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.     

Application of repair enzymes to improve the quality of degraded DNA templates for PCR amplification

PCR amplification of severely degraded DNA, including ancient DNA, forensic samples, and preparations from deeply processed foodstuffs, is a serious problem. Living organisms have a set of enzymes to repair lesions in their DNA. In this work, we have developed and characterized model systems of degraded high-molecular-weight DNA with a predominance of different types of damage. It was shown that depurination and oxidation of the model plasmid DNA template led to a decrease in the PCR efficiency. A set of enzymes performing a full cycle of excision repair of some lesions was determined. The treatment of modeldamaged substrates with this set of enzymes resulted in an increased PCR product yield as compared with that of the unrepaired samples.     

Accumulation of oxidatively generated DNA damage in the brain: a mechanism of neurotoxicity

Unrepaired or erroneously repaired DNA lesions drive genomic instability and contribute to cellular and organ decline. Since delayed neuropathologies are common in survivors of smoke inhalation injuries, we asked whether the integrity of brain DNA might be compromised by acute exposure to combustion smoke. Although many studies demonstrate that the brain is equipped to repair oxidatively damaged DNA, to date, the capacity for accurate DNA repair under conditions of disrupted oxygenation and oxidative stress has not been defined. We show that DNA adducts detectable by their ability to block PCR amplification form in the rat hippocampus after acute exposure to smoke. To identify the different types of adducts and to dissect their temporal formation and repair profiles in vivo in the brain, we used DNA-modifying enzymes to convert specific adducts into strand breaks prior to PCR amplification. Using this strategy, we detected formation of oxidative DNA adducts early on after smoke inhalation, while mismatched bases emerged at the later recovery times, potentially due to an erroneous DNA repair process. Erroneous repair can be mutagenic and because the initial smoke-induced oxidative damage to DNA is extensive, compromised fidelity of DNA repair may underlie neurotoxicity and contribute to delayed death of hippocampal neurons.     

Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR.

Fine analysis of DNA damage and repair at the subgenomic level has indicated a microheterogeneity of DNA repair in mammalian cells, including human. In addition to the well established Southern hybridization-based approach to investigate gene-specific DNA damage and repair, alternative methods utilizing the sensitivity of PCR have been evaluated. The latter technique has relied on decreased PCR amplification due to damage in template DNA. We have developed a novel quantitative assay combining the selective recovery of DNA damage containing genomic fragments with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated human skin fibroblasts was immunoprecipitated with polyclonal antibody BP-1. Recovered target sequences were amplified by PCR using primers encompassing a 149 bp target region around codon 12 of the H-ras proto-oncogene. Quantitative DNA damage specific response was observed with nanogram amounts of genomic DNA. This approach allowed analysis of the initial DNA damage at a level less than 1 anti-BPDE adduct per 6.4 kbp ras gene fragment. Repair proficient GM637 cells exposed to 2 microM anti-BPDE showed a faster removal of the adducts from the H-ras gene segment than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measurement of the repair of diverse DNA base lesions in any genomic region of known sequence.     

Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (Coelodonta antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analyses, the PCR can be biased towards the amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified.     

Molecular breeding of polymerases for amplification of ancient DNA

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.     

Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction – based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.     

Sequence-Dependent Biophysical Modeling of DNA Amplification

A theoretical framework for prediction of the dynamic evolution of chemical species in DNA amplification reactions, for any specified sequence and operating conditions, is reported. Using the polymerase chain reaction (PCR) as an example, we developed a sequence- and temperature-dependent kinetic model for DNA amplification using first-principles biophysical modeling of DNA hybridization and polymerization. We compare this kinetic model with prior PCR models and discuss the features of our model that are essential for quantitative prediction of DNA amplification efficiency for arbitrary sequences and operating conditions. Using this model, the kinetics of PCR is analyzed. The ability of the model to distinguish between the dynamic evolution of distinct DNA sequences in DNA amplification reactions is demonstrated. The kinetic model is solved for a typical PCR temperature protocol to motivate the need for optimization of the dynamic operating conditions of DNA amplification reactions. It is shown that amplification efficiency is affected by dynamic processes that are not accurately represented in the simplified models of DNA amplification that form the basis of conventional temperature cycling protocols. Based on this analysis, a modified temperature protocol that improves PCR efficiency is suggested. Use of this sequence-dependent kinetic model in a control theoretic framework to determine the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is discussed.     

Sequence-Dependent Biophysical Modeling of DNA Amplification

A theoretical framework for prediction of the dynamic evolution of chemical species in DNA amplification reactions, for any specified sequence and operating conditions, is reported. Using the polymerase chain reaction (PCR) as an example, we developed a sequence- and temperature-dependent kinetic model for DNA amplification using first-principles biophysical modeling of DNA hybridization and polymerization. We compare this kinetic model with prior PCR models and discuss the features of our model that are essential for quantitative prediction of DNA amplification efficiency for arbitrary sequences and operating conditions. Using this model, the kinetics of PCR is analyzed. The ability of the model to distinguish between the dynamic evolution of distinct DNA sequences in DNA amplification reactions is demonstrated. The kinetic model is solved for a typical PCR temperature protocol to motivate the need for optimization of the dynamic operating conditions of DNA amplification reactions. It is shown that amplification efficiency is affected by dynamic processes that are not accurately represented in the simplified models of DNA amplification that form the basis of conventional temperature cycling protocols. Based on this analysis, a modified temperature protocol that improves PCR efficiency is suggested. Use of this sequence-dependent kinetic model in a control theoretic framework to determine the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is discussed.     

A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction.

X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' region of the MYC gene of cells of the human bladder carcinoma cell line, EJ-30, by using PCR with inverted primers. When the cells were allowed to repair/misrepair for 6 or 23 h after irradiation, the frequency of rearrangements increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for unirradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(-5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy. No significant difference was observed between 6 and 23 h of repair. Sequences obtained from the products suggest that there was no homology between the two sequences involved in the recombination event and that there was no clustering of breakpoints. The procedure is relatively simple, requiring only one digestion with a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located between a pair of primer sites 120 bp apart for which the primers face in opposite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained. However, if an intrachromosomal rearrangement has occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occurred either within the same chromatid or between two sister chromatids, respectively. Digestion between the primers would linearize an episome or release a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be facing each other and would be located on either end of the linear molecule; and if they are less than approximately 5 kb apart, PCR amplification should result in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants. Also, quantification is based on the number of PCR products detected in a known amount of DNA, and not on a precise determination of the amount of PCR amplification that has occurred. Thus the inverse PCR procedure has the potential ofbeing used as an assay to detect variations in radiation-induced frequencies of DNA rearrangements.     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.     

PCR assay of DNA damage and repair at the gene level in brain and spleen of gamma-irradiated young and old rats.

The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.     

Novel amplification of DNA in a hairpin structure: towards a radical elimination of PCR errors from amplified DNA

Errors introduced during PCR amplification set a selectivity limit for microsatellite analysis and molecular mutation detection methods since polymerase misincorporations invariably get confused with genuine mutations. Here we present hairpin-PCR, a new form of PCR that completely separates genuine mutations from polymerase misincorporations. Hairpin-PCR operates by converting a DNA sequence to a hairpin following ligation of oligonucleotide caps to DNA ends. We developed conditions that allow a DNA hairpin to be efficiently PCR-amplified so that, during DNA synthesis, the polymerase copies both DNA strands in a single pass. Consequently, when a misincorporation occurs it forms a mismatch following DNA amplification, and is distinguished from genuine mutations that remain fully matched. Error-free DNA can subsequently be isolated using one of many approaches, such as dHPLC or enzymatic depletion. We present feasibility for the main technical steps involved in this new strategy, conversion of a sequence to a hairpin that can be PCR-amplified from human genomic DNA, exponential amplification from picogram amounts, conversion of misincorporations to mismatches and separation of homoduplex from heteroduplex hairpins using dHPLC. The present hairpin-PCR opens up the possibility for a radical elimination of PCR errors from amplified DNA and a major improvement in mutation detection.     

DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA

We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C→T and G→A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. In addition, ‘jumping PCR’, i.e. the occurrence of template switching during PCR, may contribute to these substitutions. When DNA sequences are amplified from ancient DNA extracts where few template molecules initiate the PCR, precautions such as DNA sequence determination of multiple clones derived from more than one independent amplification are necessary in order to reduce the risk of determination of incorrect DNA sequences. When such precautionary measures are taken, errors induced by damage to the DNA template are unlikely to be more frequent than ~0.1% even under the unlikely scenario where each amplification starts from a single template molecule.     

A rapid and inexpensive method for the direct PCR amplification of DNA from plants

? Premise of the study: We present a rapid and inexpensive alternative to DNA isolation for polymerase chain reaction (PCR) amplification from plants. ? Methods and Results: The method involves direct PCR amplification from material macerated in one buffer, followed by dilution and incubation in a second buffer. We describe the procedure and demonstrate its application for nuclear and plastid DNA amplification across a broad range of vascular plants. ? Conclusions: The method is fast, easy to perform, cost-effective, and consequently ideal for large sample numbers. It represents a considerable simplification of present approaches requiring DNA isolation prior to PCR amplification and will be useful in plant systematics and biotechnology, including applications such as DNA barcoding.     

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination^{1, 2}. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)^3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion³. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required^2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues^6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.     

Extraction, amplification and characterization of wood DNA from dipterocarpaceae

A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood if suitable information on genetic variation patterns within and among species is available. Potential applications are in forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites.     

Repair of DNA damage in a mitochondrial lysate of Xenopus laevis oocytes.

We examined DNA repair activities of a mitochondrial lysate derived from Xenopus laevis oocytes. Plasmid DNA, exposed to HCl, H2O2 or UV light, was used as the substrate for the in vitro repair reaction. DNA synthesis in the lysate was stimulated 2-8-fold by such lesions, indicating the presence of excision repair activities. This repair DNA synthesis was not affected by aphidicolin, but was sensitive to N-ethylmaleimide. Thus the mitochondrial DNA polymerase, i.e., pol gamma is indeed involved in the reaction. Actual repair of the depurinated DNA was demonstrated by using the polymerase chain reaction (PCR), where the amount of the amplified DNA fragment increased significantly if the depurinated template was incubated in the lysate prior to the PCR. UV-irradiated DNA, on the other hand, restored its ability as a PCR template only if the repair reaction was carried out under the light. Therefore, in this system, UV-induced damage is repaired mainly by photoreactivation. These results show that mitochondria of Xenopus oocytes possess excision repair as well as photolyase activities, and that the in vitro repair system described here should be useful for further molecular characterization of such DNA repair machinery.