Potential influence of the first PCR cycles in real-time comparative gene quantifications

There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1-3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.     

On the causes,consequences, and avoidance of PCR duplicates: Towards a theory of library complexity

Library preparation protocols for most sequencing technologies involve PCR amplification of the template DNA, which open the possibility that a given template DNA molecule is sequenced multiple times. Reads arising from this phenomenon, known as PCR duplicates, inflate the cost of sequencing and can jeopardize the reliability of affected experiments. Despite the pervasiveness of this artefact, our understanding of its causes and of its impact on downstream statistical analyses remains essentially empirical. Here, we develop a general quantitative model of amplification distortions in sequencing data sets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined primarily by the ratio between library complexity and sequencing depth, and that amplification noise (including in its dependence on the number of PCR cycles) only plays a secondary role for this artefact. We confirm our predictions using new and published RAD-seq libraries and provide a method to estimate library complexity and amplification noise in any data set containing PCR duplicates. We discuss how amplification-related artefacts impact downstream analyses, and in particular genotyping accuracy. The proposed framework unites the numerous observations made on PCR duplicates and will be useful to experimenters of all sequencing technologies where DNA availability is a concern.     

Antisense PCR: A simple and robust method for performing nested single-tube PCR

To overcome the disadvantages of two-round nested PCR, we developed a simple and robust closed single-tube nested PCR method (antisense PCR). The method uses antisense oligonucleotides that carry a 5′ tag and that can potentially hybridize to the 3′ ends of the outer primers, depending on the annealing temperature. During initial cycles, which are performed at a high annealing temperature, the antisense oligonucleotides do not hybridize and amplification is directed by the outer primers. During later cycles, for which the annealing temperature is decreased, the outer primers hybridize to the antisense oligonucleotides, extend to produce sequences that are mismatched to the amplicon templates, and consequently become inactivated, whereas the inner primers hybridize to the amplicon templates and continue amplification. Antisense quantitative PCR (qPCR) was compared with one-round qPCR for real-time amplification of four PCR targets (BCR, APC, N-RAS, and a rearranged IGH gene). It had equal amplification efficiency but produced much less nonspecific amplification. Antisense PCR enables both endpoint detection and real-time quantification. It can substitute for two-round nested PCRs but may also be applicable to instances of one-round PCR in which nonspecificity is a problem.     

Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications

Quantitative real-time PCR has revolutionized many aspects of genetic research, biomedical diagnostics and pathogen detection. Nevertheless, the full potential of this technology has yet to be realized, primarily due to the limitations of the threshold-based methodologies that are currently used for quantitative analysis. Prone to errors caused by variations in reaction preparation and amplification conditions, these approaches necessitate construction of standard curves for each target sequence, significantly limiting the development of high-throughput applications that demand substantive levels of reliability and automation. In this study, an alternative approach based upon fitting of fluorescence data to a four-parametric sigmoid function is shown to dramatically increase both the utility and reliability of quantitative real-time PCR. By mathematically modeling individual amplification reactions, quantification can be achieved without the use of standard curves and without prior knowledge of amplification efficiency. Combined with provision of quantitative scale via optical calibration, sigmoidal curve-fitting could confer the capability for fully automated quantification of nucleic acids with unparalleled accuracy and reliability.     

The real-time polymerase chain reaction

The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.     

Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.     

Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value

For real-time monitoring of PCR amplification of DNA, quantitative PCR (qPCR) assays use various fluorescent reporters. DNA binding molecules and hybridization reporters (primers and probes) only fluoresce when bound to DNA and result in the non-cumulative increase in observed fluorescence. Hydrolysis reporters (TaqMan® probes and QZyme? primers) become fluorescent during DNA elongation and the released fluorophore remains fluorescent during further cycles; this results in a cumulative increase in observed fluorescence. Although the quantification threshold is reached at a lower number of cycles when fluorescence accumulates, in qPCR analysis no distinction is made between the two types of data sets. Mathematical modeling shows that ignoring the cumulative nature of the data leaves the estimated PCR efficiency practically unaffected but will lead to at least one cycle underestimation of the quantification cycle (C_q value), corresponding to a 2-fold overestimation of target quantity. The effect on the target–reference ratio depends on the PCR efficiency of the target and reference amplicons. The leftward shift of the C_q value is dependent on the PCR efficiency and with sufficiently large C_q values, this shift is constant. This allows the C_q to be corrected and unbiased target quantities to be obtained.     

Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci

In this study, we have evaluated the efficacy and the validity of the AmpFISTR SGM plus multiplex PCR typing system when Low Copy Number (LCN) amounts of DNA are processed. The characteristics of SGM plus profiles produced under LCN conditions were studied on the basis of heterozygote balance, between loci balance and stutter proportion based on profiles that were obtained from a variety of mock casework samples. These experiments clearly showed that LCN DNA profiles carry their own characteristic features, which must be taken into account during interpretation. Herewith, we confirmed the data of recent other studies that a comprehensive interpretation strategy is dependent upon multiple replication of the PCR using the same extract together with the proper use of extraction and amplification controls. The limitations of LCN DNA analysis were further studied in a series of single cell PCR experiments using an amplification regime of 34 PCR cycles. The allele dropout phenomenon was demonstrated to its full extent when single cells were analysed. However, the "consensus profile" which was obtained from separate single cell PCR experiments matched the actual profile of the cell donor. Single cell PCR experiments also showed that a further increase of the number of PCR cycles did not result in enhanced sensitivity and had a highly negative effect on the balance of this multiplex PCR system which hampered correct interpretation of the profile. Also, the potential of LCN typing in analysing mixtures of DNA was investigated. It was clearly shown that LCN typing had no advantages over 28 cycles amplification in the detection of the minor component of DNA-mixtures. In addition to the 34 cycles PCR amplification regime, the utility of a new approach that involved reamplification of the 28 cycle SGM plus PCR products with an extra 6 PCR cycles after the addition of fresh AmpliTaq Gold DNA Polymerase was investigated. This approach provides the scientist with an extra typing result that enhances the reliability of the consensus profile, which is commonly retrieved from two separate 34 cycle PCR results. Furthermore, the 28 + 6 cycles approach may be used to screen LCN samples for their potential to produce a 34 PCR cycle profile. Finally and as a last resort the 28 + 6 cycles approach can be used in those cases where no further extract from the crime sample is available. Finally, the potential of LCN typing was demonstrated in typing samples from non-probative and actual casework examples. From a high proportion of samples that failed to demonstrate SGM plus typing results using the standard protocol of 28 cycles, at least partial profiles could be obtained after LCN methods were used. For example, LCN typing was applied in a case where 10-year old samples from bones and teeth that were retrieved from a mass grave had to be identified. This study resulted in the positive identification of a number of victims by comparing the LCN DNA profiles with the profiles from putative relatives. The value of LCN DNA typing was further demonstrated in a strangulation case. The throat of the victim was sampled and only after 34 PCR cycles were we able to reveal that the evidential sample contained a distinct mixture of the victim's own DNA and the DNA of the defendant.     

Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR.

The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Taq DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.     

重要肠道病原菌多重PCR-基因芯片检测方法研究

目的:建立并初步评价一种针对重要肠道病原菌的多重PCR 基因芯片检测方法。方法：对筛选出的特异引物进行多重PCR优化,将引物分别按种属内混合和种属间混合的方案排查引物间的竞争性抑制现象,再将不同菌属的模板混合,用相对应的混合引物扩增,探寻高效特异的引物组合。分别掺入和不掺入荧光素,验证其对混合PCR反应的影响,并与芯片杂交,探寻多重PCR扩增效率对芯片杂交的影响。分析不同数量引物组合产生的杂交结果,筛选出无交叉反应的最优引物组合。结果:种属内引物混合均得到特异性扩增结果。种属间混合霍乱弧菌和空肠弯曲菌得到部分预期条带,随着混合引物数量的增加,交叉抑制现象也增多。杂交信号强度随多重PCR扩增效率的增加而增强。反应中掺入荧光素的扩增条带产量低于无荧光素的产物。可将35对混合引物拆成3个体系分别标记样品,以避免假阴性结果。结论:PCR反应中掺入荧光素降低扩增效率和杂交效率,但并不影响对杂交结果的判读和数据分析。基因芯片杂交信号强度取决于多重PCR的扩增效率。肠道病原菌多重PCR 基因芯片检测方法具有较高的特异性,混合PCR可以分别按照种属内和种属间的引物组合方案用于多病原的筛检。该基因芯片检测可以采用3个引物体系完成样品标记。     

A general model of error-prone PCR

In this paper, we generalize a previously-described model of the error-prone polymerase chain reaction (PCR) reaction to conditions of arbitrarily variable amplification efficiency and initial population size. Generalisation of the model to these conditions improves the correspondence to observed and expected behaviours of PCR, and restricts the extent to which the model may explore sequence space for a prescribed set of parameters. Error-prone PCR in realistic reaction conditions is predicted to be less effective at generating grossly divergent sequences than the original model. The estimate of mutation rate per cycle by sampling sequences from an in vitro PCR experiment is correspondingly affected by the choice of model and parameters.     

PCR bias in ecological analysis: a case study for quantitative Taq nuclease assays in analyses of microbial communities

Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69-75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 10(4)-fold excess of phylogenetically similar DNA and of an up to 10(7)-fold excess of dissimilar DNA.     

Enhancing the efficiency of a PCR using gold nanoparticles

We found that the PCR could be dramatically enhanced by Au nanoparticles. With the addition of 0.7 nM of 13 nm Au nanoparticles into the PCR reagent, the PCR efficiency was increased. Especially when maintaining the same or higher amplification yields, the reaction time could be shortened, and the heating/cooling rates could be increased. The excellent heat transfer property of the nanoparticles should be the major factor in improving the PCR efficiency. Different PCR systems, DNA polymerases, DNA sizes and complex samples were compared in this study. Our results demonstrated that Au nanoparticles increase the sensitivity of PCR detection 5- to 10-fold in a slower PCR system (i.e. conventional PCR) and at least 10⁴-fold in a quicker PCR system (i.e. real-time PCR). After the PCR time was shortened by half, the 100 copies/µl DNA were detectable in real-time PCR with gold colloid added, however, at least 10⁶ copies/µl of DNA were needed to reach a detectable signal level using the PCR reagent without gold colloid. This innovation could improve the PCR efficiency using non-expensive polymerases, and general PCR reagent. It is a new viewpoint in PCR, that nanoparticles can be used to enhance PCR efficiency and shorten reaction times.     

Kinetic Outlier Detection (KOD) in real-time PCR

Real-time PCR is becoming the method of choice for precise quantification of minute amounts of nucleic acids. For proper comparison of samples, almost all quantification methods assume similar PCR efficiencies in the exponential phase of the reaction. However, inhibition of PCR is common when working with biological samples and may invalidate the assumed similarity of PCR efficiencies. Here we present a statistical method, Kinetic Outlier Detection (KOD), to detect samples with dissimilar efficiencies. KOD is based on a comparison of PCR efficiency, estimated from the amplification curve of a test sample, with the mean PCR efficiency of samples in a training set. KOD is demonstrated and validated on samples with the same initial number of template molecules, where PCR is inhibited to various degrees by elevated concentrations of dNTP; and in detection of cDNA samples with an aberrant ratio of two genes. Translating the dissimilarity in efficiency to quantity, KOD identifies outliers that differ by 1.3–1.9-fold in their quantity from normal samples with a P-value of 0.05. This precision is higher than the minimal 2-fold difference in number of DNA molecules that real-time PCR usually aims to detect. Thus, KOD may be a useful tool for outlier detection in real-time PCR.     

实时荧光PCR结果的四维杂交的核酸技术分析

本研究采用四维杂交试剂,测定PCR产物的特征熔点温度(temperature of melting point,Tmp),对实时荧光定量PCR所获得阳性信号(特异性的或非特异性的)的结果进行分析和确认,以使检测结论更客观.将荧光探针模式的实时荧光PCR检测后的标本再进行熔解曲线温度扫描,然后在4℃冰箱冷却5min,向反应管加入1μL四维杂交液,再按照温度扫描程序做熔解曲线实验.结果显示,加四维杂交试剂之后的熔解曲线中信号峰值是收敛的,且信噪比增大.相同扩增产物的Tmp的误差是在±1℃之内.实验结果证明,四维杂交试剂对荧光探针模式实时荧光PCR结果可进行更精细的分析和确认.     

Accurate and Precise DNA Quantification in the Presence of Different Amplification Efficiencies Using an Improved Cy0 Method

Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is a marked decrease in amplification efficiency, the Cy0 is impaired, hence determining reaction efficiency is essential to achieve a reliable quantification. The proposed improvement in Cy0 is based on the use of the kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. Three experimental models were used: inhibition of primer extension, non-optimal primer annealing and a very small biological sample. In all these models, the improved Cy0 method increased quantification accuracy up to about 500% without affecting precision. Furthermore, the stability of this procedure was enhanced integrating it with the SOD method. In short, the improved Cy0 method represents a simple yet powerful approach for reliable DNA quantification even in the presence of marked efficiency variations.