Polymerase Chain Reaction Conditions Research Articles

DETECTION OF A PUTATIVE VIRULENCE cadF GENE OF CAMPYLOBACTER JEJUNI OBTAINED FROM DIFFERENT SOURCES USING A MICROFABRICATED PCR CHIP

ABSTRACT A microfabricated polymerase chain reaction (PCR) chip made of epoxy‐based photoresist (SU‐8) was recently designed and developed. In this study, we tested whether the PCR chip could be used for rapid detection of a potential virulence determinant, the cadF gene of Campylobacter jejuni. PCR was performed using published PCR conditions and primers for the C. jejuni cadF gene. DNA isolated from a C. jejuni reference strain CCUG 11284, C. jejuni isolates obtained from different sources (chicken and human), and Campylobacter whole cells were used as templates in the PCR tests. Conventional PCR in tube was used as the control. After optimization of the PCR chip, PCR positives on the chip were obtained from 91.0% (10/11) of the tested chips. A fast transition time was achieved with the PCR chip, and therefore a faster cycling time and a shorter PCR program were obtained. Using the PCR chip, the cadF gene could be detected in 1.5 h in comparison to 3.4 h in the published methods. The PCR chip can be applied for rapid detection of the virulence cadF gene of C. jejuni obtained from different sources. This is the first PCR chip that has been used to detect Campylobacter virulence genes. Using the chip, the cadF gene of C. jejuni can be detected directly from bacterial whole cells without DNA isolation and purification steps.

Use of Duplex PCR-CTPP Methods for CYP2E1 RsaI/IL-2 T-330G and IL-1B C-31T/TNF-A T-1031C Polymorphisms

Two duplex polymerase chain reaction (PCR) with confronting two-pair primer (PCR-CTPP) methods were designed for cytochrome P450 (CYP) 2E1 RsaI and interleukin (IL-2) T-330G, and for IL-1B C-31T and tumor necrosis factor-alpha (TNF-A) T-1031C. The four polymorphisms are considered to be functional, and the three cytokines reportedly inhibit CYP2E1 expression. Many studies have reported associations between the above polymorphisms and risk of diseases including cancers and inflammatory diseases. The main objective of this study was to examine the applicability of the established PCR conditions to a real situation. Participants were female examinees aged from 35 to 85 years who attended health checks run by a local government in Japan. The allele frequencies among 325 female health check examinees were 0.804 for CYP2E1 c1 allele, 0.668 for IL-2-330T allele, 0.554 for IL-1B-31T allele, and 0.822 for TNF-A-1031T allele. p-Values from a Hardy-Weinberg equilibrium test were 0.658, 0.955, 0.062, and 0.806, respectively. Clear DNA bands observed with electrophoresis allowed us to genotype the four polymorphisms. The genotype frequencies were within the Hardy-Weinberg equilibrium test proportions, though the p-value for IL-1B C-31T was marginal. Both duplex PCR-CTPP methods may be useful tools for studies on the association between these polymorphisms and disease risk.

Multiplex PCR with Multichannel Microchip Electrophoresis: An Ultrafast Analysis for Genetic Diseases

A Y chromosomal polymorphic markers screening strategy using a multiplex polymerase chain reaction (PCR) and DNA microchip electrophoresis technology has recently been developed. It is a part of the human Y chromosome haplotyping system for studying Japanese population genetics and its relationship with male spermatogenic failure. This strategy is based on optimizing and modifying the primer set concentrations while keeping all other components of the PCR mixtures and conditions similar to those of a singleplex PCR. Well-balanced PCR products are obtained without changing even the DNA oligomer melting temperatures. Here, a panel of primer sets are used to amplify two groups of Y chromosome markers. The first consists of five markers and the second consists of seven markers. Both are possibly deleted in infertile men. The microchip electrophoresis technology is fast and sensitive, enables direct molecular typing of several Y chromosomal markers, and is separated by a difference of as many as six base pairs.

Type-Specific Multiple Sequencing Primers: A Novel Strategy for Reliable and Rapid Genotyping of Human Papillomaviruses by Pyrosequencing Technology

DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.

Platelet glycoprotein I(b)alpha and integrin alpha2 beta1 polymorphisms: gene frequencies and linkage disequilibrium in a population diversity panel.

Genetic variants in the GP1BA and ITGA2 genes have been proposed as potential modifiers for arterial vascular disease and bleeding disorders. Since ancestry may play an important role in the prevalence of these variants, we sought to determine their allele frequency and linkage disequilibrium in a collection of 1064 DNA samples from 51 ethnic groups. We studied haplotypes of ITGA2 defined by single nucleotide substitutions at positions -52, 807, and 1648, and GP1BA variants defined by sequence changes in positions -5 (Kozak), 1018 (T145M, HPA-2) and 1285 (VNTR A, B, C and D). Frequency of haplotypes of ITGA2 showed considerable variation across the different groups, with a higher prevalence of the haplotype -52C or T/807C/1648A observed in African compared with caucasian and Asian populations. The haplotypes 52C/807T/1648A and -52T/807T/1648A were not observed in caucasians or South Americans. While relative frequencies of the GP1BA Kozak alleles were comparable across groups, the methionine allele (HPA-2b) showed a higher frequency in Africa (0.26) than in the other groups. We also observed a high prevalence of the VNTR B allele in the African and Israeli populations. Haplotype analysis revealed incomplete linkage disequilibrium between the HPA-2 and VNTR alleles. Incorporation of GP1BA variants into the set of SNPs already genotyped by the HapMap project disrupted the pre-existing haplotype block. These data provide a valuable resource for optimal selection of variants best tailored for association studies of vascular disease or bleeding disorders when examining individuals of different ancestral origins.

Characterization and PCR multiplexing of polymorphic microsatellite loci for the invasive ant Wasmannia auropunctata

AbstractHighly polymorphic genetic markers provide a useful tool for estimating genetic parameters in studies of the evolution of sociality in insects. We isolated and characterized 12 polymorphic microsatellite loci in the invasive ant, Wasmannia auropunctata, and described experimental conditions for PCR (polymerase chain reaction) multiplexing and simultaneously genotyping these loci in two sets of five and seven markers. The number of alleles per locus ranged from two to 14 and the observed heterozygosity ranged from 0.233 to 0.967. Moreover, results of cross‐species amplification tests are reported in three other species of Wasmannia and in two species of the genus Allomerus.

Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling

A condition for multiplex polymerase chain reactions (PCRs) of which outcomes sensitively indicate the actual annealing temperature of thermal cycling is reported. The multiplex reaction was designed to produce four different amplicons of 200, 300, 400, and 480 bp. However, the degree of amplification of each amplicon sensitively responds to a small change in the annealing temperature, by which one can predict the actual annealing temperature of thermal cycling. Deviations between the actual and the designated annealing temperatures as small as 0.5 °C were manifested by the banding patterns of the multiplex PCRs in simple agarose gel electrophoresis. For prediction of temperatures in a more objective manner, capillary electrophoresis was also applied to obtain numerical expressions of the relative intensities of the amplicons. By optimizing the multiplex PCR conditions, where concentrations of buffer, dNTPs, and primer pairs were major factors, satisfactory sensitivity and reproducibility of the band patterning were achieved. Blind tests demonstrated the accuracy of the prediction of actual annealing temperatures within ±0.5 °C. The multiplex PCR approach will be further refined and tested for realization of an easily accessible alternative to a physical temperature measurement device in testing the performance of thermal cyclers for PCR.

Histone deacetylase inhibitors profoundly decrease proliferation of human lymphoid cancer cell lines

Methylation of tumor suppressor genes is frequently observed in human cancers. These genes are silenced by histone deacetylase (HDAC) recruited by methylated DNA in their promoter regions. HDAC removes acetyl groups from histones and prevents the basic transcriptional machinary access to the target gene, leading to transcriptional repression. HDAC inhibitors (HDACIs) can restore the expression of the tumor suppressor and/or cell cycle regulatory genes in cancer cells and block the cellular proliferation of these cells. In this study, we investigated the in vitro antiproliferative activities of the HDACIs, suberoylanilide hydroxamic acid (SAHA), and valproic acid against 14 human lymphoid cancer cell lines. All of these cell lines were sensitive to the antiproliferative effects of the HDACI. SAHA induced either G1 or G2-M arrest as well as apoptosis. SAHA downregulated cyclin D1 and D2, and upregulated p53, p21, and p27. Chromatin immunoprecipitation analysis revealed a remarkable increase in the level of acetylated histones associated with the p21 promoter after SAHA treatment. In nude mice, SAHA significantly inhibited growth of a mantle cell lymphoma without major toxic side effects. In summary, HDACIs are promising therapeutic agents for human lymphoid cancers.

Identification of Eimeria mitis and Eimeria praecox in broiler feces using polymerase chain reaction

There are few reports concerning the epidemiology of Eimeria praecox and Eimeria mitis in Brazil. In the present experiment, the polymerase chain reaction (PCR) was used to identify these species in 156 samples of broiler chicken feces from several Brazilian states and the Federal District. Oocysts present in feces samples were purified by sodium chloride flotation followed by addition of DNAzol reagent (Invitrogen®) for extraction of genomic DNA. DNA was precipitated and stored following DNAzol reagent manufacture's instructions. The primers and PCR conditions were as described by Schnitzler et al. (1999). In the 156 field samples analyzed by PCR, 70 and 45 were positive for E. praecox and E. mitis, respectively. In this study we have shown that DNA extraction using DNAzol followed by PCR can be a useful tool in epidemiological studies, since it provides fast and reliable detection of Eimeria sp. in field samples.

Quantitative polymerase chain reaction and microchip electrophoresis to detect major rearrangements of the low‐density lipoprotein receptor gene causing familial hypercholesterolemia

A variety of rearrangements in the low-density lipoprotein receptor (LDLR) gene cause severe forms of familial hypercholesterolemia (FH). However, current methods for searching these abnormalities in FH samples, e.g., Southern and Northern Blot, are labor-intensive and not routinely used by diagnostic laboratories. We developed a simpler approach based on the quantitative polymerase chain reaction (PCR) amplification of part or all gene's coding sequences by a series of multiplex amplifications comprising three nonadjacent gene sections plus a fourth section used as an internal reference. Thereafter, the analysis of these PCR products by microchip electrophoresis revealed either deletions or duplications in the investigated gene sections through the simple comparison of electropherograms obtained from mutant and control samples. This required primers leading to well-resolved peaks with minimal size differences among coamplified products and PCR conditions allowing a linear quantitative response to template amount variations as those caused by duplication or deletion of specific gene sections. Also, the inclusion of exon 17 amplification product as an internal reference in each multiplex PCR allowed the normalization of quantitative results by dividing the area of each amplified section by the area of exon 17. The comparison of these ratios calculated from 10 carriers of 6 LDLR known rearrangements with those obtained from 14 control samples showed that gross deletions roughly halved and duplications doubled the ratio values of exons involved in the mutation. This allowed to distinguish gross mutations from sample-to-sample differences that reached at maximum 8% variation over mean values.

Development of PCR conditions in a silicon microreactor DNA-amplification device

A silicon microsystem was developed which functions as a miniaturised DNA-amplification device. The system represents a technology platform for performing a polymerase chain reaction (PCR) with reduced volumes of 7 µL. The silicon microreactor was fabricated using silicon bulk micromachining, and a platinum heater was fabricated on a Pyrex substrate. A miniaturised DNA-amplification system permitted rapid heating and cooling, and shorter reaction times of 30 min were achieved. In this work, biocompatibility issues are addressed; conditions for efficient PCR in a silicon-based microreactor are established for the amplification of 500 bp DNA from the Escherichia coli bacteriophage Lambda; and the conditions are verified by amplifying a 255 bp region from the Mycobacterium tuberculosis rpoB gene. This work describes the PCR volume scale down experiments that were conducted and concentrations of the reactants; Taq polymerase, oligonucleotides, MgCl2 and template DNA were determined for DNA-amplification reactions with this novel device.

Rapid amplification for the detection of Mycobacterium tuberculosis using a non-contact heating method in a silicon microreactor based thermal cycler

This is the first report of a polymerase chain reaction (PCR)-based Mycobacterium tuberculosis diagnostic tool fabricated as a silicon microreactor for DNA amplification using a halogen lamp as the heating source. Our experiments demonstrate that PCR amplification can be successfully performed in silicon microreactors using a non-contact heating method. These devices have been used to amplify a specific fragment of the rpoB gene from M. tuberculosis, where mutations leading to multi-drug resistant strains are most likely to occur. Amplification was achieved in the microreactors in 34 min, compared to the 62 min amplification time required in a conventional peltier-heated PCR machine. Temperature control accuracy for our heating approach was ±1 °C. The amplification was performed from genomic DNA and produced PCR products in comparable quantities to a conventional thermal cycler, but in reduced time. This paper describes the fabrication, characterisation, control and use of these prototype devices for DNA amplification. PCR conditions were established using Lambda bacteriophage DNA as a model reaction system. Subsequently, DNA from the M. tuberculosis rpoB gene was successfully amplified.

A new method to develop internal controls for the determination of phytoplasma concentration

This study aimed to develop a quick and simple method that generates internal controls from any polymerase chain reaction (PCR) amplified phytoplasma DNA fragment. Non-specific PCR conditions were used to generate unspecific DNA bands with the same primers as the target phytoplasma DNA fragment, but different in their sizes. The method is universal enough to be adaptable for any selected primer pairs. The procedure does not require preliminary knowledge of the sequence or restriction sites of the amplified DNA fragment. Developed internal controls are ligated into a bacterial vector, which can serve as a competitor, to co-amplify with the target phytoplasma DNA in a competitive PCR reaction. Serial dilutions of the internal controls with adjusted concentration and fixed amounts of target templates from phytoplasma-infected plants were amplified together with the same primers to estimate the relative amount of phytoplasma DNA.

Accurate STR Allele Designations at the FGA and vWA Loci Despite Primer Site Polymorphisms

Polymerase chain reaction (PCR)-based STR DNA typing systems are used extensively in the field of human identification. Under optimal PCR conditions, the amplicon yield from both alleles of an STR locus is expected to be approximately equivalent. However, it is reasonable to expect that rare genomic sequence polymorphisms will co-localize with well-designed primer sets and induce allele imbalance or "dropouts". Two samples were identified in the course of genotyping thousands of individuals with AmpF/STR Profiler Plus that showed strong disparity in amplitude peak height of heterozygous peaks at the loci vWA and FGA. These samples were reamplified at reduced annealing temperature in an attempt to balance the peak heights. Nucleotide sequencing documented polymorphisms at the PCR primer binding sites of the affected alleles. The results indicate that reducing the annealing temperature to improve primer-binding efficiency at the mismatch and employing an alternative multiplex enhanced the data from both samples. Reducing annealing temperatures could provide a simple general solution to improving data quality for samples where polymorphisms are suspected to cause allele imbalance. Finally, we report on additional polymorphisms surrounding the vWA locus in a genetically diverse population.

Rapid genotyping of the osteoporosis-associated polymorphic transcription factor Sp1 binding site in the COL1A1 gene by pyrosequencing.

We describe a novel polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencingbased assay for rapid genotyping of the polymorphic Sp1 binding site in the COL1A1 gene (1). A single nucleotide G-->T substitution polymorphism at this GC-rich site has recently been reported to be a predictive genetic marker for low bone mineral density (BMD). To simplify screening for this marker, we optimized PCR conditions and subjected the amplicons to pyrosequencing, which is a convenient high-throughput sequence analysis technique, readily amenable to automation. The analysis of 200 deidentified convenience DNA samples extracted from blood revealed genotype frequences in Hardy-Weinberg equilibrium (SS 68.0%, Ss 28.5%, and ss 3.5%) in agreement with other studies of European populations. This study demonstrates for the first time that pyrosequencing can be used for rapid identification of the osteoporosis-associated single nucleotide polymorphism (SNP) in the COL1A1 gene.

Molecular profiling of microbial communities associated with seeds of Beta vulgaris subsp. Vulgaris (sugar beet)

The composition of microbial communities on and within seeds may effect their storage and field performance, whether they are indigenous or applied as biocontrol agents. In this study, we have explored the usefulness of profiling small subunit ribosomal (SSR) gene fragments for studying the microflora associated with seeds. DNA was amplified by the polymerase chain reaction (PCR) and the amplicons separated using denaturing gradient gel electrophoresis (DGGE). Primers targeting eukaryotic SSRs were used to investigate fungal communities, and primers targeting bacterial SSRs were employed to study the eubacterial microflora. As a case study, we attempted to profile the fungi and bacteria associated with seeds of Beta vulgaris (sugar beet) to permit an insight into the varying field performance of several well-characterised commercial seed lots. Serious interference with the microbial signals was observed from the plant's own nuclear 18S rRNA genes and chloroplast 16S rRNA genes using standard PCR conditions and DNA extracted from whole seeds as template. Hot-start and touchdown PCR made no appreciable improvement to these signals. Seed imbibition and dissection into operculum and fruit wall and true seed prior to DNA extraction improved signal recovery in the fruit fraction. With primer modification, bacteria and fungi were detected in an excess of plant DNA of 100:1 and 10:1, respectively. With this method, microbial communities on seeds could be profiled, however, it is likely that targeted depletion of plant rDNA targets will be a necessary extra step before this approach can be used to screen seeds routinely.

Duplex polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for genotyping alcohol dehydrogenase beta subunit (ADH2) and aldehyde dehydrogenase 2 (ALDH2).

Alcohol dehydrogenase beta subunit (ADH2) Arg47His and aldehyde dehydrogenase 2 (ALDH2) Glu487Lys were genotyped by a duplex polymerase chain reaction (PCR) with confronting two-pair primers (PCR-CTPP), which allows DNA amplification with one-tube PCR including eight primers, and subsequent electrophoresis. Several PCR conditions were tested to establish the optimal conditions for distinguishing the allele-specific bands for the two polymorphisms. Under the optimal PCR conditions, 454 Japanese health check-up examinees were genotyped. The allele-specific bands were successfully amplified under the optimal conditions of the duplex PCR-CTPP. The genotype distributions were within the Hardy-Weinberg equilibrium. The bands produced by the duplex PCR-CTPP genotyping were clearer than those produced by PCR-CTPP, conducted solely for ADH2. ADH2 Arg47His and ALDH2 Glu487Lys were successfully genotyped by this newly developed duplex PCR-CTPP, an inexpensive and time-saving genotyping tool, which will be useful in epidemiological studies on alcoholism, as well as risk estimation of alcohol-related diseases.

Application of Single‐labelled Probe‐primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

AbstractA new method based on the incorporation of a single‐labelled probeprimer into polymerase chain reaction (PCR) for the detection of PCR‐amplified DNA in a closed system is reported. The probeprimer consists of a specific probe sequence on the 5′ ‐end and a primer sequence on the 3′‐end. A fluorophore is located at the 5′‐end. The primer‐quencher is an oligonucleotide, which is complementary to the probe sequence of probe‐primer and labelled with a quencher at the 3′‐end. In the duplex formed by probe‐primer and primer‐quencher, the fluorophore and quencher are kept in close proximity to each other. Therefore the fluorescence is quenched. During PCR amplification, the specific probe sequence of probe‐primer binds to its complement within the same strand of DNA, and is cleaved by Taq DNA polymerase, resulting in the restoration of fluorescence. This system has the same energy transfer mechanism as molecular beacons, and a good quenching efficiency can be ensured. Following optimization of PCR conditions, this method was used to detect hepatitis B virus (HBV) DNA in patient sera. This technology eliminates the risk of carry‐over contamination, simplifies the amplification assay and opens up new possibilities for the real‐time detection of the amplified DNA.

Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment

We investigated biases occurring in the polymerase chain reaction (PCR) amplification of 16S rRNA genes from an environmental sample, by comparing the clone libraries that we had previously prepared from the gut homogenate of the termite Reticulitermes speratus. We detected a significant increase in the expected number of phylotypes by lowering the annealing temperature, and a significant decrease in the proportion of clones belonging to the predominant group by raising the number of PCR cycles. We also found that the Bacteria-universal primer, 63F, introduced a seriously biased amplification caused by primer mismatches, in contrast to a previous report. These results, together with suggestions from previous studies using simplified model samples, will help us to recognize the limitations of PCR-based analysis.

Polymerase chain reaction for detection of avian leukosis virus subgroup J in feather pulp.

Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture.