Set Of Primers For Amplification Research Articles

Molecular detection of glutamate dehydrogenase gene of Giardia lamblia isolated from food handlers in Erbil city

Background and objective: Giardia lamblia is the intestinal flagellated protozoan parasite that infects vertebrates, including humans. Giardiasis is the major diarrheal disease found worldwide. It can be symptomatic or may be an asymptomatic carrier that led to chronic disease. This study aimed to determine the proportion of giardiasis among food handlers and evaluate the correlation between two laboratory methods for identifying the Giardia lamblia. Methods: A total of 308 stool samples were collected from food handlers that annually attend the central laboratory in Erbil City. Wetmount microscopic examination was performed for the diagnosis of cysts and trophozoites of the Giardia parasite. Molecular analysis done for positive samples, DNA extraction performed using the QIAamp Fast DNA Stool Mini Kit (Qiagen Company, Germany). Nested PCR analysis was done targeting the Glutamate dehydrogenase gene using two sets of primers for amplification of 734bp fragment. Gel electrophoresis was performed for visualizing the amplified DNA by Ultraviolet light. Results: The mean age of food handler participants was 29 years. Most (93.8%) of the food handlers were males, and the majority (98.6%) of the participants did not have any signs and symptoms. Four (7.4%) microscopy positive sample participants were highly educated. There was no association between educational level and positive rate by microscopy (P = 0.066). The majority of participants did not receive treatments, particularly most of the microscopic positive samples. The food handlers did not take any antiparasitic treatments 9 (3.4%) (P = 0.676). From 11 (3.6%) microscopically positive samples, 10 (90.9%) Giardia lamblia gdh gene 734 bp fragments were amplified by nested PCR. Conclusion: Amplification of 734bp of gdh gene by the nested PCR is the most specific and sensitive method for identifying Giardia lamblia. Food handlers were important people to care about sanitation and preparing food, particularly for avoiding diseases transmitted by food. Keywords: Giardiasis; Genetic characterization; gdh gene; Nested PCR.

TOPO6: a nuclear single-copy gene for plant phylogenetic inference

TOPO6, a nuclear gene-marker region of subunit B of the plant homolog of archaean topoisomerase VI, occurs as single-copy locus in the haploid genome of most plant groups. The gene consists mainly of 19 exons and 18 introns, which provide conserved primer binding sites for PCR amplification in many angiosperm families combined with variable sequence stretches that can be explored in molecular systematics. Here intron/exon structure, sequence diversity, and a set of amplification primers are described to use TOPO6 as single-copy phylogenetic marker region in a wide range of plant taxa, either through PCR amplification or hybridization-based sequence capture.

Primer and platform effects on 16S rRNA tag sequencing.

Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. Beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.

Gene Cloning and Homology Modeling of the 3-Oxoacyl-ACP Synthase from Aeromonas hydrophila for Drug Discovery

Fatty acid biosynthesis in the Aeromonas hydrophila plays an important role in the formation of cell membrane and cell viability. The 3-oxoacyl- acyl carrier protein (ACP) synthase fadH and fadB from A. hydrophila have contributed in the initiation and elongation of fatty acid biosynthesis. In this study, we have designed new set of primers for amplification of the open reading frame of fadH and fadB. Cloning and sequencing of these amplified genes, analyzed that the G+C content of fadH and fadB were 63.3% and 68.79% respectively. The homology modeling was performed to generate the 3- D structure of fadH and fadB; and showed that more than 90% amino acid residues in allowed region of Ramachandranan plot. These findings provide a new avenue for better understanding, the role of genes encoding proteins involved in the fatty acid biosynthesis and thereby using as a potential and novel target for structure based drug designing to control the infections of A. hydrophila.

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis

A mutation in the gene coding for the ryanodine receptor 1 (RYR1), also known as halothane (hal) gene or swine stress gene, is associated to the porcine stress syndrome (PSS). Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N) is cut in two fragments, whereas the mutant allele (n) is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn.

MAD-DPD: Designing Highly Degenerate Primers with Maximum Amplification Specificity

This work introduces minimum accumulative degeneracy, a variant of the degenerate primer design problem, which is particularly useful when a large number of sequences are to be covered by a set of restricted number of primers. A primer set, which is designed on a minimum accumulative degeneracy basis, especially helps to reduce nonspecific PCR amplification of undesired DNA fragments, as fewer primer species are present in PCR. A Boltzmann machine is designed to solve the minimum accumulative degeneracy degenerate primer design problem, called the MAD-DPD Boltzmann machine. This algorithm shows great flexibility, as it can be determined either to solve the problem with strict fidelity to covering all input sequences or to exclude some input sequences if it results in less degenerate primers. This Boltzmann machine is successfully implemented in designing a new set of primers for amplification of antibody variable fragments from mouse spleen cells, which theoretically covers more diverse antibody sequences than currently available primers. The MAD-DPD Boltzmann machine is available online at bioinf.cs.ipm.ir/download/MAD_DPD08172007.zip.

Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli.

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.

A set of primers for amplification of mitochondrial DNA in Picea abies and other conifer species

AbstractThirteen primer pairs generating intraspecific length and/or presence‐absence polymorphism in Picea abies have been obtained from a P. abies mtDNA library, using different methodologies (agarose gel electrophoresis, polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), single‐strand conformation (SSCP). Poly‐morphism tests were extended successfully to other Picea species (P. omorika, P. engelmanii and P. glauca) and species belonging to other conifer genera (Abies alba, Larix laricina and Pinus pinaster). This set of PCR‐based mitochondrial markers can provide promising tools for studying phylogeography or phylogeny in P. abies and other conifer species in which the mt genome is generally the only one to be transmitted via the female gamete.

Rapid genotyping of Bacillus anthracis strains by real-time polymerase chain reaction.

Rapid and accurate identification of Bacillus anthracis is critical for patient care as well as outbreak control. We have developed 3 separate PCR based assays using fluorescence resonance energy transfer (FRET) to detect the presence of pXO1, pXO2 plasmids and a chromosomal marker. A set of amplification primers and probes were used in each assay. The probes were ad jacently placed inside the primer sites and were 1-bp apart. The upstream probe was labeled with fluorescein at the 3' end, and the downstream probe had Cy5 attached at the 5' end. The probes are included in the PCR reactions and hybridize with the PCR products as they are formed. Binding of probes to PCR products results in transfer of energy from fluorescein to Cy5, resulting in emission from Cy5. Increase in fluorescence, indicating amplification, was monitored in real time on a LightCycler((TM)) LC24. Initial denaturation of target sequences was accomplished at 95 degrees C for 1 min, followed by 28 cycles of denaturation at 95 degrees C for 0 sec, annealing at 58 degrees C for 15 sec, and elongation at 72 degrees C for 5 sec. These assays are specific and can be performed on as little as 25 ng of total DNA or crude cell lysate a from fresh colony. It is thus possible to deter mine the genotype of B. anthracis strains in less than 1 hour.

Group-Specific PCR Primers to Amplify 24S a-Subunit rRNA Genes from Kinetoplastida (Protozoa) Used in Denaturing Gradient Gel Electrophoresis.

We developed and tested a set of primers for amplification of a region of the 24S a-subunit rRNA genes (24S rDNA) specific to Kinetoplastida (Protozoa). The reverse primer was supplied with a GC rich region in the 5? end in order to make the PCR product suitable for analysis by denaturing gradient gel electrophoresis (DGGE). PCR product was obtained from all the kinetoplastids tested and no PCR product was obtained from any other Eukaryotes or Prokaryotes tested. It was possible to distinguish between all pure cultures of kinetoplastids by denaturing gradient gel electrophoresis in gels ranging from 20% to 60% denaturants. PCR-DGGE analysis of DNA purified from lake sediment revealed approximately 20 bands indicating high kinetoplastid diversity. Direct cloning and sequencing of 24S rDNA sequences retrieved from the lake sediment by PCR also showed high kinetoplastid diversity. Of 43 clones, 27 different sequences were found. Alignments and phylogenetic analysis showed that a majority of the sequences were most closely related to the Bodonidae. Four sequences were closer to the Trypanosomatidae, whereas three sequences fell outside both groups. The PCR-DGGE procedure developed in this study has been shown to be useful for distinguishing between different kinetoplastid species. Thus, it may be a useful tool for evaluating the genetic diversity of this group in environmental samples, e.g., as a result of perturbation. Another possible application of this method is in fast and accurate screening for the presence and identification of pathological parasitic Kinetoplastida from environmental samples and for diagnostics of human and animal infections.

A set of primers for amplification of noncoding regions of chloroplast DNA in the grasses

AbstractTwelve primer pairs, which amplify noncoding regions of chloroplast DNA, were designed for grass species. Their universality was tested on the six major grass subfamilies, and several were sequenced to examine their variability in Phragmites australis. The primers amplified well for most species tested and the majority showed intraspecific variation.

Identification of a novel FAS/ER-α fusion transcript expressed in human cancer cells

A semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to evaluate the presence of estrogen receptor-α (ER-α) in human prostate cancer cells. Unexpectedly, a novel fatty acid synthase (FAS)/ER-α fusion transcript was identified, in which the N-terminus of FAS was fused in-frame with the C-terminus of ER-α. The existence of the FAS/ER-α transcript was further confirmed by RT-PCR analysis using various sets of amplification primers and different reverse-transcribed primers in the presence of dimethyl sulfoxide to eliminate the secondary structure of RNA. The predicted FAS/ER-α protein would contain largely domain I of FAS and the entire ligand binding domain of ER-α. The FAS/ER-α was expressed in a variety of human cancer cell lines including prostate, breast, cervical and bladder cancer cell lines. Our data suggest that the presence of FAS/ER-α may complicate the FAS and the ER-α signalling pathway.

Common and Rare ABCA1 Variants Affecting Plasma HDL Cholesterol

Mutations in ABCA1, a member of the ATP-binding cassette family, have been shown to underlie Tangier disease (TD) and familial hypoalphalipoproteinemia (FHA), which are genetic disorders that are characterized by depressed concentrations of plasma high density lipoprotein (HDL) cholesterol. An important question is whether common variants within the coding sequence of ABCA1 can affect plasma HDL cholesterol in the general population. To address this issue, we developed a screening strategy to find common ABCA1 variants. This strategy involved long-range amplification of genomic DNA by using coding sequences only, followed by deep sequencing into the introns. This method helped us to characterize a new set of amplification primers, which permitted amplification of virtually all of the coding sequence of ABCA1 and its intron-exon boundaries with a single DNA amplification program. With these new sequencing primers, we found 3 novel ABCA1 mutations: a frameshift mutation (4570insA, A1484S-->X1492), a missense mutation (A986D) in a TD family, and a missense mutation (R170C) in aboriginal subjects with FHA. We also used these sequencing primers to characterize 4 novel common amino acid variants in ABCA1, in addition to 5 novel common silent variants. We tested for association of the ABCA1 I/M823 variant with plasma HDL cholesterol in Canadian Inuit and found that M823/M823 homozygotes had significantly higher plasma HDL cholesterol compared with subjects with the other genotypes. The results provide proof of principle of the effectiveness of this approach to identify both rare and common ABCA1 genomic variants and also suggest that common amino acid variation in ABCA1 is a determinant of plasma HDL cholesterol in the general population.

Identification of a Notch3 mutation in a Japanese CADASIL family. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease that is characterized by recurrent stroke episodes and focal neurologic deficits progressing to pseudobulbar palsy and dementia. The causative gene is the Notch3 gene on chromosome 19, and 22 missense mutations have been identified in Caucasian patients to date. To perform mutational analysis of the Notch3 gene, we identified its exon intron boundaries and prepared sets of primers for amplification of each exon. Using these primers, we determined the Notch3 gene in a Japanese family with CADASIL symptoms and found a missense mutation (Arg133Cys) in exon 4. The mutation was heterozygous and cosegregated with the disease. Thus, the Notch3 gene is responsible for CADASIL in patients across different ethnic groups.

Amplification Primers That Target Locus-Specific Introns in Actinopterygian Fishes

The study of interspecific and intraspecific phylogeny has benefited from the development universal amplification primers that target specific regions of the mitochondrial genome. Unfortunately, analogous primer sets for nuclear-encoded regions have not always proven reliable in targeting single loci across a wide variety of organisms. We describe a strategy for developing sets of oligonucleotide primers that flank conserved intron positions within single loci. We demonstrate this approach through development of two sets of amplification primers that target polymorphic, single-locus intron regions from a large number of actinopterygian fishes. HE study of inter- and intraspecific phylog

Evolution of DNA on Y-chromosome in hominoid primates as examined by PCR

Every species of non-human primates, especially those of hominoids, has a variety of reproductive structures and accompanying male traits, such as sexual dimorphism and relative size of testis to body weight, which may be at least partly triggered by DNA on the Y-chromosome. Recently, a panel of PCR (Polymerase Chain Reaction) primer sets were designed to amplify various DNA segments spread over the human Y-chromosome. We applied these primer sets for amplification of DNA segments on the Y-chromosome of hominoid species: chimpanzee, bonobo (Pygmy chimpanzee), gorilla, orangutan, whitehanded gibbon, agile gibbon, and Japanese monkey as an out group. The DNA segments including SRY, testis determining factor, and ZFX/ZFY could be amplified clearly in males of all species examined. These highly conserved genes may serve important biological functions. However, as the phylogenic distance from humans increased, some of the DNA segments could not be amplified. For example, DYZ1 (SY160) could be amplified only using human DNA as a template, and DYF60S1 (SY61), DYZ217 (SY126) and DYS233 (SY148) could be amplified only using human and African great ape DNA. It is interesting to note that locus DYS250 (SY17) could not be amplified in chimpanzee and bonobo but amplified in gorilla and orangutan. Locus DYS251 (SY18) was amplified in all species except the white-handed gibbon. These results indicate that a variety of evolutionary events including mutation, deletion, insertion, and rearrangement occurred in Y-chromosome DNA during primate evolution.

Coamplification of hprt cDNA and γ T-cell receptor sequences from 6-thioguanine resitant human T-lymphocytes

The nature of mutation at the HPRT locus in human T-lymphocytes in vivo is currently a subject of considerable interest. Determination of clonality in individual mutant T-lymphocytes is essential for the proper interpretation. This requires the molecular analysis of their respective T-cell receptors (TCR). We have developed a polymerase chain reaction (PCR)-based method for coamplification of hprt cDNA and the rearranged gamma T-cell receptor genes from crude cell lysates of individual 6-thioguanine resistant human T-lymphocytes. Following reverse transcription to produce hprt cDNA, the crude cell lysate is treated with proteinase K and subjected to a primary PCR with two sets of amplification primers, one specific for the hprt cDNA and the other for the rearranged gamma TCR gene. A secondary round of PCR, employing appropriate sets of nested amplification primers, are then used to produce sufficient quantities of DNA for both the sequencing and restriction fragment length analysis, of the hprt cDNA and gamma TCR gene respectively.