Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.     

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Background

Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.

Results

We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.

Conclusion

We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.     

Deconstructing the Polymerase Chain Reaction: Understanding and Correcting Bias Associated with Primer Degeneracies and Primer-Template Mismatches

The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3–12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled “Polymerase-exonuclease (PEX) PCR”, in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3’ end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.     

Effectiveness of Annealing Blocking Primers versus Restriction Enzymes for Characterization of Generalist Diets: Unexpected Prey Revealed in the Gut Contents of Two Coral Reef Fish Species

Characterization of predator-prey interactions is challenging as researchers have to rely on indirect methods that can be costly, biased and too imprecise to elucidate the complexity of food webs. DNA amplification and sequencing techniques of gut and fecal contents are promising approaches, but their success largely depends on the ability to amplify the taxonomic array of prey consumed and then match prey amplicons with reference sequences. When little a priori information on diet is available or a generalist predator is targeted, versatile primer sets (also referred to as universal or general primers) as opposed to group- or species-specific primer sets are the most powerful to unveil the full range of prey consumed. However, versatile primers are likely to preferentially amplify the predominant, less degraded predator DNA if no manipulation is performed to exclude this confounding DNA template. In this study we compare two approaches that eliminate the confounding predator template: restriction digestion and the use of annealing blocking primers. First, we use a preliminary DNA barcode library provided by the Moorea BIOCODE project to 1) evaluate the cutting frequency of commercially available restriction enzymes and 2) design predator specific annealing blocking primers. We then compare the performance of the two predator removal strategies for the detection of prey templates using two versatile primer sets from the gut contents of two generalist coral reef fish species sampled in Moorea. Our study demonstrates that blocking primers should be preferentially used over restriction digestion for predator DNA removal as they recover greater prey diversity. We also emphasize that a combination of versatile primers may be required to best represent the breadth of a generalist''s diet.     

An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

DNA analysis of predator faeces using high‐throughput amplicon sequencing (HTS) enhances our understanding of predator–prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair “ZBJ” to results using the novel primer pair “ANML.” To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single‐copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre‐ and post‐PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%), whereas ZBJ amplified 24–40 of 59 taxa (41%–68%). Furthermore, in an HTS comparison of field‐collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.     

Annealing control primer system for improving specificity of PCR amplification

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.     

The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.     

Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides

Summary DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5 terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3 terminus. Increasing annealing temperatures from 15° to 70° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.     

The choice of universal primers and the characteristics of the species mixture determine when DNA metabarcoding can be quantitative

DNA metabarcoding is a technique used to survey biodiversity in many ecological settings, but there are doubts about whether it can provide quantitative results, that is, the proportions of each species in the mixture as opposed to a species list. While there are several experimental studies that report quantitative metabarcoding results, there are a similar number that fail to do so. Here, we provide the rationale to understand under what circumstances the technique can be quantitative. In essence, we simulate a mixture of DNA of S species with a defined initial abundance distribution. In the simulated PCR, each species increases its concentration following a certain amplification efficiency. The final DNA concentration will reflect the initial one when the efficiency is similar for all species; otherwise, the initial and final DNA concentrations would be poorly related. Although there are many known factors that modulate amplification efficiency, we focused on the number of primer–template mismatches, arguably the most important one. We used 15 common primers pairs targeting the mitochondrial COI region and the mitogenomes of ca. 1,200 insect species. The results showed that some primers pairs produced quantitative results under most circumstances, whereas some other primers failed to do so. In conclusion, depending on the primer pair used in the PCR amplification and on the characteristics of the mixture analysed (i.e., high species richness, low evenness), DNA metabarcoding can provide a quantitative estimate of the relative abundances of different species.     

Detection of single DNA base differences by competitive oligonucleotide priming.

Synthetic DNA oligonucleotides can serve as efficient primers for DNA synthesis even when there is a single base mismatch between the primers and the corresponding DNA template. However, when the primer-template annealing is carried out with a mixture of primers and at low stringency the binding of a perfectly matched primer is strongly favored relative to a primer differing by a single base. This primer competition is observed over a range of oligonucleotide sizes from twelve to sixteen bases and with a variety of base mismatches. When coupled with the polymerase chain reaction, for the amplification of specific DNA sequences, competitive oligonucleotide priming provides a simple general strategy for the detection of single DNA base differences.     

在单个PCR管内快捷完成定点突变

采用大引物方法,利用质粒多克隆位点两侧的普通测序引物作为旁侧引物,在单个PCR管内,经2个步骤共34个循环进行定点突变. 该方法通过优化模板和引物的量达到降低PCR循环次数, 通过加入10个在68℃复性条件下的PCR循环达到增加突变效率而无需胶纯化.本方法达到平均62%的突变效率,而且全长扩增产物的产率很高.     

Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10°C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis.     

Target region amplification polymorphism: A novel marker technique for plant genotyping

The advent of large-scale DNA sequencing technology has generated a tremendous amount of sequence information for many important organisms. We have developed a rapid and efficient PCR-based technique, which uses bioinformatics tools and expressed sequence tag (EST) database information to generate polymorphic markers around targeted candidate gene sequences. This target region amplification polymorphism (TRAP) technique uses 2 primers of 18 nucleotides to generate markers. One of the primers, the fixed primer, is designed from the targeted EST sequence in the database; the second primer, the arbitrary primer, is an arbitrary sequence with either an AT-or GC-rich core to anneal with an intron or exon, respectively. PCR amplification is run for the first 5 cycles with an annealing temperature of 35°C, followed by 35 cycles with an annealing temperature of 50°C. For different plant species, each PCR reaction can generate as many as 50 scorable fragments with sizes ranging from 50–900 bp when separated on a 6.5% polyacrylamide sequencing gel. The TRAP technique should be useful in genotyping germplasm collections and in tagging genes governing desirable agronomic traits of crop plants.     

不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用

单细胞全基因组扩增(whole genome amplification, WGA)是指在单细胞水平对全基因组进行扩增的新技术,其原理是将分离的单个细胞的微量全基因组DNA进行扩增,获得高覆盖率的完整的基因组后进行高通量测序,用于揭示细胞异质性。目前,WGA方法主要包括引物延伸预扩增(primer extension preamplification PCR, PEP-PCR)、简并寡核苷酸引物PCR (degenerate oligonucleotide primed PCR, DOP-PCR)、多重置换扩增(multiple displacement amplification, MDA)、多次退火环状循环扩增(multiple annealing and looping-based amplification cycles, MALBAC)等。本文对不同的单细胞WGA方法的原理及应用情况分别进行了阐述,并对其扩增效率进行评价和比较,包括基因组覆盖度、均一性、重现性、SNV (single-nucleotide variants)和CNV (copy number variants)检测力等。综合对比不同单细胞WGA方法后发现,MALBAC的扩增均一性最高、等位基因脱扣率最低、重现性最好,且对于CNV和SNV的检测效果最好。本文还阐述了MALBAC技术在人类单精子减数重组、非整倍体分析以及人类卵细胞基因组研究中的应用。     

Development of a molecular diagnostic system to discriminate Dreissena polymorpha (zebra mussel) and Dreissena bugensis (quagga mussel)

A 3-primer PCR system was developed to discriminate invasive zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel. The system is based on: 1) universal primers that amplifies a region of the nuclear 28s rDNA gene from both species and 2) a species-specific primer complementary to either zebra or quagga mussel. The species-specific primers bind to sequences between the binding sites for the universal primers resulting in the amplification of two products from the target species and one product from the nontarget species. Therefore, nontarget products are positive amplification controls. The 3-primer system accurately discriminated zebra and quagga mussels from seven geographically distinct populations.     

多倍体罗汉果PCR-RFLP参数的优化与应用

以多倍体罗汉果DNA为材料,采用L16(4~5)正交组合试验和单因素梯度试验,研究Mg~(2+)、dNTP、引物、Taq DNA聚合酶、模板DNA浓度和退火温度、循环次数等对PCR扩增结果以及内切酶量、酶切时间对酶切反应的影响。结果表明,多倍体罗汉果RFLP最优PCR反应体系和扩增参数为:在25μL扩增反应体系中,10×Buffer 2.5μL,MgCl_2 1.5 mmol/L,dNTP 0.2 mmol/L,引物0.1μmol/L,Taq DNA聚合酶2.0 U,模板DNA 60 ng;退火温度为56℃,循环次数为35次。酶切反应体系:内切酶10×Buffer 2.0μL,内切酶5.0U,PCR产物15μL,超纯水补至20μL;酶切时间2 h。     

曼地亚红豆杉RAPD反应体系与程序的优化

以曼地亚红豆杉为研究对象,采用L16(45)正交组合实验和单因素梯度实验对MgCl2、dNTP、随机引物、Taq酶、模板DNA浓度和退火温度、循环次数等影响RAPD扩增的重要因素进行优化,以期建立最优的RAPD反应体系与程序。实验结果表明,各因素最适条件为:25μLPCR反应体系中10×Buffer2.5μL,MgCl21.5mmol/L,dNTP0.2mmol/L,随机引物0.6μmol/L,Taq酶1.0U,模板DNA80ng;退火温度为37℃,循环次数为45次。     

葡萄ISSR-PCR反应体系的建立与优化

采用正交设计L9(34)对影响葡萄ISSR-PCR反应体系的4个因素(dNTP、TaqDNA聚合酶、引物、模板DNA)在3个浓度水平上进行试验,并通过直观分析初步确定其反应体系;在此基础上,通过单因素试验探讨了dNTP、TaqDNA聚合酶、引物、模板DNA、退火温度及循环次数等因素或条件对葡萄ISSR-PCR扩增结果的影响,确定最佳反应水平。最终建立了葡萄ISSR-PCR扩增的最佳反应体系:在25μL的反应体系中,dNTP浓度0.2 mmol/L,TaqDNA聚合酶的用量0.5 U,引物浓度0.4mmol/L,DNA模板用量40 ng。反应程序:94℃预变性5 min;94℃变性1 min,52℃退火1 min,72℃延伸1 min 30 s,40次循环;最后72℃延伸10 min,10℃保存。     

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.     

Features of DNA fragments obtained by random amplified polymorphic DNA (RAPD) assays

Random amplified polymorphic DNA (RAPD) fragments were prepared from samples of Calonectris diomedea (Cory's shearwater, Aves) and Haemonchus contortus (Nematoda) DNA by polymerase chain reaction (PCR) using decamers containing two restriction enzyme sites as primers. Six of 19 studied RAPD fragments probably originated from traces of commensal microorganisms. Many rearranged fragments, absent in the original genomic DNA, were synthesized and amplified during the processing of all the DNA samples, indicating that interactions occur within and between strands during the annealing step of PCR. The model of interactions between molecular species during DNA amplification with a single arbitrary oligonucleotide primer was modified to include nested primer annealing and interactions within and between strands. The presence of these artefacts in the final RAPD have a major effect on the interpretation of polymorphism studies.