Rapid identification and authentication of closely related animal cell culture by polymerase chain reaction

Animal cell lines are important resources for research and diagnostic applications. Cross-contamination and misidentification of cell lines, however, can cause major problems for research (for example, false results that come from contamination cells may mislead the science). Hence, it is imperative to routinely monitor cell lines for identity and authenticity. Here, we extend our previous work on identification and authentication of animal cell culture by polymerase chain reaction (PCR) amplification and DNA sequencing. A PCR-based method for rapid identification and authentication of closely related cell lines was described. In this method, two new primers were designed based on high homology in the aldolase gene family. Used together with our previous primers, the combinations of primers were able to differentiate closely related species, including human from monkey and mouse from rat. This PCR assay provides a rapid, simple, sensitive, and cost-effective method for authentication of closely related cell lines. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency.     

Single-molecule polymerase chain reaction reduces bias: application to DNA methylation analysis by bisulfite sequencing

The treatment of DNA with bisulfite, which converts C to U but leaves 5-methyl-C unchanged, forms the basis of many analytical techniques for DNA methylation analysis. Many techniques exist for measuring the methylation state of a single CpG but, for analysis of an entire region, cloning and sequencing remains the gold standard. However, biases in polymerase chain reaction (PCR) amplification and in cloning can skew the results. We hypothesized that single-molecule PCR (smPCR) amplification would eliminate the PCR amplification bias because competition between templates that amplify at different efficiencies no longer exists. The amplified products can be sequenced directly, thus eliminating cloning bias. We demonstrated this accurate and unbiased approach by analyzing a sample that was expected to contain a 50:50 ratio of methylated to unmethylated molecules: a region of the X-linked FMR1 gene from a human female cell line. We compared traditional cloning and sequencing to smPCR and sequencing. Sequencing smPCR products gave an expected methylated to unmethylated ratio of 48:52, whereas conventional cloning and sequencing gave a biased ratio of 72:28. Our results show that smPCR sequencing can eliminate both PCR and cloning bias and represents an attractive approach to bisulfite sequencing.     

Direct sequencing of tobacco chloroplast genome by the polymerase chain reaction

We have developed a polymerase chain reaction (PCR) method for sequencing of tobacco chloroplast genome. In a mixture containing chloroplast DNA, 5-end-labeled oligonucleotide primer, Taq DNA polymerase and reaction buffer, we were able to sequence a segment of chloroplast 16S rRNA gene. The results showed that the 750 bp of DNA sequenced were identical to the sequence reported, indicating that direct sequencing method that we have developed is useful for the sequencing of chloroplast genome. To analyze the chloroplast genome more rapidly in those in vitro grown plantlets, we also developed a simple method which is applicable for the amplifications and sequencing of chloroplast 16S rRNA fragment from either 0.15 g of tobacco leaf or stem tissue. The readable sequences obtained from the presented methods were consistent with the published sequence.     

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.     

Direct sequencing of unpurified PCR-amplified DNA by semi-exponential cycle sequencing (SECS)

A simple technique for direct sequencing of PCR-amplified templates without purification of the PCR reaction product is presented. This method does not require an additional synthesis step after template amplification, and can generate sequence information form as little as 0.1 fmol of unpurified template.     

Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction

Six TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes have been developed for the detection quantitation of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples. Species-specific amplification was achieved by combining only two fluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences, decreasing the cost of the assay significantly. The limits of detection ranged from 0.03 to 0.80 pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1% of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantitation accuracy in samples containing 10-100% of beef or pork DNA was close to 90%. The system is complemented with one additional TaqMan MGB detector based on consensus sequence segments of the nuclear 18S ribosomal RNA gene. A method to evaluate the presence of unknown eukaryotic DNA in a mixture, where data derived from the species-specific detection are compared with the experimental values obtained from the general 18S detector, is presented. This method allows the validation of the quantitative measurements, providing an internal control of the total content of PCR-amplifiable DNA in the sample. The system was tested on DNA mixtures containing different shares of up to four different species and on DNA extracted from processed commercial food samples.     

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 “inhibited” crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.     

Comparison of DNA extraction methods for multiplex polymerase chain reaction

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A_260/280), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2).     

Identification of bacteria in culture negative and polymerase chain reaction (PCR) positive intraocular specimen from patients with infectious endopthalmitis

A novel Denaturing High-Performance Liquid Chromatography (dHPLC)-based technique allows rapid high-resolution analysis of PCR products. We show the application of this PCR/dHPLC approach for direct detection and identification of bacterium from the Eubacterial PCR amplified products of aqueous and vitreous aspirates from patients with endopthalmitis and to differentially identify the culture negative cases and initiate appropriate therapy. The aim of this study is to identify culture negative PCR positive cases by the application of PCR based DNA sequencing. A total of 116 intraocular specimens were subjected for the study. Sixty-nine different bacteria were identified using dHPLC based DNA sequencing of which predominant ones were Gram-positive bacteria and cannot be cultured by conventional methods. Forty eight different bacteria detected in this study is being reported for the first time in infectious endopthalmitis.     

Rapid identification of bacterial isolates from aqueous kava (Piper methysticum) extracts by polymerase chain reaction and DNA sequencing

Aim:  Fresh kava beverages have a limited shelf life under refrigerated conditions. The objective of this study was to isolate and identify bacteria in aqueous extracts of kava rhizome.
Methods and Results:  The internal part of kava rhizome was used to minimize soil contamination. Three kava extracts were prepared, serially diluted and plated on nutrient agar. Isolated colonies were identified by sequencing polymerase chain reaction amplicons targeting the eubacterial 16S rDNA and the tuf gene of Staphylococcus . Seventy-five bacterial isolates belonged to 16 genera. Bacillus , Cellulomonas , Enterococcus , Pectobacterium and Staphylococcus were identified in all kava extracts.
Conclusions:  Kava rhizome contains large amounts of starch and fibre, which justify the presence of polysaccharide-degrading bacteria in the extracts. Bacillus cereus group and Staphylococcus species may produce toxins and cause foodborne illness.
Significance and Impact of the Study:  The results of this study provide fundamental information that may be used to enhance the microbial quality and safety of kava beverages.     

几种耐热DNA聚合酶的比较

耐热DNA聚合酶广泛应用在PCR技术。本文将常用的几种耐热DNA聚合酶分为三大类,对它们的性质、差异、用途做一个简单的介绍。     

Identification of pathogenic yeast species by polymerase chain reaction amplification of the RPS0 gene intron fragment

Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (10⁷–10⁵ cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.     

任意引物PCR及其应用研究进展

任意引物PCR技术又称为随机扩增多态性DNA技术,它是在PCR技术基础上发展起来的一项分子检测技术。它具有简便、快速,一套引物可用于多个物种的分析,不需预知分析对象的核酸序列,可以显示差异表达基因等特点,已广泛应用于病原微生物的分型鉴定、物种亲源关系分析、遗传育种研究和特异表达基因的克隆与鉴定等方面。     

Computing by polymerase chain reaction

A mathematical notation is introduced to represent, at a symbolic level, different mechanisms of DNA recombination, and a 'PCR lemma' is proven by analytically describing the combinatorial properties of the polymerase chain reaction process. This approach led to the discovery of novel techniques, based on a form of PCR which we called cross pairing PCR (briefly XPCR). They were mathematically analyzed and already experimentally proven in different contexts, such as DNA extraction and recombination. Thus, a mathematical analysis of standard methodologies may highlight novel mechanisms of DNA recombination and this can provide new technologies for DNA manipulation.     

Identification and differentiation of human schistosomes by polymerase chain reaction

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.     

Comparison of proteases in DNA extraction via quantitative polymerase chain reaction

We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.     

Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia

DNA coding for the 16S rRNA of an intracellular bacterium was directly amplified from lysed cells of a host amoebae using the polymerase chain reaction and primers specific for eubacteria. The amoebae had been used to recover an uncultured bacterium observed in the sputum of a patient with pneumonia. The amplified DNA was sequenced directly and compared with published 16S rRNA sequences. The analysis revealed that the intracellular bacterium is a member of the genus Legionella and that it is different from species, including L. pneumophila, for which 16S ribosomal RNA sequence data are available.     

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.     

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.     

Parallel DNA amplification by convective polymerase chain reaction with various annealing temperatures on a thermal gradient device

We present a thermal gradient convective polymerase chain reaction (PCR) for parallel DNA amplification with different annealing temperatures. The thermal gradient for microfluidic gradient PCR is produced by an innovative fin design whose formation principle is given. Without the need for a pump, the buoyancy forces continuously circulate reagents in a closed loop through different thermal zones, which brings self-actuated convective-flow PCR. In our prototype, we measured a temperature difference of about 45 °C along the gradient direction on the copper flake (45 × 40 × 4 mm). When the temperature of the hot zone is 90-97 °C and the temperature of the cold zone is 60-70 °C, the convection triggered two-temperature amplification of 112-bp fragment of Escherichia coli DNA. The time for amplification is less than 45 min. Interestingly, parallel DNA amplification with different annealing temperatures ranging from 60 to 70 °C was performed by this method. The PCR thermocycler demonstrated herein can be further scaled down and the loop length can be further reduced, and therefore the PCR times can be further reduced. These devices are suited as a platform for a new generation of low-power, portable DNA analysis systems.