Template For Polymerase Chain Reaction Research Articles

Methodology Simplified preparation of a DNA ladder using PCR

Serving as a DNA molecular weight standard, the DNA ladder has been widely used in molecular biology applications. We developed a simple method for the preparation of a DNA marker, which involves designing primers to amplify 100- to 1000-bp DNA fragments using lambda DNA as a template for polymerase chain reaction, followed by extraction with phenol/chloroform, precipitation with ethanol and mixing. Fragments of 100- to 1000-bp DNA were successfully amplified; the sequences showed 100% identity with lambda DNA. This prepared DNA marker displayed clear bands, indicating that it can be used for molecular studies.

Coupling technique of random amplified polymorphic DNA and nanoelectrochemical sensor for mapping pancreatic cancer genetic fingerprint

ObjectiveTo review the feasibility of coupling the techniques of random amplified polymorphic DNA (RAPD) with carbon nanotube-based modified electrode for guanine/deoxyguanine triphosphate (dGTP) electrochemical sensing for mapping of the pancreatic cancer genetic fingerprint and screening of genetic alterations.MethodsWe developed a new method to study the electrochemical behavior of dGTP utilizing carbon multiwalled nanotube (MWNT)-modified glassy carbon electrodes (GCEs). RAPD was applied for amplification of DNA samples from healthy controls and patients with pancreatic cancer under the same conditions to determine the different surplus quantity of dGTP in the polymerase chain reaction (PCR), thereby determining the difference/quantity of PCR products or template strands. Using this method we generated a genetic fingerprint map of pancreatic cancer through the combination of electrochemical sensors and gel electrophoresis to screen for genetic alterations. Cloning and sequencing were then performed to verify these gene alterations.ResultsdGTP showed favorable electrochemical behavior on the MWNTs/GCE. The results indicated that the electrical signal and dGTP had a satisfactory linear relationship with the dGTP concentration within the conventional PCR concentration range. The MWNTs/GCE could distinguish between different products of RAPD. This experiment successfully identified a new pancreatic cancer-associated mutant gene fragment, consisting of a cyclin-dependent kinase 4 gene 3′ terminal mutation.ConclusionThe coupling of RAPD and nanoelectrochemical sensors was successfully applied to the screening of genetic alterations in pancreatic cancer and for mapping of DNA fingerprints.

Aptamer-Based Sensitive Detection of Target Molecules via RT-PCR Signal Amplification

In the efforts to explore an aptamer-based approach for target sensing and detection with higher sensitivity and specificity, instead of directly labeling aptamer with fluorophores, we proposed a new strategy by attaching a polymerase chain reaction (PCR) template to an oligonucleotide aptamer selected by systematic evolution of ligands by exponential enrichment (SELEX), so that after aptamer target binding, the template moiety serves as the PCR template in real-time quantitative PCR (RT-PCR), and therefore, the binding event can be reported by the following RT-PCR signals. Using the subtractive SELEX method, the oligonucleotide aptamers specific for the Fc fragment of mouse IgG were selected and subjected to coupling with the PCR dsDNA template by using overlap and the asymmetric extension PCR method. The target binding affinity of the PCR template tethered aptamer has been proven by electrophoretic mobility shift assay (EMSA), and further template tethered aptamer mediated real-time quantitative PCR (A-PCR) was conducted to validate the application for such a template tethered aptamer to be a sensitive probe for IgG detection. The results show that the protocols of A-PCR can detect 10-fold serial dilutions of the target, demonstrating a new mechanism to convert aptamer target binding events to amplified RT-PCR signal, and the feasibility of the PCR template tethered aptamer as a facile, specific, and sensitive target probing and detection is established. This new approach also has potential applications in multiple parallel target detection and analysis in a wide range of research fields.

First report of a new subgroup 16SrIX‐E (‘Candidatus Phytoplasma phoenicium’‐related) phytoplasma associated with juniper witches’ broom disease in Oregon, USA

Western juniper (Juniperus occidentalis) is a native tree indigenous to parts of Oregon, Washington, Idaho, Nevada and California (USA). The tree has increased in density since settlement of these areas, raising concern over loss of understory plants, decreased wildlife habitat and increased soil erosion. A newly recognized disease, juniper witches’ broom (JunWB), affecting at least 1% of trees in central Oregon, is characterized by abnormal proliferation of shoots, reduced size of leaves, shortened internodes, and growths having a ball-like appearance. DNA was extracted from leaf samples from ball-like growths and used as template in polymerase chain reactions primed by primer pair P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995). DNA fragments of 1·8 kb amplified from two samples were sequenced and the sequences deposited in GenBank (Accession Nos. GQ925918 and GQ925919). RFLP patterns of 16S rDNA, observed as virtual patterns using iPhyClassifier (Zhao et al., 2009), indicated that JunWB phytoplasma represents a new subgroup lineage, designated 16SrIX-E. 16S rDNA sequence similarity confirmed that JunWB is a ‘Ca. Phytoplasma phoenicium’-related phytoplasma. JunWB is one of three phytoplasmas found thus far to infect gymnosperms and is the only phytoplasma known to infect Juniperus sp. Occurrence of three distinct phytoplasmas, JunWB phytoplasma (this study), a group 16SrIII strain, and ‘Ca. Phytoplasma pini’ (Schneider et al., 2005), in gymnosperms of two different families (Pinaceae and Cupressaceae, Division Coniferae) in Europe and North America, suggests that phytoplasmal infection of conifers may be more common than previously envisioned.

Detection of Trypanosoma brucei gambiense and T. b. rhodesiense in Glossina fuscipes fuscipes (Diptera: Glossinidae ) and Stomoxys flies using the polymerase chain reaction (PCR) technique in southern Sudan

Ethanol-fixed entire bodies of the tsetse fly, Glossina fuscipes fuscipes , and unidentified stable flies, Stomoxys spp ., collected from near Juba town, southern Sudan, were tested for Trypanozoon trypanosomes infections using polymerase chain reaction (PCR) technique for the first time in Sudan. The crude target DNA sequences were extracted by incubation of entire flies in Nonindet PCR template buffer containing proteinase-K. The DNA amplification sets of conditions were adjusted for each pair of primers employed. The oligonucleotide primers used included TBR 1-2 , SRA A-E , SRA B537-B538 and TgsGP FOR-REV . The results showed that 74.4% of G. f. fuscipes and 39.36% of Stomoxys spp . were infected with Trypanozoon trypanosomes . Out of the 117 examined G. f. fuscipes , 46.2, 24.8, 35.04, 17.09 and 10.26% were due to T. b. gambiense (TgsGP FOR-REV ), T. b. rhodesiense (SRA A-E ), T. b. rhodesiense (SRA 3537-3538 ), mixed infection with T. b. gambiense and T. b. rhodesiense and T. b. brucei, respectively. However, infections in Stomoxys spp. of 2.13 and 37.2% were due to T. b. rhodesiense and T. b. brucei , respectively. Key words : Glossina fuscipes fuscipes, T. b. gambiense, T. b. Rhodesiense, vectoral capacity, infection rate, PCR technology.

Are women with lupus at higher risk of HPV infection?

Human papillomavirus (HPV) is the etiological agent of cervical cancer, the second most prevalent neoplasia among women. Although it has been proven that systemic lupus erythematosus (SLE) patients have higher frequency of cervical dysplasia, few studies have focused on HPV prevalence among them. This study aimed to investigate HPV prevalence among SLE patients and to evaluate associated risk factors, including the use of immunosuppressors (IM). Total DNA extracted from cervical samples of 173 SLE patients and 217 women (control group) submitted to routine cervical cytopathology was used as template in polymerase chain reaction (PCR)-based assays for detection of HPV DNA. HPV genotyping was performed by type-specific PCR, PCR-RFLP and/or DNA sequencing. Statistical methods included univariate analysis and logistic regression. Despite presenting significantly fewer HPV risk factors, SLE patients were found to have a threefold increase in HPV infection, mostly genotypes 53, 58, 45, 66, 6, 84, 83, 61, as compared with controls, who presented types 6, 18 and 61 more frequently. The higher rate of HPV infection was associated with immunosuppressive therapy. This study provides evidence that SLE patients have a high prevalence of HPV infection, which is even higher with the use of IM, a condition that might necessitate a more frequent cervical cancer screening program for these women.

Development and validation of a SYBR Green I–based real‐time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).

Detecção de DNA de Leishmania braziliensis em pacientes de leishmaniose tegumentar americana

Diagnostic tests for American tegumentary leishmaniasis were performed on blood samples of patients living in two endemic municipalities in the state of Pernambuco, Northeastern Brazil. DNA was extracted from 119 samples and used as template for polymerase chain reaction (PCR) analysis. The tests used primers specific for the kinetoplast mini-circle DNA (kDNA) of Leishmania braziliensis, a species circulating in Pernambuco, which amplify a 750 base pair target sequence. In total, 58 subjects (48.7%) showed positive PCR amplification and 61 (51.3%) were negative. Of the PCR-positive samples, 37 ( congruent with 64%) were from treated, lesion-free subjects. In conclusion, the PCR technique is efficacious at identifying Leishmania DNA in biopsy and venous blood samples.

Distribution ofblaTEM,blaSHV,blaCTX-MGenes Among Clinical Isolates ofKlebsiella pneumoniaeat Labbafinejad Hospital, Tehran, Iran

Extended-spectrum beta-lactamase (ESBL)-producing isolates of Klebsiella pneumoniae have been increasingly recognized in the hospital settings in Iran as well as throughout the world. The aim of this study was to detect and determine the genes encoding the ESBLs including bla(TEM), bla(SHV), and bla(CTX-M) groups among the K. pneumoniae isolates at Labbafinejad Hospital by polymerase chain reaction (PCR) and characterize them by direct sequencing of PCR products. Eighty-nine isolates were isolated from patients at different wards during March 2008-March 2009. They were identified as K. pneumoniae using biochemical tests. Susceptibility of isolates to 17 different antimicrobial agents was determined using agar disk diffusion method. The phenotypic confirmatory test was used to screen the isolates for production of ESBLs. To amplify the bla(SHV) the template DNA was extracted by boiling method. Plasmid DNA was extracted using minipreparation kit and used as template in PCR for detection of bla(TEM) and bla(CTX-M). The selected PCR products were sequenced and analyzed. All 89 strains were susceptible to imipenem. The rates of resistance to different antibiotics were in the following order: aztronam (79.7%), cefexime (67.4%), cefpodoxime (66.2%), cefotaxime (65.1%), ceftazidime (61.7%). The phenotypic confirmatory test detected 62 isolates (69.7%) as ESBL-producing K. pneumoniae. The prevalence of genes encoding ESBLs were as follows: bla(TEM) 54% (n = 48), bla(SHV) 67.4% (n = 60), bla(CTX-M-I) 46.51% (n = 40), and bla(CTX-M-III) 29% (n = 25). The bla(CTX-M-II) and bla(CTX-M-IV) were not detected. All bla(TEM) types were characterized as bla(TEM-1) and all bla(CTX-M-I) were identified as bla(CTX-M-15). The SHV types were characterized as SHV-5, SHV-11, and SHV-12. The rate of ESBL at Labbafinejad Hospital was 25% increase in a 4-year study that ended in March 2009. It appears that bla(TEM-1), bla(SHV-5), bla(SHV-11), bla(SHV-12), and bla(CTX-M-15) are the dominant ESBLs among the resistant strains of K. pneumoniae in Iran.

Quality and Quantity of DNA from Fresh and Long Term Stored Samples

Quality of genomic DNA is an important issue for the development of molecular markers because, in order for DNA to serve as a template for Polymerase Chain reaction (PCR), the entire sequence to be amplified must be present on a single DNA molecule. If the DNA is broken down into very small fragments, then the target sequence in the genomic DNA template is more likely to be disrupted by random breaks.

Distribution of Porphyromonas gingivalis fimA genotypes in primary endodontic infections

Long fimbriae (FimA) are important virulence factors of Porphyromonas gingivalis. Based on the diversity of the fimA gene, this species is classified into 6 genotypes. This study surveyed samples from primary endodontic infections for the presence of these P. gingivalis fimA variants. Genomic DNA isolated from samples taken from 25 root canals of teeth with chronic apical periodontitis and 25 aspirates from acute apical abscess was used as template in polymerase chain reaction (PCR) assays directed toward the detection of the different P. gingivalis fimA genotypes. Porphyromonas gingivalis was detected by a 16S rRNA gene-based PCR in 36% of the total number of cases sampled (44% of chronic apical periodontitis and 28% of abscess aspirates). In cases of chronic apical periodontitis, P. gingivalis variant type IV was the most prevalent (24%), followed by types I (20%), II (16%), and III (8%). In acute abscess samples, variant type II was the most prevalent (12%), followed by types III and IV (8% of each) and type I (4%). Combinations of up to 3 different genotypes were detected in a few cases. No single fimA genotype variant or combination thereof was significantly associated with symptoms. Overall, fimA types IV (16%), II (14%), and I (12%) were the most prevalent. Findings demonstrated that different P. gingivalis fimA genotypes can be present in primary endodontic infections.

Purification of Genomic DNA Extracted from Environmental Sources for Use in a Polymerase Chain Reaction: Figure 1.

INTRODUCTIONThe ability to amplify genomic DNA in a polymerase chain reaction (PCR) is dependent upon the purity of the DNA template. Environmental genomic DNA often contains contaminants (e.g., polyphenols, humic acids, polysaccharides) that reduce template purity and can be difficult to remove, thereby inhibiting PCR amplification. There is thus a need for a method to purify extracted genomic DNA without reducing DNA concentration. In this protocol, extracted genomic DNA is embedded in agarose plugs and incubated in a formamide and salt (NaCl) solution to remove contaminants. The NaCl works to deproteinize and stabilize the DNA. The formamide serves to denature the DNA (which will subsequently be renatured within the agarose plug) and any contaminants that may be bound to the DNA. The purified DNA is extracted from the agarose plug using a standard commercial agarose extraction method, and the DNA may then be used as a template for PCR. Genomic DNA purified using this method has been shown to serve as an efficient template for PCR, without significant loss of DNA yield. An additional advantage of the method is that it allows the simultaneous processing of large numbers of samples at once.

Extraction and Purification Method of Rice DNA from Rice Powder Containing Konjak Flour

Rice powder containing Konjak flour made with tuberous roots of Amorphophallus konjac is imported as a rice-processed product from China to Japan. An improved DNA purification method for the polymerase chain reaction (PCR) analysis of rice in such products is necessary, since Konjak flour constituents absorb the DNA purification buffer to form a gel, and cause problems in the subsequent purification steps. Here, we present a simple preparative system for isolation of the rice and a purification method of the rice DNA from the product. The purified DNA was confirmed to be a good template for both PCR and real-time PCR.

Characterization, detection and identification of transgenic chili pepper harboring coat protein gene that enhances resistance to cucumber mosaic virus

Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5'-flanking region of the CP gene and a Blastbased search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.

Preparation of Genomic DNA from Hawaiian Bobtail Squid (Euprymna scolopes) Tissue by Cesium Chloride Gradient Centrifugation

This procedure describes the extraction of genomic DNA from adult bobtail squid (Euprymna scolopes) tissues by cesium chloride (CsCl) gradient centrifugation. There are numerous generic methods and commercial kits for the preparation of genomic DNA based on proteolytic digestion of chromatin components, followed by selective binding of nucleic acids to ion-exchange affinity media, but many of these do not yield DNA that can be readily restricted. Also, molluscan tissues contain mucopolysaccharides, which tend to copurify with DNA under certain conditions. Although nucleic acids prepared this way can serve as a template for polymerase chain reaction (PCR), other enzymatic modifications of nucleic acids are inhibited by these contaminants. The method described here yields high-molecular-weight DNA that can be readily restricted for Southern hybridization. The procedure uses brain tissue under the assumption that its genome is unlikely to be rearranged in any way, has a high nucleic acid:protein ratio, and avoids potential sources of enzymatic contaminants and parasites from the intestinal sac. However, the method can be applied to other tissue sources and works well with other species. The purification of DNA by gradient centrifugation is an established method based on the specific buoyant density of double-stranded nucleic acids and the ability of CsCl solutions to form a salt gradient in a centrifugal field. It can also be adapted to the purification of RNA, which has a higher buoyant density than DNA. Unfortunately, this method is somewhat involved and expensive and produces large amounts of ethidium bromide waste.

Preparation of magnetite-loaded silica microspheres for solid-phase extraction of genomic DNA from soy-based foodstuffs

Solid-phase extraction has been widely employed for the preparation of DNA templates for polymerase chain reaction (PCR)-based analytical methods. Among the variety of adsorbents studied, magnetically responsive silica particles are particularly attractive due to their potential to simplify, expedite, and automate the extraction process. Here we report a facile method for the preparation of such magnetic particles, which entails impregnation of porous silica microspheres with iron salts, followed by calcination and reduction treatments. The samples were characterized using powder X-ray diffractometry (XRD), scanning electron microscopy (SEM), nitrogen adsorption/desorption isotherms, and vibrating sample magnetometry (VSM). XRD data show that magnetite nanocrystals of about 27.2 nm are produced within the pore channels of the silica support after reduction. SEM images show that the as-synthesized particles exhibit spherical shape and uniform particle size of about 3 μm as determined by the silica support. Nitrogen sorption data confirm that the magnetite-loaded silica particles possess typical mesopore structure with BET surface area of about 183 m 2/g. VSM data show that the particles display paramagnetic behavior with saturation magnetization of 11.37 emu/g. The magnetic silica microspheres coated with silica shells were tested as adsorbents for rapid extraction of genomic DNA from soybean-derived products. The purified DNA templates were amplified by PCR for screening of genetically modified organisms (GMOs). The preliminary results confirm that the DNA extraction protocols using magnetite-loaded silica microspheres are capable of producing DNA templates which are inhibitor-free and ready for downstream analysis.

Structure of the POMC promoter region in pituitary and extrapituitary ACTH producing tumors.

Proopiomelanocortin (POMC) is the common precursor of a variety of important endocrine peptides including ACTH. Transcription of the POMC gene is positively regulated by CRH through cAMP-responsive regions and is under negative feedback control by glucocorticoids which exert their inhibitory effect trough negative glucocorticoid responsive elements (nGRE). In vitro studies using the rat POMC promoter suggested that binding of the glucocorticoid receptor complex to a -63 bp binding site is correlated with repression of POMC gene transcription, and that specific mutations in this region abolish this effect. Impaired negative feedback regulation, though to a different degree, is a common feature of both corticotroph tumors (Cushing's disease) and extrapituitary ACTH producing tumors. We have analyzed the upstream promoter region of POMC gene from eleven patients with Cushing's disease, four of which had Nelson's syndrome, and from one patient with an ectopic ACTH syndrome secondary to a lung carcinoid for any possible mutations in the nGRE and/or cAMP-responsive sequences. DNA was purified from tumor tissue and was used as template for polymerase chain reaction (PCR). A segment between -371 and -19 bp of the POMC transcription start site was amplified and cloned into a plasmid vector. Sequencing was performed using the dideoxy chain termination procedure. Analysis of the 5'-flanking region revealed no defect in all tumors investigated. We conclude from our results that the defective glucocorticoid repression of POMC peptides production may be more likely due to aberrancies in other components of the complex transcriptional regulatory mechanism.

A rapid and cheap protocol for preparation of PCR templates in peanut

This paper describes a simple, low cost and reliable DNA template preparation protocol for polymerase chain reaction (PCR) using immature leaves from peanut seeds or leaves from field-grown plants. The technique may find wide utility in studies involving PCR-based molecular markers, rapid screening for transformants and gene cloning.

An improved method for purifying genomic DNA from forest leaf litters and soil suitable for PCR

Background, aim and scope An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg2+ (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine plantation of southeast Queensland, Australia.

Quick Miniprep for Plant DNA Isolation

INTRODUCTIONThis procedure describes a quick miniprep for plant DNA isolation. It yields a small quantity of poorly purified DNA. However, many samples can be processed in a short time. When used with Arabidopsis, the method generally provides DNA of sufficient quality for polymerase chain reaction (PCR) amplification of cleaved amplified polymorphic sequences (CAPS) and simple sequence length polymorphism (SSLP) markers. Repeated freeze-thaw cycles should be avoided, because they result in samples that will no longer work as PCR templates.