Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.     

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.

A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

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.     

Simplified DNA Extraction and Improved PCR Based Detection of Greening Bacterium in Citrus

Citrus greening disease caused by a fastidious bacterium is an important graft transmissible disease in commercial citrus in India and other parts of the world. Polymerase chain reaction (PCR) is a sensitive and convenient method for detection of greening bacterium. A non-phenol chloroform method of DNA extraction was evaluated for DNA quality and PCR based detection of greening bacterium. The method was comparable with a commercial DNA extraction kit (Qiagen) and better than a CTAB based DNA extraction method. To improve the reliability, three primer sets (primers A, B, and C yielding amplicons of 1160 bp, 703 bp and 451 bp, respectively) and two polymerase enzymes (Taq polymerase and Klen Taq polymerase) were evaluated. The primer set C provided better amplification when compared to primer sets A and B. Primer C in combination with Taq polymerase provided amplification band at a DNA template concentration of 100 pg but good amplification band was obtained at still lower DNA template concentration of 0.1 pg when Klen Taq polymerase was used. The standardized PCR protocol combining non-phenol chloroform method of DNA isolation, primer set C and Klen Taq polymerase enzyme was found very effective in detecting greening bacterium in citrus trees. The sequence of cloned amplicon from 16S ribosomal RNA gene had 89–100 % sequence identity with corresponding sequence of Candidatus Liberibacter asiaticus from China, Brazil, Japan and Pune isolate of India, C. Liberibacter americnus from Brazil and C. Liberibacter africanus from Africa.     

Interrogation of multimeric DNA amplification products by competitive primer extension using bst DNA polymerase (large fragment).

Linear dsDNA composed of tandem repeats may be exponentially amplified by the strongly strand-displacing Bst DNA polymerase (large fragment) and two primers specific for opposite strands. When the repetitive DNA is derivedfrom rolling circle replication of a circular template, the reaction is termed cascade rolling circle amplification (CRCA). We have developed a variant of CRCA in which one primer is attached to the surface of a microwell and the other is labeled, thus enabling detection of amplified material using an ELISA-like protocol. The circular template is derived by annealing and ligation of a padlock on target DNA. It was found that there was good correlation between the synthesis of amplified material and signal. The specificity of the reaction with respect to single-nucleotide polymorphisms was investigated, and it was found that Bst DNA polymerase is prone to extension from primers with mismatched 3' ends. Reliable single nucleotide specificity was only obtained when pre-synthesized amplified material was interrogated by competitive primer extension.     

Harnessing asymmetrical substrate recognition by thermostable EndoV to achieve balanced linear amplification in multiplexed SNP typing.

Multiplexed amplification of specific DNA sequences, by PCR or by strand-displacement amplification, is an intrinsically biased process. The relative abundance of amplified DNA can be altered significantly from the original representation and, in extreme cases, allele dropout can occur. In this paper, we present a method of linear amplification of DNA that relies on the cooperative, sequence-dependent functioning of the DNA mismatch-repair enzyme endonuclease V (EndoV) from Thermotoga maritima (Tma) and Bacillus stearothermophilus (Bst) DNA polymerase. Tma EndoV can nick one strand of unmodified duplex DNA, allowing extension by Bst polymerase. By controlling the bases surrounding a mismatch and the mismatch itself, the efficiency of nicking by EndoV and extension by Bst polymerase can be controlled. The method currently allows 100-fold multiplexed amplification of target molecules to be performed isothermally, with an average change of <1.3-fold in their original representation. Because only a single primer is necessary, primer artefacts and nonspecific amplification products are minimized.     

Amplification of target-specific,ligation-dependent circular probe

We describe a novel polymerase chain reaction (PCR)-based gene amplification method utilizing a circularizable oligodeoxyribonucleotide probe (C-probe). The C-probe contains two target complementary regions located at each terminus and an interposed generic PCR primer binding region. The hybridization of C-probe to a target brings two termini in direct apposition as the complementary regions of C-probe wind around the target to form a double helix. Subsequent ligation of the two termini results in a covalently linked C-probe that becomes `locked on to' the target. The circular nature of the C-probe allows for the generation of a multimeric single-stranded DNA (ssDNA) via extension of the antisense primer by Taq DNA polymerase along the C-probe and displacement of downstream strand, analogous to `rolling circle' replication of bacteriophage in vivo. This multimeric ssDNA then serves as a template for multiple sense primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. Subsequent thermocycling denatures the dsDNA and initiates the next round of primer extension and ramification. This model results in significantly improved amplification kinetics (super-exponential) as compared to conventional PCR. Our results show that the C-probe was 1000 times more sensitive than the corresponding linear hemiprobes for detecting Epstein–Barr virus early RNA. The C-probe not only increases the power of amplification but also offers a means for decontaminating carryover amplicons. As the ligated C-probes possess no free termini, they are resistant to exonuclease digestion, whereas contaminated linear amplicons are susceptible to digestion. Treatment of the ligation reaction mixture with exonuclease prior to amplification eliminated the amplicon contaminant, which could also have been co-amplified with the same PCR primers; only the ligated C-probes were amplified. The combined advantages of the C-probe and thermocycling have a broad applicability for the detection of both DNA and RNA. Finally, we described a novel isothermal amplification method, ramification extension amplification, utilizing circular nature of C-probe and displacement activity of DNA polymerase.     

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.     

Single base pair mutation analysis by PNA directed PCR clamping.

A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.     

Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves

We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3' end of the forward or reverse primer, and additionally incorporating a 3' mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.     

用寡核苷酸本身作为PCR引物检测其重组DNA

以待检测的寡核苷酸本身作为一个引物,加上两个载体特异引物,组成两对PCR引物。含待检测寡核苷酸片段的重组DNA用这两对引物可分别扩增出两个大小不同的片段,而载体DNA只有一对引物（即载体特异引物）可扩增出一个较小的片段。     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.     

长片段融合PCR在构建烟曲霉rho1基因回补株中的应用

目的融合PCR是一种常用的构建重组片段或重组质粒的手段,但长片段融合PCR的难度较大。文中将探讨长片段融合PCR过程中引物设计及扩增条件对产物的影响。方法以构建烟曲霉rho 1基因回补株为例,采用融合PCR的方法扩增重组片段(长达6.5 kb),在引物设计时引入不同大小的同源区,并设置不同的扩增体系。结果当设计引物的同源区为35 bp,选用具有高扩增效率、高保真性的DNA聚合酶,以及各片段在融合PCR反应体系中的浓度为15 ng/μL时,实现了长达6.5 kb的片段扩增并完成了烟曲霉rho 1回补株的构建。结论在合适的PCR引物设计、片段浓度配比及聚合酶条件下,长片段融合PCR在丝状真菌的基因敲除及回补株的构建中是一种非常有效的工具。     

痘病毒科不同病毒属特异的PCR检测方法的建立

目的:建立可准确、快速地鉴别诊断可感染人的不同属痘病毒的特异PCR方法。方法:设计针对正痘病毒属、副痘病毒属和传染性软疣病毒属的多对特异引物,并制备相应的DNA模板,针对不同的模板优化引物与反应条件,分别进行检测筛选,建立病毒属特异的单独与多重PCR方法。结果:单一模板的PCR扩增反应中,正痘病毒的检测敏感性可达101拷贝/μL(引物为OPEaL-F1880/OPEaL-R2057),副痘病毒的检测敏感性可达101拷贝/μL(引物为PP2/PP3),传染性软疣病毒的检测敏感性为100 pg/μL体系(引物为MCV1/MCV2);混合模板的PCR扩增反应中,各属特异的引物均可获得预期大小的特异片段。结论:我们建立的PCR诊断方法,可用于痘病毒科不同病毒属感染的实验室特异快速鉴别诊断。     

Detection and Identification of Brenneria nigrifluens, the Causal Agent of the Shallow Bark Canker of Walnut by, PCR Amplification

A 1 kb DNA band from strains of Brenneria nigrifluens, as shown by amplification of their genomic DNA by polymerase chain reaction (PCR) using minisatellite primer designed on the minisatellite sequence of the M13 phage, was isolated, cloned and sequenced. Specific oligonucleotides (F1–C3) were selected into this 1 kb DNA sequence and used in a PCR assay to detect and identify strains of B. nigrifluens . Several strains of B. nigrifluens were assessed with F1–C3 primers producing a specific band of approximately 250 bp pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and directly from infected walnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Brenneria, Erwinia, Agrobacterium, Pseudomonas, Ralstonia, Pectobacterium, Xanthomonas and from walnut-associated bacteria, indicating the specificity of these primers. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for B. nigrifluens and a useful tool for epidemiological studies.     

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.     

PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue.

A PCR assay which allows detection and quantification of Epichloë endophytes in tissues of the grass Bromus erectus is described. PCR with specific primers flanking a microsatellite-containing locus (MS primers) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material. When annealing temperatures were optimized, all Epichloë and Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure caused by inhibitory plant compounds present in DNA extracts. For quantitative applications, a heterologous control template in primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloë in plant DNA extracts. The fungal DNA present in infected leaves of B. erectus between 1 and 20 pg per 100 ng of leaf DNA, but the amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that leaves were colonized in a uniform way.     

Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae , the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein ( Ypt 1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae . The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt 1F/ Ypt 1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

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.