Enhancement of PCR Amplification of Moderate GC-Containing and Highly GC-Rich DNA Sequences

PCR is a commonly used and highly efficient technique in biomolecular laboratories for specific amplification of DNA. However, successful DNA amplification can be very time consuming and troublesome because many factors influence PCR efficiency. Especially GC-rich DNA complicates amplification because of generation of secondary structures that hinder denaturation and primer annealing. We investigated the impact of previously recommended additives such as dimethylsulfoxide (DMSO), magnesium chloride (MgCl₂), bovine serum albumin (BSA), or formamide. Furthermore, we tested company-specific substances as Q-Solution, High GC Enhancer, and Hi-Spec; various actively promoted polymerases as well as different PCR conditions for their positive effects on DNA amplification of templates with moderate and extremely high CG-content. We found considerable differences of specificity and quantity of product between different terms. In this article, we introduce conditions for optimized PCR to help resolve problems amplifying moderate to high GC-rich templates.     

内参基因加标法定量土壤微生物目标基因绝对拷贝数

【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因的绝对拷贝数进行荧光定量PCR检测,用来表征该样品中细菌群落总体丰度。在定量前通过双向引物扩增方法验证突变质粒中的内参基因对供试土壤的特异性。【结果】不同水稻土壤样品的DNA提取量在样品间差异较大。通过内参基因加标法对DNA提取量进行校正,显著提高了16S r RNA基因绝对定量的精确度。不同水稻土壤样品间的变异系数为17.8,与未加标处理相比降低了66.7%。在此基础上,进一步通过内参基因加标法对土壤有机质和含水率均呈现典型空间特征差异的6处亚热带湿地土壤样品中的16S r RNA基因进行绝对定量。16S r RNA基因绝对拷贝数与土壤微生物生物量碳具有显著的线性相关性(R2=0.694,P0.001),表明内参校正后的16S r RNA基因绝对拷贝数可以准确反映单位质量土壤中微生物的丰度。【结论】内参基因加标法可以对DNA提取得率以及腐殖酸对PCR扩增的抑制性进行校正,从而提高绝对定量的准确性。基于内参基因加标法的目标基因绝对定量PCR检测,可作为土壤微生物生物量测量,以及微生物功能基因绝对丰度定量的一种核酸检测方法。     

Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8

Quantitative PCR is becoming the method of choice for the detection of pathogenic microorganisms and other targets in the environment. A major obstacle when amplifying DNA is the presence of inhibiting substances like humic acids that decrease the efficiency of PCR. We combined the polymeric adsorbent Supelite™ DAX-8 with a large-volume (10 mL) nucleic acid extraction method to decrease the humic acid content prior to qPCR quantification in water samples. The method was tested by spiking with humic acid standards and the bacterial surrogate Acinetobacter baylyi ADP1. Improvements in qPCR detection of ADP1 after application of DAX-8 resin (5 and 10 w/v%) were compared with the effects of added bovine serum albumin (BSA) (50, 100 and 200 ng/μL). Both additions improved detection of ADP1 by counteracting inhibitory effects. There were no changes in mean cycle threshold difference (ΔC_T) after application of DAX-8 compared to the control despite some loss of DNA, whereas significant increases occurred for BSA, irrespective of BSA concentration applied. The use of DAX-8 leads to an increase in qPCR amplification efficiency in contrast to BSA. The commonly used method to calculate genomic sample concentrations by comparing measured CT values relative to standard curves is only valid if amplification efficiencies of both are sufficiently similar. DAX-8 can provide this efficiency by removing humic acids permanently from nucleic acid extracts and has the potential to significantly increase the reliability of reported non-detects and measured results obtained by qPCR in environmental monitoring.     

Molecular quantification of environmental DNA using microfluidics and digital PCR

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58ngμL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34ngμL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3)copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2)copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.     

BSA reduces inhibition in a TaqMan assay for the detection of Batrachochytrium dendrobatidis

A TaqMan assay for the causative agent of chytridiomycosis in amphibians (Batrachochytrium dendrobatidis) can be inhibited by phenolic compounds, including humic and tannic acids, resulting in false negatives. Bovine serum albumin (BSA) is known to reduce inhibition of PCR when samples are contaminated with these inhibitors. We assessed the effect of BSA in reducing inhibition of the TaqMan assay when analyzing skin swabs for B. dendrobatidis. We found that the addition of BSA to the TaqMan reaction reduced inhibition to insignificant levels. BSA did not appreciably affect the efficiency or analytical sensitivity of the TaqMan reaction in the analysis of standard DNA solutions free from environmental inhibitors. We recommend the addition of 400 ng microl(-1) of BSA to the standard TaqMan assay to reduce inhibition associated with sampling wild amphibians.     

Optimization of DNA Recovery and Amplification from Non-Carbonized Archaeobotanical Remains

Ancient DNA (aDNA) recovered from archaeobotanical remains can provide key insights into many prominent archaeological research questions, including processes of domestication, past subsistence strategies, and human interactions with the environment. However, it is often difficult to isolate aDNA from ancient plant materials, and furthermore, such DNA extracts frequently contain inhibitory substances that preclude successful PCR amplification. In the age of high-throughput sequencing, this problem is even more significant because each additional endogenous aDNA molecule improves analytical resolution. Therefore, in this paper, we compare a variety of DNA extraction techniques on primarily desiccated archaeobotanical remains and identify which method consistently yields the greatest amount of purified DNA. In addition, we test five DNA polymerases to determine how well they replicate DNA extracted from non-charred ancient plant remains. Based upon the criteria of resistance to enzymatic inhibition, behavior in quantitative real-time PCR, replication fidelity, and compatibility with aDNA damage, we conclude these polymerases have nuanced properties, requiring researchers to make educated decisions as to which one to use for a given task. The experimental findings should prove useful to the aDNA and archaeological communities by guiding future research methodologies and ensuring precious archaeobotanical remains are studied in optimal ways, and may thereby yield important new perspectives on the interactions between humans and past plant communities.     

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10¹ to 10² bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.     

Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen detection

Real-time PCR is a new and highly sensitive method for the quantification of microbial organisms in environmental samples. This work was conducted to evaluate real-time PCR with SybrGreen (SG) detection as quantification method for Desulfotomaculum lineage 1 organisms in samples of rice field soil. The method was optimized in several parameters like SG concentration. These allowed quantitative PCR with different primer combinations yielding PCR products with lengths up to 1066 bp and with sensitivities of 10(2) targets for all assays. The detection limit in environmental DNA extracts (rice bulk soil and rice roots) was 10(6) targets per gram dry weight according to the dilution of the DNA extracts necessary to overcome PCR inhibition of humic substances. A verification, that the fluorescence increase was due to specific PCR products, was done by agarose gel electrophoresis since melting curve analysis of the PCR products did not show a distinct peak in the first derivative, when the environmental DNA extracts were used in PCR. Amplification with a primer combination specific for Desulfotomaculum lineage 1 organisms showed an abundance of this group of approximately 2% and 0.5% of the eubacterial 16S rDNA targets in rice bulk soil and rice root samples, respectively. Approximately half of this number was obtained in both habitats with a PCR assay specific for a Desulfotomaculum sequence cluster obtained previously from rice field soil.     

Direct extraction of DNA from soils for studies in microbial ecology

Molecular analyses for the study of soil microbial communities often depend on the extraction of DNA directly from soils. These extractions are by no means trivial, being complicated by humic substances that are inhibitory to PCR and restriction enzymes or being too highly colored for blot hybridization protocols. Many different published protocols exist, but none have been found to be suitable enough to be generally accepted as a standard. Most direct extraction protocols start with relatively harsh cell breakage steps such as bead-beating and freeze-thaw cycles, followed by the addition of detergents and high salt buffers and/or enzymic digestion with lysozyme and proteases. After typical organic extraction and alcohol precipitation, further purification is usually needed to remove inhibitory substances from the extract. The purification steps include size-exclusion chromatography, ion-exchange chromatography, silica gel spin columns, and cesium chloride gradients, among others. A direct DNA extraction protocol is described that has been shown to be effective in a wide variety of soil types. This protocol is experimentally compared to several published protocols.     

Effects of the humic substances of de-inking paper sludge on the antagonism between two compost bacteria and Pythium ultimum

We investigated the in vitro influence of humic substances (HS) extracted from de-inking paper sludge compost on the inhibition of Pythium ultimum by two compost bacteria, Rhizobium radiobacter (Agrobacterium radiobacter) and Pseudomonas aeruginosa. When low concentrations (5 or 50 mg l(-1)) of HS were added to the culture medium, fungal inhibition by R. radiobacter significantly increased (P<0.01) by 2-3%. In contrast, these low levels of HS had no effect on P. ultimum inhibition by P. aeruginosa. The Fe, chelated by HS, was in part responsible for the decrease of P. ultimum inhibition by the bacteria when increasing amounts of HS were added in the culture medium. The addition of 500 mg l(-1) of humic acids isolated from de-inking paper sludge compost or from fossil origin completely eliminated the inhibition of P. ultimum by R. radiobacter. This Fe effect also stimulated growth of R. radiobacter and reduced its siderophore production in a minimal medium supplemented with HS as sole source of Fe. The results showed that HS influence microbial antagonism when added to a culture medium. However, this effect varies with different factors such as the type of bacteria, concentration of HS, molecular weight and Fe content.     

TERMINAL PHOSPHATE LABELED NUCLEOTIDES: SYNTHESIS,APPLICATIONS, AND LINKER EFFECT ON INCORPORATION BY DNA POLYMERASES

A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.     

Terminal phosphate labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases

A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.     

Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein.

The benefits of adding bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to PCR were evaluated with reaction mixtures containing substances that inhibit amplification. Whereas 10- to 1,000-fold more FeCl3, hemin, fulvic acids, humic acids, tannic acids, or extracts from feces, freshwater, or marine water were accommodated in PCR when either 400 ng of BSA per microl or 150 ng of gp32 per microl was included in the reactions, neither BSA nor gp32 relieved interference significantly when minimum inhibitory levels of bile salts, bilirubin, EDTA, NaCl, sodium dodecyl sulfate, or Triton X-100 were present. Use of BSA and gp32 together offered no more relief of inhibition than either alone at its optimal level, and neither protein had any noticeable effect on amplification in the absence of inhibitors.     

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 “inhibited” crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.     

Effects of humic substances on fluorometric DNA quantification and DNA hybridization

DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities. Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA amended with humic substances (20-80 ng/microl) using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80 ng/microl of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane. Low amounts (<2.0 microg) of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 microg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes. Therefore, we suggest that DNA samples containing low concentrations of humic substances (<20 ng/microl) could be used in quantitative membrane hybridization without further purification.     

DNA polymerases as therapeutic targets

Numerous pathological states, including cancer, autoimmune diseases, and viral/bacterial infections, are often attributed to uncontrollable DNA replication. Inhibiting this essential biological process provides an obvious therapeutic target against these diseases. A logical target is the DNA polymerase, the enzyme responsible for catalyzing the addition of mononucleotides to a growing polymer using a DNA or RNA template as a guide for directing each incorporation event. This review provides a summary of therapeutic agents that target polymerase activity. A discussion of the biological function and mechanism of polymerases is first provided to illustrate the strategy for therapeutic intervention as well as the rational design of various nucleoside analogues that inhibit various polymerases associated with viral infections and cancer. The development of nucleoside and non-nucleoside inhibitors as antiviral agents is discussed with particular emphasis on their mechanism of action, structure-activity relationships, toxicity, and mechanism of resistance. In addition, commonly used anticancer agents are described to illustrate the similarities and differences associated with various nucleoside analogues as therapeutic agents. Finally, new therapeutic approaches that include the inhibition of selective polymerases involved in DNA repair and/or translesion DNA synthesis as anticancer agents are discussed.     

Comparison and optimization of ancient DNA extraction

Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success.     

Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study

Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.     

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.     

Humic substances cause fluorescence inhibition in real-time polymerase chain reaction

Real-time polymerase chain reaction (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, that is, amplification inhibition. Humic substances (HS) are well-known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, that is, quench the fluorescence signal of double-stranded DNA (dsDNA) binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I, and SYTO 82, generating lowered amplification plots, although amplicon production was unaffected. For EvaGreen, 500 ng of HA quenched nearly all fluorescence, whereas 1000 ng of HA completely inhibited amplification when applying Immolase DNA polymerase with bovine serum albumin (BSA). Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.