Single Primer Polymerase Chain Reaction Research Articles

Intra-Discrete Typing Unit TcV Genetic Variability of Trypanosoma cruzi in Chronic Chagas' Disease Bolivian Immigrant Patients in Barcelona, Spain.

Background: Trypanosoma cruzi has a high rate of biological and genetic variability, and its population structure is divided into seven distinct genetic groups (TcI-TcVI and Tcbat). Due to immigration, Chagas disease (ChD), caused by T. cruzi, has become a serious global health problem including in Europe. Therefore, the aim of this study was to evaluate the existence of genetic variability within discrete typing unit (DTU) TcV of T. cruzi in Bolivian patients with chronic ChD residing in Barcelona, Spain.Methods: The DNA was extracted from the peripheral blood of 27 patients infected with T. cruzi DTU TcV and the fragments of the genetic material were amplificated through the low stringency single primer-polymerase chain reaction (LSSP-PCR). The data generated after amplification were submitted to bioinformatics analysis.Results: Of the 27 patients evaluated in the study, 8/27 (29.6%) were male and 19/27 (70.4%) female, 17/27 (62.9%) were previously classified with the indeterminate clinical form of Chagas disease and 10/27 (37.1%) with Chagas cardiomyopathy. The LSSP-PCR detected 432 band fragments from 80 to 1,500 bp. The unweighted pair-group method analysis and principal coordinated analysis data demonstrated the existence of three distinct genetic groups with moderate-high rates of intraspecific genetic variability/diversity that had shared parasite's alleles in patients with the indeterminate and cardiomyopathy forms of ChD.Conclusions: This study demonstrated the existence of a moderate to high rate of intra-DTU TcV variability in T. cruzi. Certain alleles of the parasite were associated with the absence of clinical manifestations in patients harboring the indeterminate form of ChD. These results support the need to search for increasingly specific targets in the genome of T. cruzi to be correlated with its main biological properties and clinical features in patients with chronic ChD.

Cloning flanking sequence by single-primer PCR in transgenic plants

The insertion position of exogenous genes in plant genomes is usually identified by adapter ligation-mediated polymerase chain reaction (PCR), thermal asymmetric interlaced PCR, and restriction site extension PCR in transgenic plant research. However, these methods have various limitations, such as the complexity of designing primers and time-consuming and multiple-step procedures. The goal of this study was to establish an easier, more rapid, and more accurate method to clone flanking sequence using single-primer PCR in transgenic plants. Unknown flanking genome sequences in transgenic plants, including those in tobacco, soybean, rice, and maize, were cloned using the single-primer PCR method established in this study, with the Bar gene as the anchor gene. The primer 1 (P1), P2, and P3 PCRs obtained 4 sequences, and the completely correct flanking sequence of 508 bp that was obtained in the P3 PCR was verified by sequencing analysis. The single-primer PCR is more rapid and accurate than conventional methods, justifying its application widely in cloning flanking sequences in transgenic plants.

Cloning of flanking sequence in transgenic plants by restriction site-anchored single-primer polymerase chain reaction.

Determining the insertion position of an exogenous gene in the target plant genome is one of the main issues in the transgenic plant field. This study introduced a simple, rapid, and accurate method to clone the flanking sequences of the transgenic bar gene as the anchoring gene in the transgenic maize genome using single-primer polymerase chain reaction (PCR). This method was based on the distribution of restriction sites in the maize genome and adopted the single-primer PCR method. Cloning the flanking sequences with the restriction site-anchored single-primer PCR simplified the experimental procedures by about 70% and reduced the experimental time by more than 80%. In conclusion, the restriction site-anchored single-primer PCR was a simple, rapid method to obtain the unknown flanking sequences in the transgenic plants.

Single-primer PCR correction: a strategy for false-positive exclusion

Polymerase chain reaction (PCR) technology plays an important role in molecular biology research, but false-positive and nonspecific PCR amplification have plagued many researchers. Currently, research on the optimization of the PCR system focuses on double-primer-based PCR products. This research has shown that PCR amplification based on single-primer binding to the DNA template is an important contributing factor to obtaining false-positive results, fragment impurity, and nonspecific fragment amplification, when the PCR conditions are highly restricted during PCR-based target gene cloning, detection of transgenic plants, simple-sequence repeat marker-assisted selection, and mRNA differential display. Here, we compared single- and double-primer amplification and proposed "single-primer PCR correction"; improvements in PCR that eliminate interference caused by single-primer-based nonspecific PCR amplification were demonstrated and the precision and success rates of experiments were increased. Although for some kinds of experiments, the improvement effect of single-primer PCR correction was variable, the precision and success rate could be elevated at 12-50% in our experiment by this way.

Problems with and a system to eliminate single-primer PCR product contamination in simple sequence repeat molecular marker-assisted selection in soybean

Polymerase chain reaction (PCR) provides a foundation for simple sequence repeat molecular marker-assisted selection (SSR MAS) in soybean. This PCR system and its various conditions have been optimized by many researchers. However, current research on the optimization of the PCR system focuses on double-primer PCR products. We compared single- and double-SSR primer PCR products from 50 soybean samples and found that the use of single-PCR primers in the reaction system can lead to amplified fragments of portions of the SSR primers in the PCR process, resulting in both false-positives and fragment impurity of double-primer PCR amplification, inconvenient for subsequent analysis. We used "single-primer PCR correction" to eliminate interference caused by single-primer nonspecific PCR amplification and improve PCR quality. Using this method, the precision and success rates of SSR MAS in soybean can be increased.

Characterization of TTAGG telomeric repeats, their interstitial occurrence and constitutively active telomerase in the mealybug Planococcus lilacinus (Homoptera; Coccoidea)

We confirmed the occurrence of the insect TTAGG telomeric repeats in the mealybug Planococcus lilacinus, a radiation-resistant coccid, by single primer polymerase chain reaction (PCR) and Southern hybridization. Analysis of Bal31 nuclease-digested DNA by Southern hybridization and chromosomes by FISH suggests that these repeats occur mainly at the ends of the chromosomes. However, sequence analysis of the PCR products of TTAGG-associated sequences from genomic DNA showed their interstitial occurrence and association with certain unrelated low-copy repeats. Because of their shorter length, the interstitial TTAGG sequences were detectable by primed in situ hybridizations but not by FISH. Analysis of chromosomes recovered after irradiation by fluorescent in situ hybridization suggested acquisition of TTAGG repeats at a majority of the healed ends. We also observed mild telomerase activity in unirradiated insects which was further enhanced after irradiation. Taken together, these results suggest that the mealybug has an efficient mechanism of formation of TTAGG repeats at radiation-induced chromosome ends and constitutively active telomerase may be a feature associated with rapid recovery of chromosome ends damaged by ionizing radiation.

An Electrochemical Assay of β-1,3-Glucanase Gene from Transgenic Capsicum Using Asymmetric PCR

5′-Thiol-derivatized specific DNA probes were added to the single primer polymerase chain reaction (asymmetric PCR) solution. In the PCR process, the DNA probes extended in the presence of target; the extended probes were then immobilized on a glassy carbon electrode (GCE) via gold nanoparticles. Finally, methylene blue and the extended probes were combined and the electrochemical signals were measured. This signal was higher than that of the GCE modified only by the original probe. When there was no target in PCR solution, the probe did not extend and the signal did not increase. The specific sequences of the β-1,3-glucanase gene were detected successfully from three targets with different length: oligonucleotide, molecule clone vector DNA, and total genome DNA of transgenic capsicum. The detection limits of 2.6 × 10−13, 7.8 × 10−13, and 9.1 × 10−13 moll−1 for oligonucleotide, molecule clone vector DNA, and total transgenic capsicum genome DNA were estimated.

Utilizing single primers as molecular markers inPoaspp.

AbstractAn approach is described whereby single simple sequence repeat (SSR) primers are utilized at high annealing temperature to identify and evaluate the presence of palindrome or quasi‐palindrome regions in polyploid genomes to provide locus specific genotyping for polyploid grass species. The procedure has been effective in identifying markers that coexpress inPoa arachniferaand interspecificPoahybrids. DNA sequence analysis of the polymerase chain reaction (PCR) amplification products identified an array of homologous loci within and among evaluated individuals. From this study, 157 single primers were identified to provide information and identify polymorphisms acrossP. arachnifera. Similar to random amplified polymorphic DNA, arbitrarily primed PCR or low‐stringency single primer PCR methods, this approach requires no prior genome information, utilizes agarose gels and can be visualized with ethidium bromide. Preliminary evaluations of additionalPoaspp.,Bromus inermis,Dactylis glomerata,Thinopyrum ponticum,Lolium perenneandAgrostisspp. suggest wide utility of this approach toward genotyping polyploid grasses.

Transmission Dynamics of Trypanosoma cruzi Determined by Low-Stringency Single Primer Polymerase Chain Reaction and Southern Blot Analyses in Four Indigenous Communities of the Sierra Nevada de Santa Marta, Colombia

This study attempted to evaluate the transmission dynamics of Trypanosoma cruzi in four indigenous communities of Sierra Nevada de Santa Marta (SNSM), Colombia. Low-stringency single primer-polymerase chain reaction (LSSP-PCR) of the minicircles and Southern blot analyses were used to characterize samples from patients, vectors, and reservoirs in these communities. The LSSP-PCR profiles revealed a high genetic variability but with similarities among the parasites present in the samples of vectors, patients, and reservoirs of the same and different communities. Cluster and analysis of molecular variance (AMOVA) analyses of data derived from LSSP-PCR and Southern blot suggest a gene flux among populations of T. cruzi circulating in patients, vectors, and reservoirs. The results support the idea that the domestic and wild transmission cycles overlap in the SNSM, with Rhodnius prolixus as the main vector and Triatoma dimidiata playing an important role in the transmission of Chagas disease in this zone, making the vector control strategy by spraying unsuccessful.

Geographical clustering of Trypanosoma cruzi I groups from Colombia revealed by low-stringency single specific primer-PCR of the intergenic regions of spliced-leader genes

A low-stringency single-primer polymerase chain reaction (LSSP-PCR) typing procedure targeted to the intergenic regions of spliced-leader genes (SL) was designed to profile Trypanosoma cruzi I stocks from endemic regions of Colombia. Comparison between SL-LSSP-PCR profiles of parasite DNA from vector faeces and cultures isolated from those faeces showed more conservative signatures than profiles using LSSP-PCR targeted to the minicircle variable regions (kDNA). This was also observed by analysing 15 parasite clones from one stock as well as serial samples of a same stock after in vitro culturing or inoculation into mice. Thus, SL-LSSP-PCR appears more appropriate than kDNA-LSSP-PCR for reliable typing of major T. cruzi I groups from in vitro cultured stocks and triatomine faeces. SL-LSSP-PCR grouped 46 of 47 T. cruzi I Colombian stocks according to their geographical procedences in four clusters: Cluster Cas from Casanare Department, Cluster Mg from Northern Magdalena department, Cluster Mom from Momposina Depression in Southern Magdalena and finally Cluster NW from northwestern Colombia, including Sucre, Chocó, Córdoba and Antioquia departments. Sequence analysis identified punctual mutations among amplicons from each cluster. Within Cluster Mg, sequence polymorphism allowed association with different sylvatic vector species. Novel SL sequences and LSSP-PCR profiles are reported from T. cruzi I infecting Eratyrus cuspidatus, Panstrongylus geniculatus and Rhodnius pallescens vectors.

Start Codon Targeted (SCoT) Polymorphism: A Simple, Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.

Molecular characterization of Avian strains of Pasteurella multocida serogroup-A:1 based on amplification of repetitive regions by PCR

Repetitive extragenic palindromic (REP)-PCR (polymerase chain reaction), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and single primer PCR assays were employed to characterize 66 strains of Pasteurella multocida serogroup A:1 isolated from avian species belonging to different regions of India. REP-PCR resulted in amplification of REP sequences from the genome which were in the range of approximately 200 to approximately 3000 bp and accounted for a total of 54 distinguishing profiles (D=0.99). ERIC-PCR analysis also generated amplified products in the range of approximately 200 to approximately 3200 bp categorizing strains into a total of 50 different profiles (D=0.98). Amplification of repetitive regions using a microsatellite primer (GTG)(5), resulted in clear distinctive bands ranging from approximately 200 to approximately 2400 bp. Strains were assigned to 43 profiles (D=0.96). No correlation could be drawn between genotypic profiles and avian hosts with their geographical area of origin. Avian strains of P. multocida serogroup A:1 were found to be highly heterogeneous with diverse profiles. REP-PCR was found to be highly discriminatory and simple method for differentiation of phenotypically similar strains. The present study also indicated that PCR based amplification of repetitive regions of P. multocida is a rapid technique with good discrimination and could be employed directly for routine typing of field isolates from fowl cholera outbreaks.

Detection of multiple strains of Pasteurella multocida in fowl cholera outbreaks by polymerase chain reaction-based typing

Applicability of molecular methods for the detection and differentiation of Pasteurella multocida strains involved in two separate fowl cholera outbreaks in a single poultry farm was investigated. A total of 12 and 18 strains of P. multocida obtained from two separate outbreaks were subjected to phenotypic and genotypic characterization. Phenotypically, all strains were similar; however, DNA-based techniques by employing polymerase chain reaction (PCR) assays were found to be highly specific and sensitive for rapid detection and differentiation of strains. All 30 strains gave amplicons of approximately 460 bp and approximately 1,044 bp specific for P. multocida and capsular serogroup A in the Multiplex Capsular PCR typing system. Molecular typing techniques such as repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus PCR and single primer PCR differentiated all 30 strains into different profiles. However, similar patterns of genome fragments were observed among all strains following restriction endonuclease analysis using the enzyme HpaII. The current investigation revealed involvement of the same and multiple strains of P. multocida in two outbreaks. The results also indicated that molecular methods of detection and typing are rapid in comparison with conventional methods for epidemiological investigations of fowl cholera outbreaks.

Molecular variability among strains of Pasteurella multocida isolated from an outbreak of haemorrhagic septicaemia in India.

The applicability of conventional and molecular methods for rapid detection and differentiation of Pasteurella multocida serogroup B isolates involved in an outbreak of haemorrhagic septicaemia affecting Indian buffaloes, was studied. Five isolates were obtained and were subjected to phenotypic and genotypic characterization. None of the five isolates could be differentiated on the basis of cultural, biochemical, pathogenicity and antimicrobial sensitivity patterns. Polymerase chain reaction (PCR)-based techniques were found to be specific and sensitive for rapid detection and differentiation of isolates. Repetitive extragenic palindromic (REP-) PCR, enterobacterial repetitive intergenic consensus (ERIC-) PCR and single-primer PCR differentiated all the five isolates into different profiles. All the isolates involved in the outbreak were found to have a genetic profile different from standard P. multocida strain (P52). However, three isolates had similar profiles, whereas each of the remaining two had a different profile. The study indicates the involvement of multiple strains of P. multocida in a single outbreak of haemorrhagic septicaemia in buffaloes. The results also indicate that molecular methods of detection and typing are superior to conventional methods for rapid epidemiological investigations of haemorrhagic septicaemia.

Linear amplification followed by single primer polymerase chain reaction to amplify unknown DNA fragments: complete nucleotide sequence of Oropouche virus M RNA segment

The whole nucleotide sequence of Oropouche virus medium (M) RNA, Orthobunyavirus genus, Bunyaviridae family, was obtained using a new genomic amplification method. This method is based on the use of a single and specific primer of high melting temperature in a linear amplification (LA), followed by a single primer polymerase chain reaction (LASP-PCR). The LASP-PCR was used to walk along the Oropouche M RNA completing the sequence in seven successive walks. The amplicons obtained in each walking step ranged from 300 to 1100 bp; however, amplicons of up to 3970 bp was obtained when the extension time of the LASP-PCR was increased from 120 to 270 s. This method was tested successfully for Escherichia coli and cytomegalovirus obtaining amplicons of up to 2130 and 6500 bp, respectively, indicating that it can be applied to amplify unknown DNA sequences adjacent to a short stretch of known sequence of more complex genomes.

Cyclooxygenase (COX)-2 and granulosa cell apoptosis in vitro.

C-myc was studied in cyclooxygenase (COX)-2 associated granulosa cell apoptosis, Granulosa cells (N = 5 cases) were incubated for 24 h in either 1 or 50 microM COX-2 inhibitor, 1 or 50 microM COX-1/COX-2 inhibitor, negative or positive controls Single primer polymerase chain reaction of c-myc exon 1 were performed. Bisbenzimide-stained control single-stranded (ssDNA) were hybridized to SYBR Gold-stained ssDNA and fluorescent images analyzed. C-myc was disrupted by the high-dose COX-2 inhibitor. Cell viability decreased with COX-1 and COX-2 inhibition. However, cell viability was similar for the positive control and at low-dose COX-2 inhibition. Inhibition of both COX-1 and COX-2 initiated apoptosis without disrupting c-myc suggesting a protective effect on c-myc. The low dosage of the COX-2 inhibitor did not disrupt c-myc and cell viability. C-myc sensitization was not part of apoptosis.

Brief communication. Development and characterization of genetic mapping resources for the turkey (Meleagris gallopavo)

The development and partial characterization of turkey genomic libraries enriched for TG, GAT, and CCT simple sequence repeats (SSR) are described. The primary library, established using conventional methods, was enriched for each of the three SSR by single-primer polymerase chain reaction (PCR). The three enriched libraries were screened by standard hybridization and washing protocols under moderate to high stringency conditions. The utility of a fraction of the markers was evaluated based on the polymorphism of PCR-amplified products in a backcross reference DNA panel. The panel consisted of genomic DNA samples from three backcrossed families developed from a cross of a wild male turkey to three inbred Orlopp line C females. A total of 181 sequences from positive clones have been characterized and deposited in GenBank. About 60% of the 60 primer pairs designed from SSR-containing sequences detected polymorphism in the reference DNA panel. The turkey genomic DNA reference panel, the enriched libraries, and the markers described here provide an opportunity to begin to characterize the turkey genome and to develop a useful public genetic map for this economically important species.

Amplification and Sequencing of End Fragments from Bacterial Artificial Chromosome Clones by Single-Primer Polymerase Chain Reaction

The presence of paralytic shellfish poisoning (PSP), diarrhetic Shellfish Poisoning (DSP), tetrodotoxin (TTX) and its analogues (11-oxoTTX, 4.9-anhydro-11-oxoTTX, 4.9-anhydroTTX, 5-deoxyTTX, 5.11-dideoxyTTX, 5.6.11-trideoxyTTX and 4.9-anhydro-5.6.11-trideoxy TTX) were initially investigated in Carcinoscorpius rotundicauda collected from south China with Liquid chromatography-tandem mass spectrometry (LC–MS–MS) and mouse bioassay. The TTX toxicity was 10.8 ± 3.9 MU/g muscle, 6.3 ± 0.6 MU/g viscera and 6.3 ± 0.6 MU/g eggs in mean value. Merely dcGTX2 and dcSTX were detected in ten Specimens, ranging from 0.01 to 0.77 μg/g. Analyses suggested that these Carcinoscorpius rotundicauda contain TTX and its analogues as the major toxin and PSPs as the minor, respectively. Besides, no DSPs were found.

Amplification of 4-9-kb human genomic DNA flanking a known site using a panhandle PCR variant.

We present a method for the in vitro amplification of > 6.0 kb of DNA flanking a known site. This is accomplished by ligating an oligonucleotide to create an inverted repeat of a portion of the known sequence, followed by single-primer polymerase chain reaction (PCR) amplifications. This method generates a panhandle template following primer extension on the strand of interest. It does not involve template-directed extension from the ligated oligonucleotide, and it is carried out without DNA extractions. We have used this method to amplify 4.5-9.4 kb of DNA flanking the original primer annealing sites directly from human genomic DNA.

Sex ratio variation among broods of great reed warblers Acrocephalus arundinaceus

The sex of 746 great reed warbler fledglings (from 175 broods) was determined by the use of single primer polymerase chain reaction. The reliability of the technique was confirmed as 104 of the fledglings were subsequently recorded as adults of known sex. The overall sex ratio did not differ from unity. Variation in sex ratios between broods was larger than expected from a binomial distribution. Female identity explained some of the variation of brood sex ratio indicating that certain females consistently produced sex ratios that departed from the average value in the population. The theory of sex allocation predicts that parents should adjust the sex ratio of their brood to the relative value of sons and daughters and this may vary in relation to the quality of the parents or to the time of breeding. In the great reed warbler, the proportion of sons was not related to time of breeding, or to any of five female variables. Of five male variables, males with early arrival date tended to produce more daughters. The sex ratio of fledglings that were a result of extra‐pair fertilizations did not differ from that of legitimate fledglings. Hence, there is currently no evidence of that female great reed warblers invest in a higher proportion of sons when mated with attractive males.