or “shotgun” strategy. This involves using the physical map of the genome, in which, overlapping cosmid or Yeast Artificial Chromosome (YAC) clones have been ordered along with the six chromosomes. These genomic clones are fragmented by mechanical forces to generate random subclones that are sequenced and assembled by computer. The chapter discusses the preparation for approximately 700 random subclones, and the sequencing for each cosmid clone. This number of subclones provides nearly complete fivefold coverage of the cosmid, thus eliminating most of the need for library screening and primer-directed sequencing. The subclones are assembled using the xbap program. After the shotgun phase is complete, the cosmid is “finished” by closing gaps, resolving compressions, and double stranding. The final sequence is analyzed by computer in order to find the genes and regions of homology with other genomes, and finally is submitted to the public sequence databases. By systematically sequencing overlapping cosmids and portions of YACs, an entire genome can be completed.
A new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state-of-the-art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with > 99% accuracy.
A major limitation in the applicability of automated DNA sequencing instruments has been the difficulty in using user-defined oligonucleotide primers which allow sequencing reactions to start at any specific point in a region of interest. Recently, new chemistries have become available for fluorescent labeling which will begin to facilitate the use of any oligonucleotide primer with automated DNA sequencers. In this report, we describe several methods for automated primerdirected DNA sequencing, and compare and discuss the relative merits and limitations of these methods.
A method is described for the rapid generation and cloning of deletion derivatives well-suited for the sequencing of long stretches of DNA. This method is based on two useful features of exonuclease III: (1) processive digestion at a very uniform rate and (2) failure to initiate digestion at DNA ends with four-base 3'-protrusions. The method was applied to a 4570-bp Drosophila genomic DNA fragment cloned in the single-stranded phage vector M13mp18. An ordered set of deletion clones was made by first cutting replicative form (RF) DNA with two restriction enzymes in the polylinker region of the vector between the Drosophila DNA and the sequencing primer binding site. One enzyme left a four-base 3 ' -protrusion that protected the remainder of the vector from exonuclease III attack, allowing unidirectional digestion of the insert sequence from the 5'-protruding end left by the other enzyme. Aliquots were removed at uniform intervals, treated with S1 nuclease, Klenow DNA polymerase, T4 DNA ligase, and then used to transfect competent cells. Most of the resulting clones derived from each aliquot were deleted to a predicted extent with only slight scatter, even for deletions of more than 4 kb. The method permits efficient isolation of clusters of deletion breakpoints within small preselected regions of large DNA segments, allowing nonrandom sequence analysis.
DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.
Fractionation of nucleic acids and their fragments with polyacrylamide gel has been widely applied in sequencing of nucleic acids. Although the conditions of electrophoresis for this purpose have previously been suggested, we have found that polyacrylamide gel electrophoresis at 5000 V (100 V/cm) is possible and effective. An apparatus consisting of a horizontal thermostated plate is used to remove the heat which was formed during the electrophoretic process. The techniques for loading samples on the horizontal thin gel and the procedure for high-voltage gel electrophoresis are described and illustrated by the fractionation of the spleen phosphodiesterase partial digest of tRNA2aVal as well as by the RNA synthesis by RNA polymerase from E. coli with poly[d(A-T)] as template in the presence of “terminator,” 3′-O-methyluridine 5′-triphosphate. This same technique was used for electrophoresis of oligonucleotides on acetylcellulose and was incorporated into a two-dimensional system which was demonstrated by fingerprinting of the guanylo-RNase digest of tRNATrp from baker's yeast. In the third part of the article a simple technique for the electric trapping of nucleic acids or their fragments from a slab gel on a DEAE-paper sheet is presented.
A simple and rapid method for determining nucleotide sequences in single-stranded DNA by primed synthesis with DNA polymerase is described. It depends on the use of Escherichia coli DNA polymerase I and DNA polymerase from bacteriophage T4 under conditions of different limiting nucleoside triphosphates and concurrent fractionation of the products according to size by ionophoresis on acrylamide gels. The method was used to determine two sequences in bacteriophage φX174 DNA using the synthetic decanucleotide A-G-A-A-A-T-A-A-A-A and a restriction enzyme digestion product as primers.
We have developed a high speed instrument for automated DNA sequence analysis. The apparatus employs laser excitation and a cooled CCD detector for the parallel detection of up to 18 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (50-100 microns) denaturing polyacrylamide gels. Four hundred and fifty bases of sequence information is obtained from 100 ng of M13 template DNA in less than one hour, corresponding to an overall instrument throughput of over 8000 bases/hr.
Pulsed fields have been remarkably useful at extending the range of DNA molecular sizes that can be separated on agarose gels by controlling the field-induced molecular orientation that often limits the resolution of large molecules. Unfortunately, the same approach seems to be much less effective for DNA sequencing on polyacrylamide gels. We present an experimental and theoretical (modelling) study of DNA sequencing which shows that molecular orientation is indeed not the main limiting factor for sequencing devices that use moderate field intensities and polyacrylamide as a separating matrix. We examine the interplay between electric field intensity, molecular size and resolution, and we suggest different approaches to increase the resolution limit of standard and automated sequencing gels. The theoretical limits of high-field electrophoretic sequencing are also discussed. We conclude that new ideas will be needed to go beyond one kilobase.
A high-resolution separation of DNA bands is achieved by electrophoresis with a long gel in DNA base sequencing using fluorescence detection. We separate 760 and 761 base DNA fragments using the 93 cm migration electrophoresis optimized for the separation of DNA bands. A T7 DNA polymerase and an Mn++ buffer are used in sequencing reactions to obtain fluorescence peaks of uniform strength, and the peak areas in the spectrum are used for recognizing the peak number in a cluster of successive peaks. This method is successfully applied to the DNA fragment spectrum obtained by 93 cm migration electrophoresis, which results in a single-band differentiation of bands of 1040 base DNA.
Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)
The Tabor and Richardson strategy for enzymatic chain termination sequencing of DNA using relative peak intensity has been adapted to high performance capillary gel electrophoresis with laser induced fluorescence detection. This approach to DNA sequencing involves the use of only a single fluor and results in significant reduction in the time required to determine a DNA sequence without the use of highly complicated and expensive instrumentation. We present a modification of the Tabor and Richardson approach employing two reactions, each containing complementary mixtures of only three ddNTP's in the concentration ratio 4:2:1. The DNA sequence is determined by relative peak height and by assigning the missing ddNTP to "gaps" between the peaks. The use of only three terminators/reaction simplifies the software task of differentiating between the termination types and makes more efficient use of the available dynamic range. Both complementary mixes generate complete sequence information and the two data files are combined in order to make a more confident sequence call. This process helps to eliminate errors caused by occasional non-uniform incorporation of ddNTP's or false terminations and also alleviates some of the difficulty associated with reading through compressed regions of the electropherogram.
When template DNA is saturated with a single-stranded DNA binding protein (SSB), strings of three or four contiguous hexanucleotides
(hexamers) can cooperate through base-stacking interactions to prime DNA synthesis specifically from the 3' end of the string.
Under the same conditions, priming by individual hexamers is suppressed. Strings of three of four hexamers representing more
than 200 of the 4096 possible hexamers primed easily readable sequence ladders at more than 75 different sites in single-stranded
or denatured double-stranded templates 6.4 kilobases to 40 kilobase pairs long, with a success rate of 60 to 90 percent. A
synthesis of 1 micromole of hexamer supplies enough material for thousands of primings, so multiple libraries of all 4096
hexamers could be distributed at a reasonable cost. Such libraries would allow rapid and economical sequencing. Automating
this strategy could increase the speed and efficiency of large-scale DNA sequencing by at least an order of magnitude.
The nucleotide sequence of a region at the 3' terminus of the murine T-cell receptor alpha/delta chain locus is presented. This region, which encodes the constant region genes for alpha and delta chain polypeptides and all 50 joining gene segments for the alpha chain polypeptide, spans 94,647 bp and includes more than 50 noncoding sequence elements important for T-cell receptor gene rearrangement and expression. DNA sequencing of this region included complete analysis of two cosmid clones and five additional restriction fragments using a random subcloning approach with various manual and automated sequencing strategies. The automated sequencing strategies hold considerable promise for future large-scale DNA sequencing efforts.
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.
The factors affecting the electrophoretic separation of DNA bands in DNA base sequencing using fluorescence detection are analyzed. All the factors contributing to DNA band spacing and band width are evaluated; DNA diffusion and thermal effects on gels are the main considerations. The dependence of the gel's electrical resistivity on gel temperature and the variation of temperature over gel thickness are associated with a broadening of DNA band width. As a result of the analyses the maximum separable base number is represented as a function of various electrophoretic variables. The best separations are possible with an electric field strength corresponding to gel thickness. The maximum separable base number increases as the gel thickness decreases. It also increases as the migration distance increases, but it becomes saturated and has an upper limit when the migration distance is long. This upper limit increases as gel thickness decreases. DNA fragments with 600 and 601 bases can be completely separated from each other under optimum conditions for a 0.2 mm thick gel plate. Furthermore, using the band spacing information, under the same conditions, 750 bases could be assigned separately.
A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate
the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating
coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive
thermal transfer methods commonly used. Bubble-free gels as thin as 25μm can be routinely cast on this device. The application
to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions
in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This
increased sharpness permits shorter autoradiographic exposure times and longer sequence reads.
A high-throughput method for the preparation of single-stranded template DNA, which is suitable for sequence analysis using fluorescent labeling chemistry, is described here. In this procedure, the asymmetric polymerase chain reaction is employed to amplify recombinant plasmid or bacteriophage DNA directly from colonies or plaques. The use of amplification primers located at least 200 base pairs 5' to the site of sequencing primer annealing removes the need for extensive purification of the asymmetric polymerase chain reaction product. Instead, the single-stranded product DNA is purified by a simple isopropanol precipitation step and then directly sequenced using fluorescent dye-labeled oligonucleotides. This method significantly reduces the time and labor required for template preparation and improves fluorescent DNA sequencing strategies by providing a much more uniform yield of single-stranded DNA.
Fluorescently labeled DNA fragments generated in enzymatic sequencing reactions are rapidly separated by capillary gel electrophoresis and detected at attomole levels within the gel-filled capillary. The application of this technology to automated DNA sequence analysis may permit the development of a second generation automated sequencer capable of efficient and cost-effective sequence analysis on the genomic scale.
A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different
chemically tuned succinylfluorescein dye distinguished by its fluorescent emission is described. Avian myeloblastosis virus
reverse transcriptase is used in a modified dideoxy DNA sequencing protocol to produce a complete set of fluorescence-tagged
fragments in one reaction mixture. These DNA fragments are resolved by polyacrylamide gel electrophoresis in one sequencing
lane and are identified by a fluorescence detection system specifically matched to the emission characteristics of this dye
set. A scanning system allows multiple samples to be run simultaneously and computer-based automatic base sequence identifications
to be made. The sequence analysis of M13 phage DNA made with this system is described.
This chapter discusses random cloning and sequencing by the M13/dideoxynucleotide chain termination method. The dideoxy chain terminator/Ml3 vector method of deoxyribonucleic acid (DNA) sequencing is considered to be the fastest method to determine the sequence of large fragments of DNA. Lengths of DNA are cloned into the bacteriophage M13 that provides a source of large quantities of single-stranded DNA. This single-stranded DNA can then be used as a template in a primer extension dideoxynucleotide sequence reaction. The chapter discusses the problems related to the simplest method of breaking a DNA molecule into subfragments, which uses restriction endonucleases. The easiest method of purifying the fragment to be sequenced from the cloning vector is cleavage with suitable restriction endonuclease(s) followed by fractionation of the restricted DNA and isolation from a low gelling-temperature agarose minigel. The chapter discusses several procedures, such as isolation of fragment, fragment self-ligation, sonication, and others. Regarding size selection for DNA purification, two factors determine the minimum size to be selected. First, the insert to be sequenced should be at least as long as the maximum, which can be read from a normal sequence gel run. Second, as the subfragments have ends generated at random and not at specific primary sequence sites as with restriction enzymes, it is not possible to detect the junctions of religated noncontiguous fragments simply by inspection. M13 is the vector of choice for dideoxy sequencing for two main reasons. First, M13 bacteriophages are packaged in single strands of DNA, which are extruded from infected Escherichia coli cells into the surrounding culture medium. This means that considerable quantities of single-stranded template DNA can be easily produced. Second, the M13 mp vectors have a quick color assay to identify bacterial cells infected with phage containing an insert.
A simple procedure has been developed for sequencing long (40-kb) DNA fragments by the dideoxy method. The fragment is cloned in the sequencing cosmid pAA113X by in vitro packaging, and subdivided by a series of overlapping IS1-promoted deletions. The deletions are isolated by positive selection for galactose resistance. Plasmids from several thousand galactose-resistant colonies are fractionated on an agarose gel, and DNA from each fraction is restricted with enzymes (such as SphI and SalI) to shorten each deletion from the opposite end. As a result, a series of short overlapping segments, spread across the entire length of the fragment, are fused to IS1. The plasmids are extracted by a rapid method, arranged according to size, and used for supercoil sequencing with an IS1 primer. Sequences of IS1-promoted deletions contain extensive overlaps that are connected further by restriction enzyme-generated deletions to give the complete 40-kb sequence.
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.
We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA are detected near the bottom of the tube, and the sequence information is acquired directly by computer.
The RNAs and proteins specified by five early genes of bacteriophage T7 have been identified by electrophoresis on sodium dodecyl sulfate, polyacrylamide gels. Extracts of cells infected by different deletion strains and point mutants of T7 are analyzed on a slab gel system in which 25 samples can be run simultaneously and then dried for autoradiography. The high capacity of this system makes it possible to do many types of experiment that would be extremely tedious by other means.The five early genes are designated 0.3,0.7, 1, 1.1 and 1.3, in order from left to right on the T7 genetic map. The stop signal that prevents host RNA polymerase from transcribing into the late region of T7 DNA is located to the right of gene 1.3 (ligase). Most deletions that affect gene 1.3 also delete the stop signal, and some of them affect at least one late protein, the 1.7 protein. Several small, early RNAs can be resolved that are not affected by any of the deletions. These small RNAs could not come from between the five early genes or from the right end of the early region, and other work (Dunn & Studier, 1973) indicates that at least some of them come from the region to the left of gene 0.3.Deletions have been found that enter either end of the gene 1 RNA or the right ends of the 0.3 or 1.1 RNAs without seeming to affect the proteins specified by these RNAs. Perhaps all of the early messenger RNAs of T7 have untranslated regions at both ends. Some deletions that enter the left end of the gene 1 RNA reduce the amount of gene 1 protein that is synthesized, presumably by interfering with initiation of protein synthesis.
High molecular weight ribonucleic acid (RNA) from rat liver, kidney, and brain has been fractionated by polyacrylamide gel electrophoresis. A large number of new species of RNA have been resolved with electrophoretic mobilities and sedimentation coefficients intermediate between 30S and 18S RNA and intermediate between 18S and 4S RNA. The number and relative amount of each of these species were constant from preparation to preparation from the same tissue as well as from different tissues. Evidence is presented that these RNA components are present in vivo and are not the result of in vitro artifacts. The results suggest that the current classification of RNA into three major types (30, 18, and 4 S) is inadequate to describe the true heterogeneity of cytoplasmic RNA, and therefore hinders studies of the functional role played by different RNA species.
A method for DNA sequencing has been developed that utilises libraries of cloned randomly-fragmented DNA. The DNA to be sequenced
is first subjected to limited attack by a non-specific endonuclease (DNase I in the presence of Mn++), fractionated by size and cloned in a single-stranded phage vector. Clones are then picked at random and used to provide
a template for sequencing by the dideoxynucleotide chain termination method. This technique was used to sequence completely
a 4257 bp EcoRI fragment of bovine mitochondrial DNA. The cloned fragments were evenly distributed with respect to the EcoRI fragment, and completion of the entire sequence required the construction of only a single library. In general, once a clone
library has been prepared, the speed of this approach (>1000 nucleotides of randomly selected sequence per day) is limited
mainly by the rate at which the data can be processed. Because the clones are selected randomly, however, the average amount
of new sequence information per clone is substantially diminished as the sequence nears completion.
A method for producing random subclones using sonication to fragment the DNA is presented. The sonication is combined with enzymatic repair of the fragment ends and a rigorous size fractionation step to prepare subclones of relatively homogeneous and specific size. Under some conditions sonication is shown to shear A + T-rich sequences preferentially, although under most conditions it will create a random subclone library. The use of these subclone libraries for an improved "shotgun" DNA sequencing strategy is tested on a 17.2-kb (kilobase) fragment of Epstein-Barr virus.
A method is described for casting and operating ultrathin wedge-shaped field-strength gradient gels for DNA sequencing. One of the gradients results in a uniformly-spaced oligonucleotide banding pattern from 60 to 300 nucleotides in a 53 cm long 4% polyacrylamide gel. From these field-strength gradient gels, more sequencing data can be obtained with greater confidence than from gels with uniform thickness. The gel-casting method is fast and simple. At 2.5 kV two gels can be run simultaneously in 2 h with the same power supply.
A simple field gradient technique which leads to sharpening of bands and to an increase in the number of resolvable bases per gel is described. About 300 bases per sample application could be resolved on a 53-cm-long field gradient 6% gel, compared with about 200 on a standard gel. Gels of increasing cross-sectional area, producing the field gradients, are prepared by varying the thickness of spacers along the gel. Compared with the ionic strength gradient method, the field gradient method described here uses the standard sliding technique for preparation of very thin gels and does not require any special skills or preparation of more than one gel solution. Other possible gradient techniques are discussed.
Two methods for increasing the length of DNA sequence data that can be read off a polyacrylamide gel are described. We have developed a rapid way to pour a buffer concentration gradient gel that, by altering the vertical band separation on an autoradiograph, allows more sequence to be obtained from a gel. We also show that the use of deoxyadenosine 5'-(alpha-[35S]thio)triphosphate as the label incorporated in dideoxynucleotide sequence reactions increases the sharpness of the bands on an autoradiograph and so increases the resolution achieved.
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.
We describe three simple modifications of DNA sequencing gels which all result in improved oligonucleotide resolution as visualized by autoradiography. First, it was possible to reduce the thickness of the gel to 0.2 mm by using new gel molding techniques. Second, the gel could be dried without any distortions of its dimensions by prior binding of the gel to the surface of the glass plate. Third, a uniform high temperature was obtained in all parts of the gel during electrophoresis by replacing one of the glass plates with an inexpensive thermostating plate with circulating water. The use of this heating plate resulted in a straight band pattern all over the gel and also in the resolution of such bands which were not resolved in other electrophoresis systems.
