Polymerase chain reaction-based methods of DNA methylation analysis

DNA methylation is the main epigenetic modification in humans, and changes in methylation patterns play an important role in tumorigenesis. Hypermethylation of normally unmethylated CpG islands in the promoter regions often occurs in important tumor suppressor genes, DNA repair genes, and metastasis inhibitor genes. The changes of methylation status of various gene promoters seem to be a common feature of malignant cells and these changes can occur early in the progression process. Therefore detection of aberrant promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or detection of cancer recurrence. The purpose of this review is to provide a summary of the most commonly used techniques for the study of DNA methylation. Current scientific literature involving methylation detection methods was reviewed with an emphasis on polymerase chain reaction (PCR)-based detection methods. The current methodologies may be broadly classed into PCR-based methylation assays and non-PCR-based methylation assays. The problems and advantages of the different methods for detecting aberrant methylation are discussed. As the number of genes known to be hypermethylated in cancer is growing, the detection of aberrant promoter region methylation will be a promising approach for using DNA-based markers for the early detection of human cancers. Many techniques, especially PCR-based methylation assay techniques, make it practical to use these new methylation biomarkers in early cancer diagnosis.     

Polymerase chain reaction-based polymorphisms in the porcine cholecystokinin (CCK) gene and assignment to chromosome 13

Polymorphisms were identified in the porcine cholecystokinin (CCK) gene by digestion of products from polymerase chain reaction (PCR) with the restriction enzyme Dpn II. Individuals from the European pig gene mapping project (PiGMaP) consortium reference families (eight full-sib families, 91 total progeny) were genotype to determine linkage relationships between the CCK gene and previously mapped loci. Linkage analysis revealed that the CCK gene is located on porcine chromosome 13.     

Effects of bisulfite (sulfur dioxide) on DNA synthesis and fidelity by DNA polymerase I

In an attempt to explain the mechanism of comutagenesis by bisulfite, the extent and accuracy of DNA synthesis by E. coli DNA polymerase I was examined in the presence of sodium bisulfite. Bisulfite concentration of 100 mM caused nearly complete inhibition of dNTP incorporation into activated calf thymus DNA. Other salts (NaCL, Na2SO4) at the same concentration had no effect on enzyme activity. Preincubation of the various DNA synthesis assay components in 100 mM bisulfite showed that only preincubation of DNA polymerase I caused inhibition of DNA synthesis. Exonuclease functions of DNA polymerase I were unaffected by up to 100 mM bisulfite. Accuracy of DNA synthesis in the presence of bisulfite was determined using poly (dA-dT) as a template-primer. Concentrations of bisulfite greater than 50 mM caused a progressive decrease in enzyme accuracy. At 100 mM bisulfite there was an approximate 7.5-fold decrease in the fidelity of DNA synthesis, compared to control values, as measured by the ratio of noncomplementary (dGTP) to complementary (dTTP) nucleotide incorporated. Based on the known chemistry of bisulfite, it is hypothesized that sulfitolysis of the one disulfide group in DNA polymerase I by bisulfite might be responsible for the reduced polymerase activity and accuracy. The exonuclease functions of DNA polymerase I do not seem to require the disulfide linkage. These results suggest that the effects of bisulfite on mutation frequency might be mediated by effects on the fidelity of DNA repair systems.     

Mutagenicity and cytotoxicity of 2-butoxyethanol and its metabolite, 2-butoxyacetaldehyde,in Chinese hamster ovary (CHO-AS52) cells

2-Methoxyethanol (2-ME) is being substituted by 2-butoxyethanol (2-BE) as a solvent for the preparation of industrial and consumer products. Since we have shown that a metabolite of 2-methoxyethanol, methoxyacetaldehyde (MALD), is mutagenic in a subline of Chinese hamster ovary cells (CHO-AS52), we have conducted a similar study using 2-BE and its metabolite, butoxyacetaldehyde (BALD). The results indicate that 2-BE and BALD are not mutagenic to CHO-AS52 cells. However, 2-BE is more cytotoxic than 2-ME. In comparison of our study with others on glycol ethers, the data indicate that, for glycol ethers, cytotoxicity increased with chain length of the alkyl groups. For their metabolites, mutagenicity increases with reduced chain length. Therefore, we suggest that safer solvents should be developed for use in preparation of products.     

Polymerase chain reaction-based detection of Fusarium circinatum, the causal agent of pitch canker disease

Pitch canker is a highly damaging disease of Pinus radiata and the New Zealand forest industry is concerned by the potential impact of the disease, should it arrive, in New Zealand. To provide a rapid identification technique for this pathogen, a polymerase chain reaction-based diagnostic method has been developed. The method is able to detect the presence of the pathogen within infected host tissue, as well as infested soil and the reliability of the test has been estimated using Bayesian statistics.     

Polymerase chain reaction-based analysis using deaminated DNA of dodecamer expansions in CSTB, associated with Unverricht-Lundborg myoclonus epilepsy

Progressive myoclonus epilepsy of the Unverricht-Lundborg type is an autosomal recessive disorder that is characterized clinically by myoclonic seizures and ataxia. The majority of affected individuals carry repeat expansions of a dodecamer in the promoter region of the cystatin B gene. The unusually high GC content of this tract is refractory to conventional polymerase chain reaction (PCR), and, as a result, a circumventive procedure involving the deamination of DNA with sodium bisulfite has been proposed. This study evaluates the effectiveness of this deamination modification for the detection of dodecamer repeat variants. An analysis of 258 healthy Japanese individuals revealed an allele with four copies of the dodecamer repeat with a frequency of 0.01, in addition to the more commonly observed two and three copy repeat alleles. Homozygous repeat expansions 600 and 680 base pairs in length were detected in the analyses of two affected individuals. For these cases, sequencing, along with an alternative PCR-stutter formation, revealed 41 and 48 copies, respectively, of the dodecamer repeat. The complete conversion of C to T was observed in the expanded tracts, indicating that no methylation occurred at the CpG sites. Based on these results, it was concluded that the use of deaminated DNA allows for a precise analysis of consecutive GC tracts.     

Bisulfite (sulfur dioxide) is a comutagen in E. coli and in Chinese hamster cells

The mutagenic and comutagenic effects of bisulfite were investigated. Under moderate exposure conditions (high survival) it was found that bisulfite is not mutagenic to either eukaryotic cells (Chinese hamster V79), or prokaryotic cells (Escherichia coli). However, bisulfite does act as a comutagen with UV irradiation. Bisulfite approximately doubles the mutation frequency in UV-irradiated Chinese hamster V79 cells, and it causes a greater than 8-fold increase in Trp⁺ revertants in UV-irradiated E. coli. The comutagenic effect occurs whether cells are exposed to bisulfite during or immediately after UV irradiation. Kinetic studies of the comutagenic effect in E. coli shows that it decays in a biphasic manner, with an apparent half-life of 15 min and a persistence of the comutagenic effect for up to 120 min after UV irradiation.Experiments with several strains of E. coli of varying DNA-repair capacities indicate that excision repair is necessary for a comutagenic effect by bisulfite. It is thought that bisulfite acts to inhibit excision repair, perhaps by effects on DNA polymerase I, or DNA ligase.     

Polymerase chain reaction (PCR) techniques for site-directed mutagenesis

Polymerase chain reaction (PCR) technology has revolutionized the process of isolating and amplifying segments of DNA. One powerful application of PCR is its use in precise site-directed mutagenesis (SDM). SDM provides an elegant tool for scientists and engineers to explore biocatalytic mechanisms and processes to understand the structural-functional relationships of enzymes and other proteins. This article reviews techniques and methodology used in site-directed mutagenesis of genes by PCR.     

Emission of ethylene and ethane by leaf tissue exposed to injurious concentrations of sulfur dioxide or bisulfite ion

Leaf tissues injured with SO₂ gas or bisulfite ion in solution emit ethylene and ethane. The amounts of these gases produced by the tissues depend on the degree of exposure to SO₂ or bisulfite. The amount of ethylene produced in response to SO₂ fumigation correlates positively with SO₂ exposure (0 to 5.5 microliters per liter for 16 hours), SO₂ absorbed, and the amount of visible injury sustained by the leaf tissues. Ethane production is correlated positively with the injury resulting from treatment with bisulfite ion. The rate of emission of ethane from leaf discs of cucurbit cultivars as a result of exposure to bisulfite solutions is in agreement with the order and the degree of their resistance to injury by SO₂. Thus, exposure to bisulfite and the subsequent release of ethane can be used to determine the relative resistance of different species and cultivars to SO₂ gas.     

Polymerase chain reaction (PCR) amplification of hypervariable genomic sequences.

This paper describes a rapid method for VNTRs (variable number of tandem repeats) typing using polymerase chain reaction (PCR). Three VNTRs (YNZ22, Apo B, MCT118) were amplified and alleles mendelian segregation was confirmed. We also demonstrate their applicability to paternity testing and forensic purposes.     

Polymerase chain reaction-based genotyping for allotypic markers of immunoglobulin kappa shows allelic association of Km with kappa variable segment.

Allotypic markers of immunoglobulin kappa (Km) may be determined using a novel method of amplification of the constant segment (C kappa) (IGKC) by polymerase chain reaction (PCR) followed by restriction enzyme digestion. Restriction sites in the C kappa PCR product correlate with allotypic differences among Km(1), Km(1,2), and Km(3) alleles. An AccI site in the PCR product correlates with Km(3); and presence or absence of a MaeII site correlates with the Km(1) or Km(1,2) allele, respectively. Km allelic frequencies were determined in a Caucasian population and compared to genotypic frequencies of nearby polymorphic markers. Among unrelated individuals with rheumatoid arthritis (RA) and controls, there is no evidence of allelic association between CD8A and polymorphic markers of the immunoglobulin kappa region [a V kappa (IGKV) BglII polymorphism about 24 kb centromeric to C kappa, Km allotype, and a SacI polymorphism 3.5 kb telomeric to the C kappa segment]. Similarly, there is no allelic association of the SacI C kappa polymorphism with Km or with the BglII V kappa polymorphism. However, there is evidence of allelic association of V kappa B3 and Km, specifically between the V kappa BglII 2.2-kb allele and Km(3) and also between the V kappa 3.5-kb allele and Km(1,2). Therefore, Km typing by PCR-based methods suggests the presence of allelic association between polymorphisms within the coding region of the C kappa segment and the nearest V kappa segment.     

Palladium (II)-catalyzed alternating copolymerization of alkenes with sulfur dioxide

Cationic palladium(II) complexes of the type [L₂Pd(CH₃)(CH₃CN)]BF₄ (L₂ = 1,3-bis(diphenylphosphino)propane (Dppp) or 1,2-bis(2,5-dimethylphospholano)benzene (Me-Duphos)) were found to catalyze the alternating copolymerization of ethene, propene and cyclopentene with sulfur dioxide, as well as the terpolymerization of ethene, propene and sulfur dioxide. The resultant materials are high melting solids with an alternating alkene-SO₂ structure and an exclusive head-to-tail enchainment for propene.     

Polymerase chain reaction (PCR) amplification with a single specific primer

A method is described for amplification of DNA fragments flanking a single known sequence that is sufficiently long to enable synthesis of a functional primer in polymerase chain reactions.     

Synthesis and antimycobacterial activity of prodrugs of sulfur dioxide (SO2)

Here, we synthesized and studied a library of 2,4-dinitrophenylsulfonamides that closely resembled N-benzyl-2,4-dinitrophenylsulfonamide (1), a thiol-activated prodrug of sulfur dioxide (SO(2)) which has shown high potency as a Mycobacterium tuberculosis (Mtb) inhibitory agent. The ability of these compounds to generate SO(2) in the presence of a thiol was evaluated. A good correlation between pK(aH) of the corresponding amine and reactivity with thiols to generate SO(2) was found suggesting that the rate determining step of SO(2) generation involved protonation of the amine. Amongst analogues with measurable MICs, we also found a correlation between ability to generate SO(2) and Mtb growth inhibitory activity. Together, we report several thiol-mediated prodrugs of SO(2) which strongly inhibited Mtb growth (MIC <1 μg mL(-1)) with potential for further development as tuberculosis drug candidates.     

Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803

Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp. PCC 6803 genome and show very strong homologies to those found in higher plants. The gene knock-outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728- 29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock-outs were evaluated in terms of Na+ and pH, and light-induced acidification and alkalization that are asso-ciated with inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance. On the other hand, the mutation of one of the two copper-trafficking ATPases produces a cell line that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+-ATPase and a gene cluster encoding a putative Na+-ATPase subunit also cause high NaCl sensitivity. The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.