Sequencing of large double-stranded DNA using the dideoxy sequencing technique

The dideoxy sequencing technique has been applied to the direct sequencing of large double-stranded DNA molecules with a small single-stranded primer. For instance, the method was applied to the lambda genome, which contains 48 502 base-pairs (Sanger F, Coulson AR, Hong GF, Hill D & Petersen GB, 1982, J. Mol. Biol., in press), and the coding region for gene W identified. The procedure proves useful in the sequence analysis of a large number of different mutations in a particular region and in the analysis of eukaryotic DNA cloned in plasmids, phages, and cosmids.     

Rapid and reliable dideoxy sequencing of double-stranded DNA

We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector-specific primers, the M 13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands.     

T7 DNA polymerase in automated dideoxy sequencing.

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.     

Inhibition of Taq DNA polymerase by catalpol.

DNA polymerases have recently emerged as important cellular targets for chemical intervention in the development of anti-cancer agents. This report describes a PCR assay as a method to investigate the action mechanism of the inhibition of Taq DNA polymerase by catalpol. This inhibition was not primer or template specific, nor was it due to chelation of Mg2+ ions. In assays of hyperchromicity of double-stranded DNA, catalpol did not affect melting profile. The inhibitory effect of catalpol does not appear to depend on DNA concentration. In contrast, increasing dNTP concentration rescue the Taq DNA polymerase activity, suggestingthat catalpol acts in a competitive way with dNTPs at the binding site of the enzyme. Theoretical calculations reinforce the experimental data and the proposed mode of action of catalpol.     

Development of a new DNA sequencing method: 3'-ester cleavage catalyzed by Taq DNA polymerase.

A new 3'-esterified dTTP is incorporated into DNA by Taq DNA polymerase but does not act as a chain terminator. The esterase activity of the polymerase seems to be template dependent and occurs only if the next correct nucleotide is present.     

Thermal cycle dideoxy DNA sequencing

Thermal cycle dideoxy DNA sequencing eliminates the requirements for independent primer annealing and double-stranded DNA denaturation steps. The method enables sequencing from nanogram amounts of DNA from double-stranded and single-stranded PCR products, and plasmid or phage DNA templates. Thermal cycle sequencing also enables direct sequencing from bacterial colonies or phage plaques. Protocols using the Vent exo⁻ DNA polymerase, helpful suggestions, and a troubleshooting guide are also presented.     

Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing.

Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5'-32P-labeled. The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3'-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.     

Affinity generation of single-stranded DNA for dideoxy sequencing following the polymerase chain reaction

A method to rapidly generate single stranded DNA for dideoxy sequencing following the polymerase chain reaction is described. By incorporating biotin in one of the amplification primers, we are able to physically separate the two DNA strands produced in the polymerase chain reaction. After amplification, the mixture is passed through a column containing streptavidin agarose. The strand produced by the biotinylated primer is bound in this matrix. The unbiotinylated strand is eluted with 0.2 N NaOH and sequenced by the dideoxy method. This method was utilized to sequence mitochondrial DNA from crude genomic DNA and to determine the sequences of four clones containing human mitochondrial DNA as a test of its accuracy. The use of biotin-facilitated separation permitted us to amplify and sequence DNA samples in a single day.     

Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase.

A polymerase stop assay has been developed to determine the DNA nucleotide sequence specificity of covalent modification by antineoplastic agents using the thermostable DNA polymerase from Thermus aquaticus and synthetic labelled primers. The products of linear amplification are run on sequencing gels to reveal the sites of covalent drug binding. The method has been studied in detail for a number of agents including nitrogen mustards, platinum analogues and mitomycin C, and the sequence specificities obtained accord with those obtained by other procedures. The assay is advantageous in that it is not limited to a single type of DNA lesion (as in the piperidine cleavage assay for guanine-N7 alkylation), does not require a strand breakage step, and is more sensitive than other primer extension procedures which have only one cycle of polymerization. In particular the method has considerable potential for examining the sequence selectivity of damage and repair in single copy gene sequences in genomic DNA from cells.     

One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography

The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities.