Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya

Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures ~100°C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases δ and ε for nuclear DNA and polymerase γ for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.     

Characteristics of microspheres formed in PCR with bacterial genomic DNA or plasmid DNA as templates

Earlier, we discovered that, along with linear DNA fragments, nano- and microparticles of DNA and their aggregates are formed in the PCR with yeast genomic DNA used as a template and gene-specific or partially complementary primers. The size of the microparticles (microspheres) varied in the range of 0.5 to 3–4 μm. Only thermostable KlenTaq polymerase but not Taq polymerase could effectively generate microspheres. In this work, we demonstrate that KlenTaq polymerase can produce microspheres of variable size (1 to 7 μm in diameter) if genomic DNA of the bacterium Acidithiobacillus ferrooxidans and partially complementary primers are present in the PCR mixture. Conditions for generation of DNA microparticles in PCR with Taq-polymerase and bacterial genomic DNA as template were also elaborated. It was also found that mainly large microspheres of up to 7 μm accumulated in PCR with plasmid DNAs used as templates and gene-specific primers in the presence of KlenTaq polymerase or mixtures of KlenTaq and Pfu polymerases. Besides, small aggregates, as well as linear branched structures and three-dimensional conglomerates of fused microspheres, were also revealed in the PCR mixtures. UV absorption spectra of native DNA microspheres and microspheres that had undergone heating at 93°C were registered. The key role of Mg²⁺ cations in the formation and stabilization of the microsphere structure was established.     

Biochemical properties and base excision repair complex formation of apurinic/apyrimidinic endonuclease from Pyrococcus furiosus

Apurinic/apyrimidinic (AP) sites are the most frequently found mutagenic lesions in DNA, and they arise mainly from spontaneous base loss or modified base removal by damage-specific DNA glycosylases. AP sites are cleaved by AP endonucleases, and the resultant gaps in the DNA are repaired by DNA polymerase/DNA ligase reactions. We identified the gene product that is responsible for the AP endonuclease activity in the hyperthermophilic euryarchaeon, Pyrococcus furiosus. Furthermore, we detected the physical interaction between P. furiosus AP endonuclease (PfuAPE) and proliferating cell nuclear antigen (PCNA; PfuPCNA) by a pull-down assay and a surface plasmon resonance analysis. Interestingly, the associated 3′–5′ exonuclease activity, but not the AP endonuclease activity, of PfuAPE was stimulated by PfuPCNA. Immunoprecipitation experiments using the P. furiosus cell extracts supported the interaction between PfuAPE and PfuPCNA in the cells. This is the first report describing the physical and functional interactions between an archaeal AP endonuclease and PCNA. We also detected the ternary complex of PfuPCNA, PfuAPE and Pfu uracil-DNA glycosylase. This complex probably functions to enhance the repair of uracil-containing DNA in P. furiosus cells.     

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.     

An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene

In this study, overlap-PCR, an efficient and rapid method, was used to clone cattle Ghrelin gene CDS (coding sequence) from genomic DNA. The procedure included seven primers and three-step PCRs. Cattle Ghrelin gene consists of four exons and the CDS contains 351 bps. In the first step three PCRs were performed to generate extended exon1, exon2, and exon3 that contained overlapped nucleotides and were used as the templates for second ligation PCR. Secondly, exon1 and exon2 were spliced together. And it was same with exon3 and exon4. Lastly, the four exons were linked together with outermost primers and the templates from the second step. Comparison analysis on the obtained CDS of Ghrelin gene and cDNA by RT-PCR showed that the two sequences were same. As an efficient and rapid method, overlap-PCR is feasible and acceptable for gene cloning from genomic DNA.     

Excision repair of uracil incorporated in DNA as a result of a defect in dUTPase.

Mutants of Escherichia coli that are severely defective in the enzyme dUTPase (dut) accumulate short (4 to 5 S) Okazaki fragments following brief pulses with [³H]thymidine. The transient appearance of DNA fragments in these mutants is plausibly explained by the misincorporation of uracil in DNA as a result of an increase in available dUTP, followed by its rapid excision and repair. The evidence in support of this interpretation is the following: (1) accumulation of short DNA fragments can be partially suppressed by a mutation in dCTP deaminase, presumably by decreasing the intracellular level of dUTP relative to dTTP; (2) accumulation of the short DNA fragments can be almost completely suppressed by a mutation in uracil N-glycosidase, probably by preventing the introduction of nicks at the sites of uracil incorporation; (3) introduction of DNA polymerase I or DNA ligase mutations into dUTPase-defective strains results in the persistence of the 4 to 5 S fragments and rapid cessation of DNA synthesis. Uracil N-glycosidase, DNA polymerase I and DNA ligase must therefore be involved in the excision repair of uracil-containing DNA.     

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99°C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or Taq plus Pwo DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.coli exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.     

Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments.

We describe a sensitive method for mapping replication initiation sites near regions of sequenced genomic DNA in vivo. It is based on selective amplification of sets of segments in purified nascent DNA strands and subsequent determination of the lengths of these strands required to include each member of the set. We demonstrate the ability of this method to accurately map a well-defined origin, that of replicating SV40 DNA. Pulse-labeled DNA from infected CV-1 cells was size-fractionated on an alkaline sucrose gradient and newly-synthesized strands purified by immunoprecipitation using anti-BrdU antibodies. Three pairs of synthetic oligonucleotide primers were used to amplify three SV40 segments, using the polymerase chain reaction (PCR), at known distances from the origin. Lengths of the nascent DNA strands that allow amplification were determined by hybridization to probes homologous to the amplified segments and used to calculate position of the origin. Experiments with a mix of SV40 and human HeLa cell DNA demonstrate the applicability of the method to mapping origins present at the level of single-copy genomic sequences in mammalian cells.     

Deconstructing the Polymerase Chain Reaction: Understanding and Correcting Bias Associated with Primer Degeneracies and Primer-Template Mismatches

The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3–12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled “Polymerase-exonuclease (PEX) PCR”, in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3’ end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.     

Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates

PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.     

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.     

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.     

一种改良的启动子序列克隆的染色体步查法

利用染色体步行法,从已知DNA序列克隆侧翼未知序列是非常有效的方法之一,但由于所选用的特定限制性内切酶对目标基因组不能酶解成合适大小的片段,因而受PCR扩增能力的局限,往往扩增不出有效产物. 针对这一点,这里我们介绍一种简单有效的改良方法,它包括以下步骤：首先用不同的限制性内切酶(包括平末端和粘性末端) 酶解目标基因组DNA,接着,选择能将基因组酶切成弥散、分布均匀的限制性内切酶,如DraⅠ和HindⅢ,合成相对应的接头;然后,选择弥散的、分布均匀的限制性内切酶的酶解产物,构建成含相应接头的基因组DNA文库,用作PCR的模板;最后,用接头引物和特异引物,通过巢式PCR扩增目的片段,获得了理想的扩增效果.采用改进后的染色体步查法,有效地从较复杂的棉花核DNA中克隆出6个棉花启动子序列.     

Rapid detection of mycoplasma contamination in cell cultures using sybr green-based real-time polymerase chain reaction

Summary We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluated the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, in suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.     

Specific detection of residual CHO host cell DNA by real-time PCR.

Chinese hamster ovary cells have been widely used to manufacture recombinant proteins for human therapeutic use. A sensitive quantitative real-time polymerase chain reaction assay for the detection of residual Chinese hamster (Cricetulus griseus) DNA is presented in this paper. The assay is reasonably affordable and can be adapted for high-throughput screening using 96-well format. Real-time PCR primers were designed to amplify a 150bp region of a genomic fragment from hamster DNA. The specificity of the probe was evaluated in real-time PCR reactions using genomic DNA from mouse fibroblast, human kidney and hamster ovary cell lines as template. Sensitivity of real-time PCR was compared on genomic DNA from hamster cell line CHO DG44. These primers can be used in real-time PCR reactions to detect presence of contaminating hamster DNA in purified protein samples down to sensitivity of 300fg genomic DNA.     

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.     

Isolation and Characterization of a Bacillus subtilis Mutant with a Defective N-Glycosidase Activity for Uracil-Containing Deoxyribonucleic Acid

Crude cell extracts of Bacillus subtilis 168T exhibit enzyme activity capable of releasing free uracil from phage PBS1 deoxyribonucleic acid (DNA) in the presence of ethylenediaminetetraacetate. By measuring the enzyme activity in 300 clones that emanated from mutagenized cells, we obtained a mutant strain that did not show this N-glycosidase activity. The mutant strain, designated as TKJ6901 (urg-1) exhibited no physiological abnormalities. We observed the intracellular action of the enzyme by following the fate of uracil-containing DNA in cells from wild-type and mutant cultures. When infection with phage PBS1 was allowed in the presence of chloramphenicol, extensive degradation of phage DNA was observed only in the wild-type cells. When bromouracil residues were converted to uracil residues by ultraviolet light irradiation in the presence of cysteamine, the DNA was extensively fragmented in the wild-type cells. These single-strand breaks were rejoined upon postirradiation incubation. In contrast, such fragmentation of the DNA was not observed in the mutant cells, indicating that the uracil residues were not removed from the DNA. This demonstrated that the N-glycosidase activity was involved in the excision of uracil in DNA. A transformation assay with four types of recipient strains with combinations of N-glycosidase and DNA polymerase I deficiencies indicated that DNA polymerase I was involved in the later steps of this base excision repair pathway initiated by the action of the N-glycosidase.     

A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.     

DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight

Alterations in cytosine-5 DNA methylation are frequently observed in most types of human cancer. Although assays utilizing PCR amplification of bisulfite-converted DNA are widely employed to analyze these DNA methylation alterations, they are generally limited in throughput capacity, detection sensitivity, and or resolution. Digital PCR, in which a DNA sample is analyzed in distributive fashion over multiple reaction chambers, allows for enumeration of discrete template DNA molecules, as well as sequestration of non-specific primer annealing templates into negative chambers, thereby increasing the signal-to-noise ratio in positive chambers. Here, we have applied digital PCR technology to bisulfite-converted DNA for single-molecule high-resolution DNA methylation analysis and for increased sensitivity DNA methylation detection. We developed digital bisulfite genomic DNA sequencing to efficiently determine single-basepair DNA methylation patterns on single-molecule DNA templates without an interim cloning step. We also developed digital MethyLight, which surpasses traditional MethyLight in detection sensitivity and quantitative accuracy for low quantities of DNA. Using digital MethyLight, we identified single-molecule, cancer-specific DNA hypermethylation events in the CpG islands of RUNX3, CLDN5 and FOXE1 present in plasma samples from breast cancer patients.