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.     

PCR and DNA sequencing

Specific DNA segments defined by the sequence of two oligonucleotides can be enzymatically amplified up to a millionfold using the polymerase chain reaction (PCR). One of the most significant uses of this technique is for generation of sequencing templates, either from cloned inserts or directly from genomic DNA. To avoid the problem of reassociation of the linear DNA strands in the sequencing reaction, ssDNA templates can be produced directly in the PCR or generated directly from dsDNA by enzymatic treatment, electrophoretic separation or affinity purification. By combining PCR with direct sequencing, both the amplification and the sequencing reaction can be performed in the same vial. Finally, use of fluorescently labeled terminators or sequencing primers will allow the whole procedure to be amenable to complete automation.     

Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA.

A specific segment of mitochondrial DNA from 18 people was examined by two methods of direct DNA sequencing. This segment includes a small noncoding region (V) shown before by restriction analysis to exhibit length polymorphism. All 11 of the human mtDNAs previously reported to have a deletion in this region proved to lack one of the two adjacent copies of a 9-base-pair sequence normally present in human mtDNAs. Phylogenetic analysis suggests that this deletion occurred only once during the evolution of modern types of human mtDNA and that it will be a valuable anthropological marker for peoples of East Asian origin. The one human mtDNA reported to have an addition in region V differs from the wild type by two mutations in the first copy of the 9-base-pair sequence: one transition and an addition of four cytosines, thereby producing a run of 11 cytosines. One of the direct DNA sequencing methods uses a single oligonucleotide primer to facilitate dideoxy sequencing from purified mtDNA templates. The second, more successful, method first amplifies this mtDNA segment enzymatically with two flanking primers (the "polymerase chain reaction") and then uses a third primer for DNA sequencing. This latter method, which works on the DNA extracted from small amounts of blood as well as on purified mtDNA, is shown to be a rapid means of defining sequence variants without purifying and cloning the same DNA segment from many individuals.     

Interaction of terminal transferase with single-stranded DNA

A 58-kDa monomer of terminal transferase was isolated from calf thymus using a monoclonal antibody affinity column. The enzymatic activity was comparable to that of the 44-kDa alpha beta dimer isolated by conventional methods. Binding of the two enzyme forms to single-stranded DNA was monitored by fluorescence. The site size of both forms was approximately 11 +/- 2 nucleotides. Binding of the 44-kDa alpha beta dimer to polydeoxyadenosine was examined under several conditions. The cooperativity parameter increased from about 90 in the presence of Mg2+ to 300-400 in the absence of Mg2+. The observed dissociation constant of 3-5 microM was essentially independent of salt concentration, whereas the intrinsic dissociation constant decreased about 5-fold in the presence of Mg2+. The binding parameters of the 58-kDa monomer were independent of buffer composition and were similar to those of the 44-kDa alpha beta dimer in the presence of Mg2+. These results indicate that the additional 14-kDa peptide sequences present in the high molecular mass monomer form are not part of the DNA-binding site of terminal transferase.     

DNA containing the base analogue 2-aminoadenine: preparation, use as hybridization probes and cleavage by restriction endonucleases.

The base analogue 2-aminoadenine (2,6-diaminopurine, D) has been introduced at selected positions into synthetic oligodeoxyribonucleotides and DNA by the combined use of chemical and enzymatic methods. 2-aminoadenine substitution for adenine introduces changes in the minor groove of DNA and creates an additional hydrogen bond in the Watson-Crick base pair with thymine. Oligonucleotide hybridization probes containing 2-aminoadenine showed increased selectivity and hybridization strength during DNA-DNA hybridization to phage or genomic target DNA. Properties of the base analogue with respect to DNA modifying enzymes were examined. 2-aminoadenine was used to probe minor groove determinants during the treatment of DNA by 12 restriction endonucleases. Inhibition of cleavage was found for several restriction enzymes.     

Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue

This article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tC^O, is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tC^O is thoroughly characterized with respect to its photophysical properties and structural performance in single- and double-stranded oligonucleotides. The lowest energy absorption band at 360 nm (ε = 9000 M⁻¹ cm⁻¹) is dominated by a single in-plane polarized electronic transition and the fluorescence, centred at 465 nm, has a quantum yield of 0.3. When incorporated into double-stranded DNA, tC^O shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tC^O, on average, the brightest DNA-incorporated base analogue so far reported. Furthermore, it base pairs exclusively with guanine and causes minimal perturbations to the native structure of DNA. These properties make tC^O a promising base analogue that is perfectly suited for e.g. photophysical studies of DNA interacting with macromolecules (proteins) or for determining size and shape of DNA tertiary structures using techniques such as fluorescence anisotropy and fluorescence resonance energy transfer (FRET).     

Non-radioactive chemical sequencing of biotin labelled DNA.

Methods for the nonradioactive chemical sequencing of DNA are described. A biotin marker molecule, attached chemically to an oligonucleotide primer or enzymatically in an endfilling reaction of restriction enzyme sites, is stable during the base-specific chemical modification and strand scission reactions. Following fragment separation by direct blotting electrophoresis, the membrane bound sequence pattern can be visualized by a streptavidin-bridged enzymatic color reaction. The biotin labeling is also applicable for DNA sequencing by random degradation of phosphothioates, thus showing to be a universal label for nonradioactive DNA sequencing.     

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).     

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg²⁺ or Mn²⁺ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.     

The linear PCR reaction: a simple and robust method for sequencing amplified rRNA genes

The linear polymerase chain reaction was used to sequence amplified RNA genes from strains of Bacillus, Thermus and Legionella. The technique described is simple and reproducible and it works well with double standard product which has been PEG precipitated directly from PCR reactions.     

Initiator role of double stranded DNA in terminal transferase catalyzed polymerization reactions.

Binding of the 58 kDa monomer and 44 kDa alpha beta dimer forms of terminal deoxynucleotidyl transferase to double stranded DNA was demonstrated by gel retardation and tryptophan fluorescence quenching. The dissociation constants and cooperativity parameters were similar to those that have been determined for binding of these two forms of terminal transferase to single stranded DNA. However, the double stranded DNA binding site size of 10 nucleotides was half the size expected. The efficacy of blunt ended DNA as an initiator in the polymerization reaction catalyzed by terminal transferase was demonstrated by radiometric assays and product analyses on agarose gels. The initial reaction kinetics indicated that dGTP but not dATP was added efficiently to a blunt double stranded DNA 3' end. These results are correlated with current models for in vivo terminal transferase function.     

Strand annealing and terminal transferase activities of a B-family DNA polymerase

DNA replication polymerases have the inherent ability to faithfully and rapidly copy a DNA template according to precise Watson-Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in the hyperthermophilic archaeon, Sulfolobus solfataricus, is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, Dpo1 also displays a competing terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerase. Dpo1 is shown to elongate single-stranded DNA in template-dependent and template-independent manners. Experiments with different homopolymeric templates indicate that initial deoxyribonucleotide incorporation is complementary to the template. Rate-limiting steps that include looping back and annealing to the template allow for a unique template-dependent terminal transferase activity. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures.     

Differentiation of Mycobacterium species by direct sequencing of amplified DNA

Nucleotide sequences specific for a range of Mycobacterium species were defined by computer-assisted sequence comparisons of small subunit ribosomal RNA. A polymerase chain reaction-based sequencing strategy was used to demonstrate that the 16S rRNA sequence can be used for the rapid identification of mycobacterial isolates. Identification at the species level can be obtained within 2 d, requiring less than 10,000 bacteria. This procedure reliably differentiates Mycobacterium spp. which are difficult to identify by classical methods, such as M. malmoense, M. szulgai and M. flavescens.