Conventional Primer Research Articles

DNA Polymerase Binding to the Primer/template Duplex Affects the Efficiency of PCR

Polymerase chain reaction(PCR) for DNA and RNA amplifications in vitro has a profound impact on modern molecular biology. Since design of proper primers is crucial for the success in PCR,many parameters have been used for primer design. However,the effect of DNA polymerase binding to primer /template duplex on PCR efficiency was not taken into account in conventional primer design programs. To reveal whether or not the DNA polymerase-primer /template binding affects the PCR efficiency,here we built structural models for the Taq DNA polymerase in complex with different primer /template sequences,and designed PCR primers according to relative binding free energies calculated by MM /GBSA method. We verified our primer design approach using Human Serum Albumin(HSA) gene and Mycobacterium tuberculosis pyrF gene,and found that the PCR efficiencies of different designed primers for both tested genes correlated well with the calculated binding free energies. Our finding indicates clearly that the DNA polymerase-primer /template binding affects the PCR efficiency significantly. Thus,the calculated binding free energy could be used as a new parameter to design efficient PCR primers.

Comparison of low molecular weight hydroxyethyl starch and human albumin as priming solutions in children undergoing cardiac surgery

Human albumin is the conventional cardiopulmonary bypass circuit primer. However, it has high manufacturing costs. Crystalloid and colloid solutions have been developed as alternatives, including a new generation of non-ionic hydroxyethyl starch (HES). The efficacy of hydroxyethyl starch with a 130 molecular weight and substitution degree of 0.4 (hydroxyethyl starch 130/0.4) was compared with human albumin for use in cardiopulmonary bypass surgery in American Society of Anesthesiologists' grade I-II pediatric congenital heart disease patients. Efficacy was evaluated by comparing perioperative hemodynamic parameters, including plasma colloid osmotic pressure, renal function, blood loss, allogeneic blood volumes and plasma volume substitution. The hydroxyethyl starch group exhibited significantly higher preoperative colloid osmotic pressure (p<0.01) and significantly lower operative renal function and postoperative allogeneic blood volumes than the human albumin group. No significant differences were observed in serum creatinine, glucose, hematocrit or lactic acid levels (p>0.05). Our results indicate that hydroxyethyl starch may be a viable alternative to human albumin in pediatric patients undergoing relatively simple cardiopulmonary bypass surgeries.

Bartonella henselaeandB. koehleraeDNA in Birds

<i>Bartonella henselae</i>and<i>B. koehlerae</i>DNA in Birds

Getting Things Backwards to Prevent Primer Dimers

This Commentary highlights the article by Satterfield that describes a new class of primer technology-cooperative primers, which prevent primer-dimer amplification.

English

Designed degenerate primers unlike conventional primers are superior in matching and amplification of large number of genes, from related gene families. DPPrimer tool was designed to predict primers for PCR amplification of homologous gene from related or diverse plant species. The key features of this tool include platform independence and user friendliness in primer design. Embedded features such as search for functional domains, similarity score selection and phylogebetic tree further enhance the user friendliness of DPPrimer tool. Performance of DPPrimer tool was evaluated by successful PCR amplification of ADP-glucose phosphorylase genes from wheat, barley and rice.AvailabilityDPPrimer is freely accessible at http://202.141.12.147/DGEN_tool/index.html

Clinical bond failure rates of adhesive precoated self-ligating brackets using a self-etching primer

To comparatively assess the failure rate of adhesive precoated (APC) self-ligating metal brackets bonded with two different enamel surface preparation techniques: self-etching primer (SEP) and conventional two-step etch and primer method (CM). Fifty-seven patients with complete permanent dentition were included in this study. A total of 1140 APC self-ligating brackets (3M Unitek, Monrovia, Calif) were bonded using a split-mouth design. For each patient, SEP (Transbond Plus SEP, 3M Unitek) and CM (37% phosphoric acid) were used in alternate quadrants. All brackets were bonded by the same investigator after pumicing and rinsing of all of the teeth. The number, site, and date of first-time bracket failures were monitored throughout orthodontic treatment (mean, 22 months). The survival rates of the brackets were estimated by Kaplan-Meier and log-rank tests (P < .05). The adhesive remnant index was used to determine the bond failure interface. The bond failure rates were 2.97% and 2.18% for the CM and SEP, respectively. No statistically significant difference in failure rates was found between the groups. The bond failure sites were predominantly at the enamel-adhesive interface in both groups. This long-term in vivo study showed that the combined use of SEP and the APC bracket system can be used effectively for bonding brackets after pumicing the enamel surfaces in clinical orthodontics.

A comparative clinical study of the failure rate of orthodontic brackets bonded with two adhesive systems: conventional and self-etching primer (SEP)

This study compared the clinical performance of orthodontic brackets bonded with Transbond adhesive paste after two primer systems: a two-stage conventional system (acid etching + Transbond XT adhesive primer) and a single-stage self-etching primer (SEP) (Transbond Plus). The sample comprised 480 metal brackets bonded to the teeth of 24 consecutive patients treated for 36 to 48 months. A split-mouth design was used for bonding, and both systems were used in each patient. Bracket failure rates for each system were analyzed; and failure causes as reported by the patients and the quadrant of teeth for which brackets failed were recorded. The conventional system group had a failure rate of 5.41%, whereas the rate for SEP was 4.58%. In this group, there were 5 failures (38.4%) in the right maxillary quadrant, 2 (15.4%) in the left maxillary quadrant, 4 (30.8%) in the right mandibular quadrant, and 2 (15.4%) in the left mandibular quadrant. In the SEP group, there were 4 (36.4%) failures in the right maxillary quadrant, 1 (9%) in the left maxillary quadrant, 3 (27.3%) in the right mandibular quadrant, and 3 (27.3%) in the left mandibular quadrant. Results of descriptive statistical analysis and odds ratio did not show any significant differences between rates (p = 0.67). The clinical efficiency of SEP was similar to that of the conventional system.

Development of a versatile TaqMan™ real-time quantitative PCR (RT-qPCR) compliant anchor sequence to quantify bacterial gene transcripts from RNA samples containing carryover genomic DNA

BackgroundIn bacterial systems, the sequence congruence of genomic DNA (gDNA) and cDNA obtained following reverse transcription of RNA, makes gDNA an automatic target for qPCR primers. This could lead to aberrant gene expression quantification. This is why a rigorous treatment of bacterial RNA with DNase I is usually required to remove any traces of carryover gDNA. As bacterial RNA is known to be extremely labile, any procedure that affects RNA yield, such as DNase I treatment, can be logically assumed to also influence detection and quantification of gene transcripts, leading to either an underestimation or no detection at all. To address such problems, we have developed a novel and versatile TaqMan RT-qPCR compliant anchor sequence (MYT4) for quantifying bacterial gene transcripts without the need for DNase I treatment.ResultsA non-genomic anchor sequence, henceforth referred to as MYT4 was designed using a synthetic DNA sequence called myIC, previously shown to share no significant homology to any known accession in the GenBank database. The sequence characteristic of MYT4 was kept within the design parameters required for the TaqMan RT-qPCR platform. The specificity and robustness of the novel MYT4 sequence was validated on RNA extracted from the bacterium Pseudomonas sp. LBUM300, grown under liquid culture and spiked soil conditions. Two transcripts, namely hcnC and phlD, were quantified from these two experimental systems. Using the MYT4 anchor, no RT-qPCR signal was detected from non-DNase I treated RNA, while strong signals were obtained using conventional reverse primers and RT-qPCR, indicating the presence of carryover gDNA in the RNA, extracted from either liquid culture or soil. Serial treatment of the RNA samples with DNase I (required to achieve absolute gDNA elimination) resulted in 50-70% loss of RNA which, when submitted to conventional RT-qPCR, significantly altered the transcript numbers detected when compared to the MYT4-based approach.ConclusionsImplementation of the versatile approach described in this study, which can be “retrofitted” to any existing TaqMan RT-qPCR system, should contribute to reducing the time and lowering the costs required to perform adequate bacterial RNA purification for downstream quantification of gene transcripts.

Cytotoxic and Genotoxic effects of Orthodontic Adhesives on Human lymphocyte - An In-vitro Study

Aim of this study was to evaluate the in vitro genotoxicity and cytotoxicity of two orthodontic adhesives and to determine the type of cell death they induce on human lymphocytes. The materials tested were 1.Light cure orthodontic adhesive with conventional primer (Transbond XT3M) and 2. Self cure orthodontic adhesive (Unite, 3M). Cured sterile individual masses were immersed in DMEM and left at 370C for 24 h. Then a volume of 200 μL of the extract medium was mixed with human peripheral blood lymphocyte tested for comet assay by Single cell DNA Damage assay and Apoptosis by DNA diffusion agar assay. Evaluation of Cytotoxicity was carried out by Hemolysis assay method. Hemolytic activity of Self cure orthodontic adhesive (52.9± 1.82) was significantly more than that of Light Cure orthodontic adhesive (48.9±1.23). The results showed all parameters studied by comet assay were significant (P>0.05).In Case of Apoptosis, Light cure orthodontic adhesive (155.11±6.03) and self cure orthodontic adhesives (154.77±13.17) showed slightly increased diffusion of DNA compared to normal lymphocyte (111.22±8.78).However the level of DNA diffusion was not significantly different between the two adhesives. Light Cure orthodontic and self cure orthodontic adhesives were cytotoxic and induced apoptosis. The self cure orthodontic adhesive was found to be significantly more toxic than that of Light Cure orthodontic adhesive. Both the adhesives had no significant effect on the percentage of DNA tail and Tail length of the human lymphocyte.

Comparative Analysis of Polymerase Chain Reaction Assay for Herpes Simplex Virus 1 Detection in Tear

PurposeTo comparatively analyze the methodological efficacy of the polymerase chain reaction (PCR) assay for herpes simplex virus 1 (HSV) detection in tears.MethodsThis retrospective study reviewed the medical records of 115 patients who were clinically diagnosed with herpes keratitis, and their tear samples were collected for HSV detection. PCR positive rates were analyzed for their dependence on the PCR primers used (conventional PCR primer vs. nested PCR primer), the tear collecting method used (micropipetting vs. collection with schirmer strip), the disease manifestation and the patient's previous medication history.ResultsHSV DNA was detected in 23 out of 115 (20%) tear samples. The PCR positive rate in tear samples did not differ depending on the PCR primer or tear collection method used. Typical epithelial lesions showed a higher positive rate (31.4%) than atypical epithelial lesions (10.9%). The previous history of the antiviral agent seemed to affect the PCR positive rate.ConclusionsAlthough the PCR positive rate was not dependent on the tear collection method or primers, HSV detection in tears using PCR was shown to be a supplementary diagnostic test in typical and atypical herpes epithelitis.

Primers for amplification of nitrous oxide reductase genes associated with Firmicutes and Bacteroidetes in organic-compound-rich soils

The nosZ gene encodes nitrous oxide reductase, a key enzyme in the nitrous oxide reduction that occurs during complete denitrification. Many conventional approaches have used Proteobacteria-based primers to detect nosZ in environmental samples. However, these primers often fail to detect nosZ in non-Proteobacteria strains, including Firmicutes (Gram-positive) and Bacteroidetes. In this study, newly designed nosZ primers successfully amplified this gene from five Geobacillus species (Firmicutes). The primers were used to construct nosZ clone libraries from DNA extracted from sludge and domestic animal feedlot soils, all with high organic carbon contents. After DNA sequencing, phylogenetic analysis identified many new nosZ sequences with high levels of homology to nosZ from Bacteroidetes, probably because of the high sequence similarity of nosZ from Firmicutes and Bacteroidetes, and a predominance of Bacteroidetes in feedlot environments. Three sets of new quantitative real-time PCR (qPCR) primers based on our clone library sequences were designed and tested for their specificities. Our data showed that only Bacteroidetes-related nosZ sequences were amplified, whereas conventional Proteobacteria-based primers amplified only Proteobacteria-related nosZ. Quantitative analysis of nosZ with the new qPCR primers recovered ~10(4) copies per 100 ng DNA. Thus, it appears that amplification with conventional primers is insufficient for developing an understanding of the diversity and abundance of nosZ genes in the environment.

Trypanosome Diversity in Wildlife Species from the Serengeti and Luangwa Valley Ecosystems

BackgroundThe importance of wildlife as reservoirs of African trypanosomes pathogenic to man and livestock is well recognised. While new species of trypanosomes and their variants have been identified in tsetse populations, our knowledge of trypanosome species that are circulating in wildlife populations and their genetic diversity is limited.Methodology/Principal FindingsMolecular phylogenetic methods were used to examine the genetic diversity and species composition of trypanosomes circulating in wildlife from two ecosystems that exhibit high host species diversity: the Serengeti in Tanzania and the Luangwa Valley in Zambia. Phylogenetic relationships were assessed by alignment of partial 18S, 5.8S and 28S trypanosomal nuclear ribosomal DNA array sequences within the Trypanosomatidae and using ITS1, 5.8S and ITS2 for more detailed analysis of the T. vivax clade. In addition to Trypanosoma brucei, T. congolense, T. simiae, T. simiae (Tsavo), T. godfreyi and T. theileri, three variants of T. vivax were identified from three different wildlife species within one ecosystem, including sequences from trypanosomes from a giraffe and a waterbuck that differed from all published sequences and from each other, and did not amplify with conventional primers for T. vivax.Conclusions/SignificanceWildlife carries a wide range of trypanosome species. The failure of the diverse T. vivax in this study to amplify with conventional primers suggests that T. vivax may have been under-diagnosed in Tanzania. Since conventional species-specific primers may not amplify all trypanosomes of interest, the use of ITS PCR primers followed by sequencing is a valuable approach to investigate diversity of trypanosome infections in wildlife; amplification of sequences outside the T. brucei clade raises concerns regarding ITS primer specificity for wildlife samples if sequence confirmation is not also undertaken.

Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle

A one-step multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection of five viruses causing diarrhea in adult cattle: bovine group A rotavirus (GAR), bovine group B rotavirus (GBR), bovine group C rotavirus (GCR), bovine coronavirus (BCV), and bovine torovirus (BToV). The detection limit of the one-step multiplex RT-PCR for GAR, GCR, BCV, and BToV was 102, 100, 101, and 102 TCID50/ml, respectively, and that for GBR was 106 copies/ml. The one-step multiplex RT-PCR with newly designed primers to detect GAR had higher sensitivity than a single RT-PCR with conventional primers, with no false-positive reactions observed for ten other kinds of bovine RNA viruses To assess its field applicability, 59 of 60 fecal samples containing one of these five viruses from all 25 epidemic diarrhea outbreaks in adult cattle were positive in the one-step multiplex RT-PCR assay. Furthermore, using four additional fecal samples containing two viruses (GBR and BCV or BToV), two amplified products of the expected sizes were obtained simultaneously. In contrast, all 80 fecal samples lacking the five target viruses from normal adult cattle were negative in the multiplex assay. Taken together, our results indicate that the one-step multiplex RT-PCR developed here for the detection of GAR, GBR, GCR, BCV, and BToV can be expected to be a useful tool for the rapid and cost-effective diagnosis and surveillance of viral diarrhea in adult cattle.

Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens.

Sensitive and Specific Detection of Xanthomonas oryzae pv. oryzae by Real-Time Bio-PCR Using Pathovar-Specific Primers Based on an rhs Family Gene.

The present study describes bio-polymerase chain reaction (PCR) assays to detect bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae in rice. Successful control of X. oryzae. pv. oryzae requires a specific and reliable diagnostic tool. However, other X. oryzae pathovars are detected by currently available molecular and serological methods. In this study, SYBR Green real-time and conventional PCR primer sets were designed based on an rhs family gene of X. oryzae pv. oryzae KACC10331 because these genes are structurally diverse. The specificity of the primers was evaluated using purified DNA from 11 isolates of two X. oryzae pathovars, 21 other Xanthomonas species, and 4 other reference phytopathogenic bacteria and fungi. The assay was also able to detect at least two genome equivalents of cloned amplified target DNA using purified DNA. Thus, the SYBR Green real-time PCR-based method can be used for the rapid and specific detection of X. oryzae pv. oryzae and will potentially simplify and facilitate diagnosis and monitoring of this pathogen and guide plant disease management.

Abstract 3870: Improved PCR amplification of DNA biomarkers with the use of chimeric oligonucleotides

Abstract Methylation of the Twist1 gene has been linked to various cancers, including bladder cancer. We are developing a non-invasive diagnostic assay utilizing urinary protein and DNA biomarkers, including Twist1, as triage monitors in high-risk bladder cancer populations. Since final clinical assay performance is dependent on the optimal technical performance of each individual marker, we incorporated the use of chimeric PCR primers to establish PCR conditions for all DNA markers that would greatly simplify the process of optimizing PCR performance. Amplification of difficult DNA templates can involve significant optimization of PCR conditions, and is often complicated by mispriming and other non-specific amplification events. The chimeric primers we designed have a variable 3’ target-specific sequence, and a common 22 nucleotide 5’ sequence that is unrelated to any human DNA. The common 5’ sequence generates similar melting temperatures for all primers (Tm 68-72oC) that permits uniform PCR conditions regardless of the characteristics of the template specific sequence. In addition, the chimeric primers permit a higher annealing temperature, which minimizes non-specific PCR amplification. In the study described here, conventional methylation specific primers were designed against a highly methylated region of the Twist1 promoter with and without the 5’ chimeric DNA sequence, and PCR conditions were optimized for each primer pair. Performance was assessed using DNA isolated from 50 bladder cancer tissues. After bisulfite conversion, PCR amplifications were carried out using the Bio-Rad C1000 platform and subsequently analyzed by agarose gel electrophoresis. Using traditional primers, Twist1 sensitivity was comparable to previous reports (68%, Renard et al, 2010), whereas using the chimeric primers, sensitivity increased to 90%. Incorporation of chimeric primers greatly simplifies PCR optimization and permits the use of higher annealing temperatures, resulting in higher specificity. We have applied this method to the detection of Twist1 methylation in bladder cancer tissues and demonstrate significant improvement in performance, when compared to traditional, non-chimeric oligonucleotide primers. In addition, this primer modification can be applied to multiple assay formats and DNA targets to improve sensitivity for use in non-invasive diagnostic assays, where mutant DNA is expected to be limiting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3870. doi:10.1158/1538-7445.AM2011-3870

Enhanced Detection of Coxiella burnetii with a Complementary Locked Primer-Based Real-Time PCR Method

Coxiella burnetii is the bacterial causative agent of Q fever in humans. Because Q fever can establish itself with an initial inoculation of fewer than ten C. burnetii cells, a sensitive detection method for C. burnetii infection is needed for early detection. We aimed to evaluate the effectiveness of a complementary locked primer (CLP)-based real-time PCR method for sensitive detection of C. burnetii infection. To evaluate the ability of CLPs to enhance the efficiency of the real-time PCR assay for the C. burnetii IS1111 insertion sequence, the mean threshold cycle values from 20 real-time PCR replicates with either CLPs or conventional primers were determined using tenfold serial dilutions (102-108) of purified C. burnetii Nine Mile genomic DNA. In addition, the cross-reactivity between C. burnetii and 31 non-Coxiella species was examined. The CLP-based real-time PCR allowed specific and reliable detection of as few as 59 copies of the IS1111 element present in the genome of C. burnetii, which represents approximately 2.96 genome equivalents or three cells of C. burnetii. These results demonstrate the effectiveness of CLP-based real-time PCR for sensitive detection of C. burnetii infection. It can be concluded that the CLP-based real-time PCR assay is a more appropriate method for sensitive detection and quantification of C. burnetii than previously reported methods.

Effect of orthodontic bonding steps on the initial adhesion of mutans streptococci in the presence of saliva

To test the hypothesis that orthodontic bonding has no effect on the initial adhesion of mutans streptococci (MS) in the presence of saliva. Hydroxyapatite (HA) and orthodontic adhesive (AD) disks were prepared to a uniform size. HA disks were etched with 37% phosphoric acid (HE, etched group). Some of the HE disks were coated with Transbond XT primer and light cured (HP, primed group). Transbond Plus SEP was applied to a third set of HA disks, dried, and light cured (SEP, self-etching primer group). Adhesion assays were performed using two MS strains in the presence of fluid-phase or surface-adsorbed unstimulated whole saliva (UWS). The MS adhesion patterns were examined by scanning electron microscopy. MS adhesion was influenced by the bonding steps and the presence of UWS. UWS treatment decreased MS adhesion. However, surface-adsorbed UWS resulted in slightly less inhibition of MS adhesion than fluid-phase UWS. MS adhesion was significantly greater for HE than for the other groups. There were interaction effects between the UWS treatment and surface groups. MS adhesion to HP and AD was significantly diminished in the presence of surface-adsorbed or fluid-phase UWS compared with adhesion to HA, HE, or SEP. The hypothesis is rejected. Our results suggest that MS adhesion is significantly influenced by the bonding procedure used, and the application of conventional primers for the bracket bonding can inhibit MS adhesion to tooth surfaces in the presence of UWS.

A New Method for Human ABO Genotyping Using a Universal Reporter Primer System

We developed a new method for forensic ABO genotyping based on a universal reporter primer (URP) system. This allows for the simultaneous detection of six single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 297, 526, 703, 796, and 803). This URP system provides obvious peaks, ranging from 82 to 151 bp in length. ABO genotypes were classified and successfully genotyped by our method, including minor alleles that may cause a discrepancy between the genetic data and serological phenotypes. Full profiles were identified using as little as 0.1 ng (0.05 ng ⁄ reaction) of standard K562 and 9947A DNA. Moreover, the success rate of genotyping from a URP system was much higher than that from a conventional primer extension method in degraded DNA. This method enables simple and rapid detection of multiple SNP sites on human ABO genes and is highly specific and sensitive when using limited and degraded DNA.

Use of a High-Resolution Melt Assay To Characterize Codon 54 of the cyp51A Gene of Aspergillus fumigatus on a Rotor-Gene 6000 Instrument

A high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument was developed to characterize the codon for glycine 54 in the cyp51A genes from 13 reference isolates and 12 clinical isolates of Aspergillus fumigatus. Mutations in this codon confer reduced susceptibility to itraconazole and posaconazole. The assay is simple to perform, and a result of "wild type" or "mutant" is available after approximately 1 h following DNA extraction using commercially available reagents and conventional primers.