Exonuclease activity of proofreading DNA polymerases is at the origin of artifacts in molecular profiling studies

CE fingerprint methods are commonly used in microbial ecology. We have previously noticed that the position and number of peaks in CE-SSCP (single-strand conformation polymorphism) profiles depend on the DNA polymerase used in PCR [1]. Here, we studied the fragments produced by Taq polymerase as well as four commercially available proofreading polymerases, using the V3 region of the Escherichia coli rss gene as a marker. PCR products rendered multiple peaks in denaturing CE; Taq polymerase was observed to produce the longest fragments. Incubation of the fragments with T4 DNA polymerase indicated that the 3'-ends of the proofreading polymerase amplicons were recessed, while the Taq amplicon was partially +A tailed. Treatment of the PCR product with proofreading DNA polymerase rendered trimmed fragments. This was due to the 3'-5' exonuclease activity of these enzymes, which is essential for proofreading. The nuclease activity was reduced by increasing the concentration of dNTP. The Platinum Pfx DNA polymerase generated very few artifacts and could produce 85% of blunted PCR products. Nevertheless, despite the higher error rate, we recommend the use of Taq polymerase rather than proofreading in the framework for molecular fingerprint studies. They are more cost-effective and therefore ideally suited for high-throughput analysis; the +A tail artifact rate can be controlled by modifying the PCR primers and the reaction conditions.     

Biochemical Characterization of Translesion Synthesis by Sulfolobus acidocaldarius DNA Polymerases

To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarchaeota, two DNA polymerases of B family(polB1 and polB3), and one DNA polymerase of Y family(polIV) were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polB1(Saci_1537) and polB3(Saci_0074) possessed DNA polymerase and 3' to 5' exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV(Saci_0554) was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn²⁺ and Mg²⁺. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KCl with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.     

Preparation of single-stranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease

Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.     

Guanine-thymine intrastrand cross-linked lesion containing oligonucleotides: from chemical synthesis to in vitro enzymatic replication

An intrastrand cross-link lesion, in which two neighboring nucleobases are covalently tethered, has been site-specifically synthesized into defined sequence oligonucleotides in order to perform in vitro replication studies using either bacterial replicative or translesional synthesis polymerases. The investigated tandem base lesion that involves a cross-link between the methylene group of thymine and the C8 of an adjacent guanine residue has been prepared by UV-photolysis under anaerobic condition of the photolabile precursor 5-(phenylthiomethyl)-2'-deoxyuridine that has been site-specifically incorporated into a 9-mer oligonucleotide. After ligation, the lesion-containing modified oligonucleotide was used as a DNA template in primer extension reactions catalyzed by several DNA polymerases including the fragment Klenow exo-(Kf-) of E. coli polymerase I, the Thermus aquaticus polymerase (Taq pol) and the E. coli translesional DNA polymerase Pol IV (dinB). It was found that the primer extension reaction was stopped after the incorporation of the correct nucleotide dAMP opposite the 3'-thymine residue of guanine(C8-CH2) thymine lesion by Kf- and Pol IV; however it was noted that the efficiency of the nucleotide incorporation was reduced. In contrast, the Taq polymerase was totally blocked at the nucleotide preceding the tandem lesion. These results are strongly suggestive that the present intrastrand cross-link lesion, if not repaired, would constitute a blocking lesion for prokaryotic DNA polymerases, being likely lethal for the cell.     

Discrimination of A1555G and C1494T point mutations in the mitochondrial 12S rRNA gene by on/off switch

The objective of this study was to apply the “on/off” switch consisting of 3′ phosphorothioate-modified allele specific primers and exo⁺ polymerase in single base discrimination of A1555G and C1494T mutations in the highly conserved sites of the mitochondrial 12S rRNA. The two point mutations are the hotspot mutations associated with either aminoglycoside antibiotics induced deafness or inherited nonsyndromic hearing loss. The PCR products of mitochondrial DNA (mtDNA) 12S rRNA gene were inserted into the pMD19-T vector for transformation into Escherichia coli JM109 competent cells for preparing wild-type pMD19-T/mt vector. Inverse PCR was carried out for mtDNA 12S rRNA gene C1494T and A1555G mutagenesis and DpnI endonuclease degradating methylated pMD19-T/mt vector existing in the inverse PCR products was carried out to construct the mutation-type pMD19-T/mtM vector. These constructed vectors were confirmed by DNA sequencing. Allelic specific primers targeting wild-type and mutation-type templates were designed with 3′ terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. Amplified by exo⁺ polymerase, allelic specific primers perfectly matching wild-type allele were extended while no products were produced from primers targeting point-mutated deafness-related allele. Similarly, allelic specific primers perfectly matching point-mutated deafness-related mutation-type allele were extended and no products were yielded from primers targeting wild-type allele. No specific product was observed in the primer extension reaction mediated by on/off switch in screening the mtDNA 12S rRNA gene harboring either C1494T or A1555G mutation in 40 healthy volunteers tested. These data suggest that the “off switch” mediated by exo⁺ polymerase is highly reliable in the diagnosis of monogenic diseases and the novel “on/off” switch has enormous applications in systematic and extended screening of the12S rRNA gene A1555G and C1494T mutations. The established assay can be widely used not only for hearing loss patients but also for normal subjects before the use of aminoglycoside antibiotics.     

Cloning and Sequence Analysis of Novel DNA Polymerases from Thermophilic Geobacillus Species Isolated from Hot Springs in Turkey: Characterization of a DNA Polymerase I from Geobacillus kaue Strain NB

The complete coding sequences of the polA genes from seven thermophilic Geobacillus species, isolated from hot springs of G?nen and Hisaralan in Turkey, were cloned and sequenced. The polA genes of these Geobacillus species contain a long open reading frame of 2,637 bp encoding DNA polymerase I with a calculated molecular mass of 99 kDa. Amino acid sequences of these Geobacillus DNA polymerases are closely related. The multiple sequence alignments show all include the conserved amino acids in the polymerase and 5'-3' exonuclease domains, but the catalytic residues varied in 3'-5' exonuclease domain of these Geobacillus DNA polymerases. One of them, DNA polymerase I from Geobacillus kaue strain NB (Gkaue polI) is purified to homogeneity and biochemically characterized in vitro. The optimum temperature for enzymatic activity of Gkaue polI is 70 °C at pH 7.5-8.5 in the presence of 8 mM Mg(2+) and 80-100 mM of monovalent ions. The addition of polyamines stimulates the polymerization activity of the enzyme. Three-dimensional structure of Gkaue polI predicted using homology modeling confirmed the conservation of all the functionally important regions in the polymerase active site.     

Synthesis of photolabile dUTP analogues and their enzymatic incorporation for DNA labeling

<正>~~     

Determination of photomodified oligodeoxynucleotides by exonuclease digestion, matrix-assisted laser desorption/ionization and post-source decay mass spectrometry

A fast method to detect and sequence photomodified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is reported. Upon treatment of modified ODNs with both phosphodiesterase I and phosphodiesterase II, the digestion stops at the sites of photomodification. Post-source decay (PSD) of MALDI-produced ions from two enzymatic digestion end products distinguishes isomers such as 5'-d(T[cis-syn]TAAGC) and 5'-d(CGAAT[cis-syn]T), which have symmetrical or identical compositions at the 3' and 5' ends, respectively. Studies have also been done to follow the kinetics for enzyme degradation of photomodified ODNs. The calculated rate constants from a mathematical treatment of the time-dependent MALDI data clearly show that the enzymatic digestion rate slows as the enzyme approaches the modified site.     

Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses

The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.     

Synthesis and Some Biochemical Properties of a Novel 5,6,7,8‐Tetrahydropyrimido[4,5‐c]pyridazine Nucleoside

A novel nucleoside analogue is described based on the pyridazine ring system. The nucleoside was successfully incorporated into DNA via both its phosphoramidite and 5′‐triphosphate derivatives. Enzymatically, the analogue behaves essentially as thymidine: it is a good substrate for the DNA polymerases Taq and exonuclease‐free Klenow fragment, leading to full‐length products when present in either the primer or template strands. In hybridisation studies, the nucleoside displays ambiguous base‐pairing properties, including universal base properties.     

Microarray-based resequencing by apyrase-mediated allele-specific extension

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.     

Matrix-assisted laser desorption/ionization detection of polymerase chain reaction products by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase

The 5'-3' exonuclease activity of DNA polymerase was utilized in the polymerase chain reaction system to generate a specific signal concomitant with amplification. These signals were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method obviates the need to perform extensive DNA purification of reaction products that is often necessary for detecting larger DNA molecules by mass spectrometry. Oligonucleotides complementary to the internal region of the amplicon are degraded by the 5'-3' exonuclease activity and the degradation products are analyzed by MALDI mass spectrometry. We refer to this assay as the Exo-taq assay or probe degradation assay. This method should be amenable to automation.     

A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity

We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3′ (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the “wrong” genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3′ exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3′-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3′-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.     

An efficient site-directed mutagenesis using polymerase chain reaction

We report here a simple and efficient method for site-directed mutagenesis using polymerase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restriction gene, we made two point mutations. While one created a new SalI site prior to the SD sequence, the other replaced Glu144 with Lys. A 1.5 kb SalI-PstI fragment isolated from pER101 was used as the template. Two 25 mer oligonucleotide primers containing the desired mutations were synthesized and used to direct PCR amplification with Taq DNA polymerase. About 0.5 microgram of the 0.49 kb fragment was obtained from 0.05 microgram of the 1.5 kb fragment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically, 99% of the product contained the desired mutations. The product was cloned into pUC19 using SalI and PstI, two of the transformed colonies were randomly chosen for sequence analysis, and both of them were shown to contain the desired mutations. Finally, the amplified fragment was cloned into pER304 to place the EcoRI (Lys144) gene directly under the control of the lambda PL promoter.     

Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs

Expansion of the genetic alphabet with an unnatural base pair is a long‐standing goal of synthetic biology. We have developed a class of unnatural base pairs, formed between d 5SICS and analogues of d MMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, we report the synthesis of five new d MMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d 5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, d NaM –d 5SICS , to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examined. The resulting structure–activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addition, we identify an analogue (d NMO1 ) that is a better partner for d 5SICS than any of the previously identified d MMO2 analogues with the exception of d NaM . We also found that d NaM –d 5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.     

Identification of smoked Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) using PCR-restriction fragment length polymorphism of the p53 gene

Raw and smoked samples of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the p53 gene. DNA from S. salar and 0. mykiss was amplified by using primers flanking exons 5 to 6 of the p53 nuclear gene. PCR products of different length were obtained for each species (532 and 518 base pairs, respectively). Sequences of PCR products obtained from S. salar and O. mykiss were compared in the search for polymorphic restriction sites. The restriction fragments obtained with Eco RV, Hinf I, and Taq I endonucleases showed interspecific polymorphism, making it a useful method for identification of Atlantic salmon and rainbow trout.     

Molecular cloning, expression and sequence analysis of DNA polymerase I from an Iranian thermophilic bacterium, Bacillus sp. G (2006)

Thermostable DNA polymerases are widely used in DNA amplification reactions such as the Polymerase Chain Reaction (PCR), requiring the activity of the enzymes at high temperatures. The aim of the present study was to assess the potential biotechnological capabilities of Iranian thermostable DNA polymerases. To this end, we cloned the gene encoding a DNA polymerase from a novel thermophilic eubacterium, Bacillus sp. G (2006). Phylogentic analysis of this gene revealed that the new isolate belongs to the genera Bacillus. Sequence analysis of the fragment produced by degenerate primers also showed that it consists of 2,631 bp encoding an 876 amino acid protein, and subsequent amino acid sequence analysis of this DNA polymerase showed that it belongs to family A-type DNA polymerases. The expression vector pET28a (+) was chosen for expression of the gene fragment in the mesophilic host bacterium E. coli BL21. This expression vector has some advantages such as attachment of a Poly-His tag to the N-terminus of the protein for the ease of purification and a powerful promoter of lac-Z induced by IPTG. The band corresponding to the protein product was observed in the molecular weight range of about 100KDa on the SDS-PAGE gel after heat and Ni⁺²-NTA column chromatography. Using the dot blot technique, the polymerase activity of the enzyme was qualitatively confirmed at 70 °C. Therefore, it is suggested that optimizations of this activity could make this enzyme appropriate for PCR processes in future.     

Effects of DNA mismatches on binding affinity and kinetics of polymerase-DNA complexes as revealed by surface plasmon resonance biosensor

In this study, surface plasmon resonance (SPR) biosensor techniques were used to obtain quantitative information on the kinetics of the DNA and polymerase I (Klenow fragment) interaction. DNA duplexes containing different base compositions at the binding site were immobilized on the SPR sensor surface via biotin-streptavidin chemistry and the subsequent binding of the polymerase was measured in real time. Various kinetic models were tested and a translocation model was shown to provide the best fit for the binding and dissociation profiles. The results revealed that the enzyme binds to DNA at both the polymerase and the exonuclease domains with different association and dissociation rates as well as affinity constants, depending on the presence of mismatches near the primer 3'-end. Introduction of unpaired bases increases the DNA binding affinity towards the exonuclease domain and promotes the translocation of DNA from the polymerase site to the exonuclease site. The results also demonstrated that SPR biosensors may be used as a sensitive technique for studying molecular recognition events such as single-base discrimination involved in protein-DNA interaction.     

Faithful PCR Amplification of an Unnatural Base‐Pair Analogue with Four Hydrogen Bonds

In vitro replication of an unnatural imidazopyridopyridine:naphthyridine base pair, (i.e., ImN^N:NaO^O), having four hydrogen bonds was investigated. Kinetic studies of single‐nucleotide insertion revealed that ImN^N and NaO^O were recognized as complementary bases by an exonuclease‐deficient Klenow fragment with higher specificity and efficiency than two previously described pairs (ImN^O:NaO^N and ImO^N:NaN^O) because of higher thermal and thermodynamic stabilities and the DAAD:ADDA (D=donor, A=acceptor) hydrogen‐bonding pattern of the ImN^N:NaO^O pair. Faithful polymerase chain reaction (PCR) amplification of a DNA fragment containing the ImN^N:NaO^O pair was achieved by using DNA polymerases possessing 3′→5′ exonuclease activity (≈99.5 % per doubling).