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We have compared sequencing of cloned “polymerase chain reaction” (PCR) products and the direct sequencing of PCR products
in the examination of individuals from six families affected with α1-antitrypsin (AAT) deficiency. In families where paternity was in question we confirmed consanguinity by DNA fingerprinting
using a panel of locus-specific minisatellite probes. We demonstrate that direct sequencing of PCR amplification products
is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes
and homozygotes in a single, rapid and facile assay. Furthermore, we demonstrate the reproducibility of the PCR and a rapid
DNA isolation procedure. We have also shown that two loci can be simultaneously amplified and that the PCR product from each
locus can be independently examined by direct DNA sequencing.
In the decade since their inception, manual DNA sequencing techniques have made only minor inroads into the primary biological data–base. Attempts have been made to automate the process in order to achieve an enhanced rate of data–capture to allow access to the larger genome. Progress in instrumenting these techniques has been restricted because of their complexity and the multidisciplinary nature of the procedures involved. This article outlines the manual methods presently employed and reviews current sequencing methodology. A rationale for the possible successful automation of DNA sequencing incorporating a potential for improvements in related areas is presented. Recent sequence instrumentation projects are assessed and an account is provided of contemporary national and international proposals to implement accelerated DNA sequence data–capture.
A set of bacteriophage vectors was generated to combine the well-established method of M13 DNA sequencing with the high throughput multiplex strategy.
In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled
primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described.
Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without
subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The
fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with
the enzymatic polymerization. High sensitivity of the detection device, down to 10−18 moles, corresponding to less than 106 molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with
about 5×104 copies of recombinant DNA.
A multipurpose plasmid, pUR222, was constructed. It contains six unique cloning sites (PstI, SalI, AccI, HindII, BamHI and EcoRI) in a small region of its lac Z-gene part. Insertion of foreign DNA into the plasmid can be easily detected. Bacteria harbouring recombinant plasmids generally
give rise to w hite colonies, while those containing only vector DNA form blue colonies on indicator plates. Plasmid DNA purified
by a rapid method (Birnboim, H. C. and Doly, J. (1979) Nucl. Acids. Res. 7, 1513–1523) can be used for chemical sequencing of the cloned insert DNA. Labeled fragments need not be isolated after cutting
with the proper restriction enzymes and are treated directlyaccording to the sequencing protocol of Maxam and Gilbert.
Substitution of inosine for granosine in the nucleic acid fragments synthesized for the sequencing of RNA effectively prevents the formation of secondary structures during electrophoretic analysis. Consequently, the mobility of each fragment in the sequencing gel is a strict function of its molecular weight. Inosine substitution should markedly improve the resolution that can be obtained in the sequencing of DNA as well as RNA.
Evidence is presented that ColE1 hybrid plasmids carrying the cohesive-end site (cos) of lambda can be used as gene cloning vectors in conjunction with the lambda in vitro packaging system of Hohn and Murray [(1977) Proc. Natl. Acad. Sci. USA 74, 3259--3263]. Due to the requirement for a large DNA molecule for efficient packaging, there is a direct selection for hybrids carrying large sections of foreign DNA. The small vector plasmids do not contribute a large background in the transduced population, which is therefore markedly enriched for large hybrid plasmids (over 90%). The efficiency of the in vitro packaging system is on the order of 10(5) hybrid clones per microgram of foreign DNA for hybrids in the 20--30 million dalton range.
A new procedure for automated Sanger sequencing with internal fluorescent labelling of DNA is described. In a primer extension/labeling step dTTP is substituted by fluorescein-12-dUTP, which is continuously incorporated during chain elongation by T7 DNA polymerase. Compared to the end-labeling protocols, DNA sequencing with internal label results in stronger peak intensities due to the multiple labeling of the fragments, allowing sequencing of small amounts of DNA. A further advantage of this procedure is the use of standard unlabeled primers, increasing the speed of the sequencing method and reducing the cost, particularly for the 'walking primer' strategy. Mobility shifts of the bands in the sequence pattern which could result from multiple internal fluorescent labeling were not observed. The observed sequence reading length on single stranded templates and plasmids is 400-500 bases and appears to be limited only by the resolution of the gel.
DNA consists of a continuous chain of deoxyribose units linked via phosphodiester bonds at their 5′ and 3′ positions and joined to one of the four nucleotide bases, Thymine (T), cytosine (C), the pyrimidines, guanine (G), or adenine (A), the purines, at their 1′ positions. It exists in organisms in the form of very long chains (5,000 base-pairs long, in the case of bacteriophage øX174, to 240,000,000 bp long, in the case of the largest human chromosome). Thus, in order to determine the sequence of a DNA molecule, the order of nucleotide bases along its sugar-phosphate backbone, it is first necessary to fragment the molecule into segments of a manageable size and to purify each fragment type. This is most readily accomplished by cloning into a plasmid or viral DNA vector. After amplification of the resulting hybrid DNA molecule, the cloned DNA segments (inserts) can be released for sequencing by cleavage with a restriction endonuclease.
The standard protocol for purification of M13 single-stranded templates, the most critical step for efficient DNA sequencing, is tedious and prone to errors and cannot be easily automated. A robot, with a few parts added, was programmed to automate the preparation and purification of the sequencing templates according to a new method developed in our laboratory. The automated simple and rapid procedure, the first reported in the literature, does not involve phenol extractions and alcohol precipitations. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. The yield is comparable to or higher than that obtained by standard methods. The system is flexible and allows variations and optimization of the procedure. In one cycle (about 30 minutes) 24 templates are processed. The method is applicable to fluorescent and radioactive labeling, and the sequence resolution is as good as that obtained by the standard procedure.
Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5â² region of human x-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.
At the ends of bacteriophage λ DNA, the 5′-terminated strands are 12 nucleotides longer than the 3′-terminated strands. The complete sequence of deoxynucleotides in both the protruding 5′-terminated single strands of λ DNA has been determined by partial repair and by complete repair followed by sequencing of isolated oligonucleotides. Starting from the 5′-end of the left-hand cohesive end, the 12 nucleotides are in the sequence dpGpGpGpCpGpGpCpGpApCpCpT. The sequence from the right-hand cohesive end is exactly complementary to that from the left-hand end.
A simple technique and device for the preparation of gels are described that complement the conventional “pouring” technique, which fails in the preparation of bubble-free gels with thicknesses less than about 0.4 mm. Included are methods for processing and manipulation, design of a thermostating platen and gel casting device, and chemical treatment of glass and polyester substrates to which the polyacrylamide gel adheres during processing. The gel is cast directly between the glass cover plate and the thermostating platen, and this assembly provides isothermal conditions in the gel layer during the electrophoresis. Further, techniques for introducing spacers, stacking gel, sample slot forming and sample application have been developed and applied to DNA sequencing and protein sodium dodecyl sulphate electrophoresis. The results obtained indicate that the technique is simple to use, has many analytical applications (DNA sequencing, sodium dodecyl sulphatepolyacrylamide gel electrophoresis, isoelectric focusing) and has various advantages.
The construction and evaluation of a novel type of chemical sequencing vectors (pCSV) is described. These small plasmids bear a unique asymmetric restriction site suitable for direct single-end labeling by filling-in polymerization with a selected radiolabeled nucleoside triphosphate. Thus, a secondary cleavage reaction or segregation step is rendered superfluous. Sequencing gels can be read unambiguously starting from the 3′ penultimate nucleotide. pCSV03 and pCSV27 are prototypes of such sequencing plasmids based on BstEII as the labeling site. In its proximity, restriction sites are present that allow subcloning of the DNA fragments to be sequenced. Approaches to random and progressive sequencing using these vectors are discussed. pCSV31 has two Tth111I sites that allow single-end labeling of inserts on either strand by the use of different labeled nucleoside triphosphates. Thus, bidirectional sequencing of larger inserts (>500 bp) is possible.
Akiyoshi Wada describes a system that takes the grind out of DNA sequencing
The direct sequencing of DNA generated by the polynucleotide chain reaction, via the incorporation of phosphorothioate nucleotides
and followed by treatment with an alkylating reagent that cleaves specifically at the phosphorothioate positions, is described.
The Taq polymerase used in the amplification reaction incorporates the Sp-diastereomer of the deoxynucleoside 5'-0-(1-thiotriphosphates)
as efficiently as the natural nucleotides. Chemical degradation of the phosphorothioate-containing DNA fragment can be performed
with either 2-iodoethanol or 2,3-epoxy-1-propanol. The higher reactivity of 2,3-epoxy-1-propanol allows less reagent to be
used to obtain the same amount of degradation as with 2-iodoethanol.
Nature and stability of secondary structures formed from DNA-fragments during electrophoresis in urea containing polyacrylamide-gels
were investigated. Duplices and especially hairpin loops were found to be surprisingly stable. Polyacrylamide-gels containing
high concentrations of formamide effectively melt these structures. Such gels will improve nucleotide sequence analysis in
regions of dyad symmetry that often cause ‘band compressions’ under standard electrophoresis conditions.
We have developed a rapid method for sequencing supercoiled plasmid DNA and bacteriophage λ DNA. The plasmid DNA is obtained from a 5-ml overnight culture using a rapid alkaline extraction method. The double-stranded lambda DNA is extracted from 0.75 ml of a standard liquid phage lysate using a modification of the diethylaminoethyl cellulose method. A synthetic oligonucleotide primer (12–18 bases) is annealed to the DNA, and the DNA is rapidly sequenced using the Sanger dideoxy chain-termination technique and reverse transcriptase. The advantages of these methods are 1) no cloning of the DNA is necessary, 2) one can rapidly and easily extract DNA, sequence it, and obtain the sequence in one day, and 3) both strands of the DNA can be sequenced from one molecule.
We have developed an automated DNA sequenator using a real-time fluorescence detection method, and achieved excellent sensitivity and rapid analysis by optimizing operating conditions. The sequenator uses unicolor labeling and four-track electrophoresis. Fluoresceine isothiocyanate(FITC)-labeled primers are used to produce labeled DNA families. The fluorescent bands are excited by an Ar laser (488 nm, 10 mW) introduced from the side of a thin gel during electrophoresis. Analysis, with an accuracy greater than 99%, consumes 0.01–0.04 picomole sample for each DNA family, which is almost comparable to the amount used in conventional autoradiography. With the optimized electrophoresis conditions reported here, four hundred bases of DNA can be analyzed within two and a half hours using a four percent polyacrylamide gel.
We describe the construction of a laboratory workstation for the micromanipulation of liquids. The performance of the workstation is demonstrated by its use in a DNA sequencing protocol; although its operational units form the backbone of a robotic system with wide applications in molecular biology and biochemistry.
DNA methylation is believed to be involved in the control of gene expression1,2. In higher plants, up to 30% of the cytosine residues can be methylated—at C-X-G trinucleotides as well as C-G dinucleotides—compared with only 2–8% of total cytosines appearing exclusively as 5-methyl-C-5-methylcytosine in higher animals1–4. We have used the genomic sequencing technique of Church and Gilbert5 to study the methylation of an alcohol dehydrogenase gene, Adhl, from maize. Adhl is one of two unlinked maize alcohol dehydrogenase genes6,7 and appears in several tissues, including the scutellum of the kernel and the primary root of the seedling. It is further induced in primary roots subjected to anaerobiosis8–10. However, this gene is totally repressed in maize leaves11. Nonetheless, we have now found that a 900-base-pair (bp) region 5′ to the ATG is not methylated in leaves, even though it is rich in potential methylation sites. The first methylations occur 965 bp 5′ to the ATG. Thus, methylation does not appear to be involved in repressing this plant gene.
To improve the technique for DNA sequencing, large numbers of stable isotopes could be used to label DNA, thereby multiplexing the separation process and providing a new and much faster procedure for localizing DNA after electrophoresis. Sputter-initiated resonance ionization spectroscopy (SIRIS) was used to demonstrate detection of subattomole quantities of isotopically enriched iron- and tin-labeled DNA with excellent lateral and mass resolution. The organometallic compounds, ferrocenecarboxylic acid and (triethylstannyl)-alkanecarboxylic acid, were synthesized and attached through the amine group of a 5'-hexylamine on the terminal position of an oligonucleotide. The adaptation of SIRIS for detecting samples containing 50 pmols of either iron or tin attached to DNA is described. Using high repetition rate lasers in SIRIS, it should eventually be possible to detect more than 10(7) bases per day if other preparation steps in the procedure do not become rate limiting. The results of analysis of iron- and tin-labeled DNA on polyacrylamide gels and Nylon membrane indicate that SIRIS has the potential to make a strong contribution to DNA sequencing and also for other methods that requires detection of the location and amount of DNA or an oligonucleotide that hybridizes to DNA.
The determination of the complete nucleotide sequence of large DNA molecules is a tedious and time-consuming process. The repetitive nature of the task makes it an ideal subject for automation. We report here a system designed and constructed to perform the chain termination reactions of the Sanger sequencing procedure automatically. It is capable of carrying out these reactions for from five to twelve DNA templates at one time. The system takes 12 minutes to dispense 12 templates, add the reagents and mix them; the complete cycle time, including incubations, is 45 minutes.
A direct sequencing approach has been used to analyze the polymorphism in the human apolipoprotein E gene. A method is described, in which the DNA is amplified by the polymerase chain reaction, immobilized, and sequenced by a semi-automatic procedure adaptable to clinical diagnosis. The three alleles of the apolipoprotein E gene, which differ from each other by two nucleotide substitutions and which influence serum cholesterol levels, were analyzed. The solid-phase method was able to resolve the correct nucleotide sequence in samples from both homozygous and heterozygous individuals. No cloning steps are needed and the immobilization and separation of the DNA is accomplished using magnetic beads.
The complete sequence of 57 kb of the human HPRT locus has been determined using automated fluorescent DNA sequencing. The strategy employed increasingly directed sequencing methods: A randomly generated M13 library was sequenced to generate contigous overlapping sets of sequences (contigs). M13 clones at the ends of these contigs were further sequenced using M13 (universal and reverse) and custom oligonucleotide primers to order the contigs and to complete the sequencing project. The human HPRT sequence includes 1676 bp 5′ and 15,238 bp 3′ to exons 1 and 9, respectively. The sequence contains 49 representatives of the Alu repeat, along with several other types of repetitive sequences. The Alu sequences exhibit a biased orientation, with those sequences in the first half of the locus oriented in the minus direction relative to transcription of the gene (3′ → 5′ = 77%, P < 0.005) and those sequences in the latter half of the locus oriented randomly (5′ → 3′ = 67%, P < 0.5). The development and performance of the sequencing strategy and the features of the human HPRT gene are presented.
A method is described for synthesis of a tin reagent, triethylstannylpropanoic acid (TESDA), and its attachment to oligonucleotide primers. Except for the expected mobility retardation, the presence of [116Sn]-TESDA did not affect the sequencing ladder on electrophoresis gels. By using [120Sn]-TESDA and [35S]-dTTP simultaneously in the Sanger procedure, DNA bands on an electrophoresis gel were first located by autoradiography and then by resonance ionization spectroscopy to demonstrate the coincidence of the signals. Previous results using stable isotopes as labels on model compounds are now confirmed by their use in actual DNA sequencing products.
The sequencing of DNA by current procedures involves the use of radioisotopic or fluorescent labels. We propose that stable isotopes can be used as such labels and that the large number of stable isotopes available would allow multiplexing so that many DNA segments could be sequenced simultaneously. We have developed methods to use 57Fe2O3 to synthesize ferrocene and to attach the ferrocene to the 5′ end of oligonucleotides. The 57Fe-labeled M13 universal primer functioned normally in a Sanger sequencing procedure. When a 57Fe-labeled oligonucleotide had migrated on a polyacrylamide gel it was readily located on the dried gel by scanning with resonance ionization spectroscopy (RIS) coupled with mass spectrometry. Using a 57Fe-labeled primer in a PCR reaction a 2000-bp DNA was produced that was detected by RIS on nylon membrane after agarose electrophoresis. The rapid analysis features of RIS coupled with the multispectral multiplexing possibilities of stable isotopes should significantly increase the rate of determination of DNA sequences.
A solid-phase method for simultaneous sequencing often or more long DNA fragments has been developed, using as support the cellulose matrix for chemical sequencing (CCS), anion-exchange paper [Rosenthal et al., Nucl. Acids Res. 13 (1985) 1173–1184]. We optimized several of the seven steps which include: (i) immobilization; (ii) washing; (iii) modification; (iv) washing; (v) sorting of the paper segments; (vi) piperidine reaction and chemical elution, and (vii) lyophilization. During carrier-supported chemical cleavage with dimethylsulfate (DMS) (G), HCOOH (A + G), KMnO4 (T > Pu) and NH2OH (C), losses of immobilized DNA are very low. DNA fragments ranging in length from several hundred bp up to 6 kb can be effectively chemically eluted from CCS paper during the piperidine reaction with an efficiency of more than 90%. Because no DNA salt elution and ethanol precipitation steps are necessary the method is rapid, convenient and allows complete automation.
Nucleotide sequencing by the chemical degradation method requires the labeling of a single nucleotide at the 3′ or 5′ terminus of the fragment to be sequenced. In the present report, I described the construction of two new sequencing plasmids, pSP64CS and pSP65CS, which are ideally suited for use when sequencing a nested set of deletion fragments by the chemical degradation method. Plasmids pSP64CS and pSP65CS contain the sequencing element derived from pGV403 required for specific labeling of individual 3′ ends, and the polylinker site derived from pSP64 or pSP65, respectively. The plasmids also retain the SP6 promoter element which allows for the efficient in vitro synthesis of complementary RNA. Any DNA fragment having a blunt end and a second end which is any of the following restriction sites, HindIII, PstI, SalI, AccI, XbaI, BamHI, SacI, BstEII or EcoRI, can be directionally sequenced in these plasmids. Fragments having two blunt ends can also be sequenced. pSP64CS and pSP65CS differ only in the orientation of the polylinker/sequencing element relative to the SP6 promoter.
A simple procedure has been developed for sequencing long fragments of DNA. The fragment (which can be several kb in length) is cloned in pAA3.7X, and subdivided into many overlapping segments by TnP-promoted deletions. The deletions are isolated by positive selection for galactose resistance. A rapid plasmid preparation from several hundred galactose-resistant colonies is fractionated by agarose gel electrophoresis to pick a series of deletions terminating at approx. 200-bp intervals across the entire length of the fragment. Selected plasmids are purified by rapid alkaline extraction, and used directly for supercoil sequencing with a primer derived from IS1. Sequences of adjacent deletions contain overlaps which are used to connect individual sequences to give the complete sequence.
An improved rapid method for sequencing a target DNA is described. A new plasmid, pAA-PZ1, which contains the origin of replication from phage M13 and a portion of the Tn9 transposon was constructed. A long fragment of target DNA cloned into this vector is progressively shortened in vivo from one end by transposon-mediated deletions. The plasmids carrying different lengths of target DNA are then made into single-stranded DNA in the same host upon infection with an M13 phage and their sequence is determined using the dideoxynucleotide chain-termination method. This method bypasses the in vitro enzymatic manipula- tions for progressive deletions and requires no subcloning. Using this strategy, we sequenced 1.3 kb of rice DNA containing a histone 3 gene within three weeks.
The polymerase chain reaction (PCR) catalyzed by Taq DNA polymerase has been used to amplify genomic and plasmid DNA sequences. In all reported cases, the template has been extensively purified. We show here that genomic and plasmid DNA sequences can be amplified by the PCR directly from single colonies of both Escherichia coli and Bacillus subtilis without any purification of the template. This technique makes the use of the PCR even more applicable in the automation processes, screening and diagnosis.
A systematic DNA sequencing strategy is presented. Instead of the sequencing of randomly selected DNA fragments (the “shotgun” method), the nucleotide order is progressively determined along a DNA chain using the dideoxynucleotide termination sequencing system and the single-stranded bacteriophage vector M13 derivatives. The length of DNA along which the sequence data can be progressively read appears to be limited only by the insertion capacity of the vector. As an example of this strategy a recombinant replicative form with a 2327 nucleotide long HindIII fragment from the restriction enzyme digest of bacteriophage λ DNA was prepared. The replicative form of the recombinant was partially digested with DNAase I in the presence of Mn2+. As the replication origin of the phage vector was located near one end of the inserted DNA and the priming site of the vector at the other, the breaks outside the inserted DNA either destroyed the phage or removed the priming site. With the use of a unique restriction site close to the priming site, the breaks within the inserted DNA gave rise to a recombinant mixture with the inserted DNA fixed at one end and sequentially shortened at the other. Using the ddT reaction screening procedure, 11 recombinants were identified in which the inserted DNA varied in length by about 200 nucleotides. Sequencing these recombinants by the dideoxynucleotide sequencing system covered the whole 2327 nucleotides of the HindIII DNA fragment. The average number of nucleotides read from a gel was 210, which fell into the most readable region of a sequencing gel: the overlapping regions between two gels were of 33 to 48 nucleotides.
The strategy of shotgun cloning with M13 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project. In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for M13 shotgun sequencing. Two new M13 vectors, M13mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (+) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M13mp8 and M13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.
We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.
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.
The restriction fragment Hind H contains 5·2% of the genome of simian virus 40 (SV40) and is located near the middle of the early region. It can be split by the Arthrobacter luteus (Alu) enzyme into fragments Hind H-A1 and Hind H-A2. The nucleotide sequence of fragment Hind H is reported here. It has been established by analysis of transcription products, synthesized by Escherichia coli RNA polymerase and nucleoside triphosphates, one of which was (α-32P)-labeled. These products are very heterogeneous in size and may even exceed the length of the template. Strand assignment was possible by hybridizing the asymmetric, labeled transcript of total SV40 DNA to filter-bound Hind H DNA. Very clean, discrete products were obtained under appropriate conditions where transcription was dependent on an added primer such as (Ap)5A. These transcripts were derived from one strand only, except in the case of Hind H-A2, where the product (in a single chain) contained information derived from both strands. Unambiguous confirmation of the sequence was obtained by experiments directly on the terminally labeled DNA fragments.
The message strand is particularly Ap-rich (37%) and purine rich. The dinucleotide CpG occurs only once but UpC is also rare. The nucleotide sequence can be translated unambiguously into an amino acid sequence. This polypeptide, which is part of the gene A-protein, is neutral, rich in methionine, cysteine and tyrosine, and has a high lysine to arginine ratio. All serine codons are of the A-G-Py type; A and U are clearly preferred as third bases.
Exposure of DNA to methylene blue and visible or ultraviolet light causes guanine-specific modification, and subsequent treatment
with piperidine leads to chain cleavage at each guanine residue. Treatment of DNA with osmium tetraoxide in dilute pyridine
leads to thymidine-specific modification, and subsequent treatment with piperidine leads to chain cleavage at the modified
thymidine residues. Both reactions can be used in conjunction with other base specific modifications described by Maxam and
Gilbert (1) for the determination of the nucleotide sequence in DNA.
The determination of the total 5,224 base-pair DNA sequence of the virus SV40 has enabled us to locate precisely the known genes on the genome. At least 15.2% of the genome is presumably not translated into polypeptides. Particular points of interest revealed by the complete sequence are the initiation of the early t and T antigens at the same position and the fact that the T antigen is coded by two non-contiguous regions of the genome; the T antigen mRNA is spliced in the coding region. In the late region the gene for the major protein VP1 overlaps those for proteins VP2 and VP3 over 122 nucleotides but is read in a different frame. The almost complete amino acid sequences of the two early proteins as well as those of the late proteins have been deduced from the nucleotide sequence. The mRNAs for the latter three proteins are presumably spliced out of a common primary RNA transcript. The use of degenerate codons is decidedly non-random, but is similar for the early and late regions. Codons of the type NUC, NCG and CGN are absent or very rare.
Comparison of the human mitochrondial DNA sequence of the cytochrome oxidase subunit II gene and the sequence of the corresponding beef heart protein shows that UGA is used as a tryptophan codon and not as a termination codon and suggests that AUA may be a methionine and not an isoleucine codon. The cytochrome oxidase II gene is contiguous at its 5' end with a tRNAAsp gene and there are only 25 bases at its 3' end before a tRNALys gene. These tRNA'S are different from all other known tRNA sequences.
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.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
A HindII restriction fragment comprising the Escherichia coli lac regulatory region and the genetic information for the alpha peptide of beta-galactosidase (beta-D-galactosidegalactohydrolase, EC. 3.2.1.23) has been inserted into 1 of the 10 Bsu I cleavage sites of M13 by blunt end ligation. A stable hybrid phage was isolated and identified by its ability to complement the lac alpha function. Further characterization of the hybrid phage includes retransformation studies, agarose gel electrophoresis, DNA-DNA hybridization, and heteroduplex mapping. The insertion point has been localized at 0.083 map unit on thewild-type circular map-i.e., within the intergenic region. The results prove that part of the intergenic region is nonessential and that the phage can be used as a cloning vehicle.