Improved Polymerase Chain Reaction Method Research Articles

An improved single-round PCR leads to rapid and highly sensitive detection of Pneumocystis spp.

In order to standardize a polymerase chain reaction (PCR)-based method of Pneumocystis detection, we describe the development of an improved PCR method that targets the Pneumocystis mtLSUrRNA gene. Design of a new primer pair and PCR program with suitable parameters and optimization resulted in a simpler and faster single-round amplification assay. The sensitivity of the novel Pneumocystis genus-specific PCR proved comparable to the reference nested PCR. The improvement that this new PCR assay offers in the detection and epidemiological studies of Pneumocystis spp. infection in research laboratories is discussed.

The Improved Polymerase Chain Reaction Method Applied for Sex Identification of Crested Ibis, Nipponianippon

Given the necessity for increased conservation of endangered birds such as the Crested ibis, it is crucial to identify the gender of individuals fro m species with similar external mo rphologies among females (ZW) and males(ZZ) to establish a self-sustainable population for restoration of the species. Moreover, raising young chicks during the juvenile period based on sex determination encourages familiarity, wh ich might increase mating success rates. Thus, this study was carried out to determine the sex of Crested ibis chicks using three primers designed to check their identity. A se t of poly- merase chain reaction(PCR) primers was used to amplify a 276 bp chro mo-helicase-DNA b inding protein region(CHD) on the Z chro mosome in both sexes, whereas the other set was used to amp lify a 169 bp female -specific CHD sequence on the W chromosome. The sequences have been submitted to NCBI(GenBank Accession Number: AB620020, AB620021). Th is method will enable researchers to determine the gender of Crested ibis more simp ly and quickly.

One-stop polymerase chain reaction (PCR): An improved PCR method with speedy operation and comparable efficiency

The widely used polymerase chain reaction (PCR) protocol requires several post-cycling steps to visualize amplicons, decelerating PCR sample processing and result calling. “One-stop PCR” was developed by including both the loading buffer and nontoxic staining dye within a single PCR tube, allowing direct loading and simultaneous staining of amplicons for visualization. The efficiency and sensitivity of one-stop PCR were comparable to commercially available PCR kits and it is compatible with downstream uses and is not mutagenic. One-stop PCR robustly minimizes the time and labor required for PCR reaction setup and product visualization. Key words : Polymerase chain reaction (PCR), post-cycling, compatible dye, Gelred.

An improved polymerase chain reaction method for genetic testing of spinocerebellar ataxia type 3

The development of a reliable PCR assay for genetic testing of spinocerebellar ataxia type 3. Touchdown PCR conditions were tested and different primer sets were evaluated with genomic DNA from blood sample of patients suffering from spinocerebellar ataxia type 3 (SCA3). An improved PCR assay was developed with a new set of primers and using the optimized touchdown PCR protocol. This new assay had been successfully employed in the screening of one identificated SCA3 family. Results from the present study document a simple and reliable PCR assay for genetic testing of SCA3. Strategies used in the present study may find applications in the optimization of PCR assay for triplet expansion with GC rich in the sequence context.

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

Improved PCR Method for Amplification of GC-Rich DNA Sequences

Most housekeeping genes, tumor-suppressor genes, and approx 40% of tissue-specific genes contain G+C sequences in their promoter region that were very difficult to amplify. In this report, we propose an improved polymerase chain reaction (PCR) method to be used for successful amplification of the tissue factor pathway inhibitor (TFPI)-2 gene promoter region that exhibit >70% G+C content in a sequence of approx 300 bp and a complete CpG island region spanning exon 1, the three transcription initiation sites, and the translation start site. Therefore, this method can be recommended to amplify other GC-rich genomic templates.

Demonstration That Mast Cells, T Cells, and B Cells Bearing the Activating Kit Mutation D816V Occur in Clusters within the Marrow of Patients with Mastocytosis

Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering.

Improving diagnosis of Huntington's disease by analysis of an intragenic trinucleotide repeat expansion.

To develop an accurate presymptomatic test for Huntington's disease. An improved polymerase chain reaction method was used to investigate the pattern of expansions of a CAG repeat sequence located in the 5' region of a gene recently found to produce the protein called Huntingtin. We documented the range of trinucleotide repeat expansions in the responsible gene in 82 affected individuals compared with 80 control subjects from a Western Australian population. The number of expanded repeats ranged from 40 to 73 in affected individuals and from 13 to 38 in normal controls. Polymerase chain reaction analysis of a CAG repeat sequence in the Huntington's disease gene clearly differentiated between normal and mutated alleles, providing an accurate diagnostic test for the disorder in individuals at risk. This predictive test has met with greater acceptance and demand than methods using family based linkage studies.

Detection and identification of Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica by an improved polymerase chain reaction method.

We developed a polymerase chain reaction method in order to detect and identify both Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica. Polymerase chain reaction was performed by using a mixture of primers against the inv gene from Y. pseudotuberculosis and the ail gene from pathogenic Y. enterocolitica. Further addition of primers against the plasmid-coded virF gene from Y. enterocolitica made it possible to detect a virulence-associated gene of both species at the same time. This method was proved to be an adequate and convenient procedure for routine detection and identification of these bacilli.

Rapid, sensitive, specific, and quantitative detection of human T-cell leukemia virus type 1 sequence in peripheral blood mononuclear cells by an improved polymerase chain reaction method with nested primers.

Improving on the nested double polymerase chain reaction (PCR) described previously, we have developed a new two-step PCR (TS-PCR) method for detecting more specifically the human T-cell leukemia virus type 1 (HTLV-1) proviral sequences in peripheral blood mononuclear cells (PBMC). In our TS-PCR method, the point of modification is to use optimal concentrations of primers in the first amplification step in the range of 0.01-0.025 microM. This increases sensitivity and specificity enough to detect from 1 to 10(5) copies of template DNA without radioisotopes. This method is rapid because of completion in 1 day and is also applicable for quantitative detection of clinical specimens. The data show that the quantitative detection of HTLV-1 proviral sequences by this method correlates with the anti-HTLV-1 antibody titers from serologic analysis of seropositive healthy carriers. Moreover, the TS-PCR method using each specific primer was also attempted for successful detection of other viral genomes; therefore, the principle of this method is widely suitable for routine detection of genomes in the basic and clinical microbiological fields.