A method for determining DNA sequence by labeling the end of the molecule and cleaving at the base. Isolation of DNA fragments, end-labeling, cleavage, electrophoresis in polyacrylamide gel and analysis of results

We elaborate basic chemical principles and current laboratory procedures for sequencing end-labeled DNA by partial cleavage and gel electrophoresis (A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA, 1977, v. 74, p. 560-564). We provide step-by-step protocols for 32P-labeling DNA ends, segregating the labeled ends by cutting with a second restriction enzyme or separating strands, partially cleaving the DNA at specific bases with reagents, electrophoresing the labeled products of cleavage on sequencing gels, and interpreting sequencing band patterns. Many of these procedures have been condensed, to make them faster and easier, and some are new. We also discuss sequencing strategies, and suggest a technique which will reduce plasmid or viral DNA to a collection of singly-end-labeled fragments in one day, for efficient sequencing of these chromosomes in 250-nucleotide blocks.     

Mechanism of DNA strand breakage by piperidine at sites of N7-alkylguanines

The volatile, secondary amine piperidine is used in the Maxam-Gilbert chemical method of DNA sequencing to create strand breaks in DNA at sites of damaged bases. As such it is often used in generalized studies of DNA damage to identify 'alkali-labile lesions'. We confirm the mechanism proposed by Maxam and Gilbert (Maxam, A. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) by which aqueous piperidine creates strand breaks at sites of N7-guanine alkylations: alkaline conditions catalyze rupture of the C8-N9 bond, forming a formamido-pyrimidine structure which is displaced from the ribose moiety by piperidine. In keeping with this mechanism, the tertiary amine, N-methylpiperidine, does not catalyze the formation of strand breaks in alkylated DNA. Our data confirm the prediction that high pH in and of itself will not create strand breaks at sites of N7-alkylguanines.     

A solid phase method of determining DNA sequence

A solid-phase method for DNA sequencing has been developed which involves immobilization of the terminally labeled DNA fragment on the DEAE-paper followed by chemical modification and cleavage at G, A + G, C + T, and C sites. As compared to the Maxam and Gilbert method, the new technique is more rapid and less laborious, being of the same efficiency.     

Purification to homogeneity and characterization of a redoxyendonuclease from calf thymus.

A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0-8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono-S, Mono-Q and Mono-P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560] the purified enzyme was found to incise ultraviolet-light-irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N-terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins.     

An adaptation of the maxam and gilbert sequencing method to the study of human metaphase chromosomes

We have adapted the chemical DNA sequencing method developed by Maxam and Gilbert to study human metaphase chromosomes. Particularly, the in situ cytosine-specific chemical cleavage has been assayed. After exhaustive treatments, a C-like banding pattern was consistently shown. Since methyl groups interfere with the cytosine-specific chemical cleavage reaction, we assume that heterochromatin remains resistant as a result of the high level of methylation. On the other hand when moderate treatments are applied, T-bands remain as resistant regions, implying that these domains would be the most intensely methylated regions within euchromatin.     

Pyrimidine-specific chemical reactions useful for DNA sequencing.

Potassium permanganate reacts selectively with thymidine residues in DNA (1) while hydroxylamine hydrochloride at pH 6 specifically attacks cytosine (2). We have adopted these reactions for use with the chemical sequencing method developed by Maxam and Gilbert (3).     

Nucleotide sequence studies of polyoma DNA. The Hpa II 3/5 junction to the Hpa II 4/Hae III 18 junction, encoding the origin of DNA replication and the 5' end of the early region.

The nucleotide sequence of polyoma DNA, from near the Hpa II 3/5 unction to the Hpa II 4/ae III 18 junction has been determined by the chemical method of Maxam and Gilbert (Maxam, A., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560--564). The sequence contains 878 base paris, including the origin of DNA replication and the region known to encode the hr-t function. The region corresponding to the origin of DNA replication contains several short-repeated sequences and palindromes. There is a 30-base-pair region with striking similarity to the corresponding region of SV40, and, as in SV40, a portion of that sequence is capable of forming a stable hairpin loop. In the region encoding the hr-t function, there is apparently a single open reading frame extending from position 188 to theHpa III 4/Hae III 18 junction. The potential translation product of this open frame begins with an initiation codon starting at position 188, and the first five amino acids of this product are Met-Asp-Arg-Val-Leu. This sequence is similar to the NH2-terminal five amino acids of SV40 small t-antigen known from nucleotide and amino acid sequencing to be Met-Asp-Lys-Val-Leu.     

pUR222, a vector for cloning and rapid chemical sequencing of 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 white 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 directly according to the sequencing protocol of Maxam and Gilbert.     

DNA sequences of the integration sites and inverted repeated structure of transposon Tn3.

The nucleotide sequence of the "inverted repeat" structure of the transposon Tn3 was determined by the DNA sequencing procedure developed by Maxam and Gilbert(1). The sequence, 38 base pairs long, is as follows: 5'-GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG..(Tn3) 3'-CCCCAGACTGCGAGTCACCTTGCTTTTGAGTGCAATTC.. The integration of Tn3 is associated with a directly repeated sequence of 5 nucleotides appearing at each end of Tn3. The two directly repeated sequences so far determined are not the same. Furthermore, there is no homologous structure around the integration point of Tn3.     

Alteration in DNA cross-linking and sequence selectivity of a series of aziridinylbenzoquinones after enzymatic reduction by DT-diaphorase.

DT-diaphorase (DTD) mediated reduction of a series of 2,5-bis-substituted-3,6-diaziridinyl-1,4-benzoquinones was found to increase the level of DNA interstrand cross-linking (ISC) formed at neutral pH with an enhancement observed as the pH was decreased to 5.8. The analogues used were symmetrically alkyl-substituted carbamoyl ester analogues of AZQ (D1-D7), 3,6-diaziridinyl-1,4-benzoquinone (DZQ), the 2,5-dimethyl derivative (MeDZQ), and a 2,5-bis[(2-hydroxyethyl)amino] analogue (BZQ). At pH 5.8, the level of DNA ISC induced by enzymatic reduction was as follows: DZQ greater than MeDZQ much greater than D1 (methyl) greater than D3 (n-propyl) greater than D2 (AZQ; ethyl) greater than D5 (n-butyl) greater than D7 (sec-butyl) greater than D4 (isopropyl) D6 greater than (isobutyl). A similar trend was observed at pH 7.2. The level of DNA ISC induced by BZQ, which is not a substrate for DTD, was not increased by enzymatic reduction. Dicumarol, a known inhibitor of DTD, was capable of inhibiting the DNA ISC induced by these quinones upon enzymatic reduction. MeDZQ and DZQ reacted with guanines, as measured by Maxam and Gilbert sequencing, with a sequence selectivity similar to that of the nitrogen mustard class of antitumor agents. Enzymatic reduction of DZQ and MeDZQ by DTD was found to alter their sequence-selective alkylation. Reduced DZQ showed enhanced guanine alkylation in 5'-GC-3' sequences and new sites of adenine alkylation in 5'-(A/T)AA-3' sequences. Reduced MeDZQ only showed new sites of adenine alkylation at 5'-(A/T)AA-3' sequences but no enhancement of guanine alkylation. The new sites of adenine alkylation were found to be inhibited in the presence of magnesium and rapidly converted into apurinic sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nucleotide sequence of the right-hand terminus of adenovirus type 5 DNA: implications for the mechanism of DNA replication.

The nucleotide sequence of the right-hand terminal 3% of adenovirus type 5 (Ad5) DNA has been determined, using the chemical degradation technique developed by Maxam and Gilbert (1977). This region of the genome comprises the 1003 basepair long HindIII-I fragment and the first 75 nucleotides of the adjacent HindIII-F fragment, extending from the right-hand terminus to the sequences from which the main body of the mRNA of early region 4 is transcribed. One of the origins of adenovirus DNA replication is located within this part of the genome. The sequencing results are discussed in relation to several models proposed for the mechanism of replication of linear DNA molecules, which invariably depend on the presence of specific arrangements of nucleotides at the termini of those linear DNAs.     

A review of DNA sequencing techniques

The four best known DNA sequencing techniques are reviewed. Important practical issues covered are read-length, speed, accuracy, throughput, cost, as well as the automation of sample handling and preparation. The methods reviewed are: (i) the Sanger method and its most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other chemical methods; (iii) the Pyrosequencing method--DNA sequencing in real time by the detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with exonuclease (exonuclease digestion of a single molecule composed of a single strand of fluorescently labelled deoxynucleotides). Each method is briefly described, the current literature is covered, advantages, disadvantages, and the most suitable applications of each method are discussed.     

Nearest neighbor effects on carcinogen binding to guanine runs in DNA.

A synthetic DNA fragment was constructed to determine the effect of 5' and 3' neighbors of guanine runs on the binding of chemical carcinogens. Determinations were made on the relative intensity of reactivity between aflatoxin B1 or benzo(a)pyrene and methylnitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with various guanine positions in an endlabeled DNA fragment of known sequence. After reaction, the fragments were depurinated to produce strand breaks to allow Maxam and Gilbert sequencing for guanine positions. Relative reaction intensities were compared densitometrically. 3' neighbors exerted greater influence on carcinogen binding than did 5' neighbors, the influence extended only to the adjacent guanine and depended upon the chemical nature of the carcinogen. In addition, the presence of one carcinogen adduct in the guanine run influenced the formation of a subsequent adduct when the DNA was exposed to a second carcinogen, and this effect also depended on the nature of the second carcinogen. The results suggest that DNA adduct formation in the presence of multiple carcinogens is more complicated than an additive mechanism would suggest.     

Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus.

A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to immunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA.     

Use of solubilizable acrylamide disulfide gels for isolation of DNA fragments suitable for sequence analysis

Acrylamide gels which are crosslinked by disulfide bridges are found to be an excellent electrophoretic medium for DNA restriction fragments over a wide size range. The gels are solubilized by mercaptoethanol and the fragments are rapidly and conveniently recovered on DEAE-cellulose columns. Fragment recovery appears nearly quantitative. The gel formulation described here requires polymerization at a temperature of about 40°C. Recovered fragments are active in all systems tested, including subsequent restriction, kinasing, ligation, nick translation, and sequencing by the method of A. M. Maxam and W. Gilbert (1980, in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 499–560, Academic Press, New York).     

Direct DNA sequencing of PCR amplified genomic DNA by the Maxam-Gilbert method

We present a method to directly sequence PCR amplification products utilizing the chemical method of Maxam and Gilbert. This procedure yields clearly readable DNA sequences of 100-400 base pairs in length derived from human genomic DNA in four days' time.     

Synthesis of a DNA fragment containing dihydro-5,6 thymine

The preparation of a pentadecanucleoside tetradecaphosphate containing the modified base 5,6-dihydrothymine (DHT) is reported herein. The synthesis was performed with a mixture of diastereoisomers (5R and 5S), obtained by catalytic hydrogenation of thymidine. The phosphorylated protected monomer was characterized by proton NMR and FAB mass spectrometry. It was introduced into the DNA fragment (poly dT) by the solid phase phosphotriester approach. After deprotection, the determination of the site of the modified base in the chain was made using different methods. The classical chemical sequencing method of Maxam and Gilbert showed no difference with respect to thymine moieties in the pentadecamer. To characterize the presence and the location of the modified base DHT, the 32P 5'-end labelled pentadecamer was heated in formic acid at 90 degrees C and length separated by polyacrylamide gel electrophoresis. The Electronic Impact mass spectrometry under pyrolytic conditions of the 15-mer showed that the modified base was present in the deprotected DNA fragment.     

Radiation-induced DNA damage and its repair

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes. A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment 32P-labelled at one end, it has been shown that the alkali-labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.