Genomic fingerprints produced by PCR with consensus tRNA gene primers.

The polymerase chain reaction using only a single 'consensus' tRNA gene primer, or a pair of primers facing outward from tRNA genes, amplifies a set of DNA fragments in bacterial, plant and animal genomic DNAs. Presumably, these PCR fingerprints are mainly derived from the regions between closely linked tRNA genes. The pattern of the PCR products is determined by which genomes and which primer(s) are used. Genomic fingerprints are largely conserved within a species and, in bacteria, most products in the fingerprint are conserved between closely related species. Thus, PCR with tRNA gene consensus primers helps to identify species and genera.     

Genomic sequencing by ligation-mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited due to the complexity of the mammalian genome. Ligation-mediated PCR (LMPCR) is a sensitive genomic sequencing procedure that generates high quality, reproducible sequence ladders starting with only 1 μg of uncloned mammalian DNA per reaction. This genomic sequencing procedure can be adapted for various methylation, in vivo footprinting and DNA adduct mapping procedures. We provide a detailed protocol for genomic sequencing by LMPCR and discuss the principles and applications of the method.     

Specific PCR product primer design using memetic algorithm

To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150–300 bps and 500–800 bps, and two different methods of calculating T_m for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near‐optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma‐pd/ . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Rapid identification of Triticeae genotypes from single seeds using the polymerase chain reaction

An easy and quick protocol has been developed for DNA analysis via PCR. Single cereal endosperm or small leaf pieces can be separately processed in several PCR reactions. The resultant PCR patterns are equivalents to those obtained with standard DNA extraction protocols using either specific or random primers. Intra-and inter-specific variability can be detected. This method allows the analysis of a large number of individuals in early stages prior to the plant sowing.     

Detection of the low-germination-rate resting oospores of Pythium myriotylum from soil by PCR

AIMS: To establish a sensitive and specific polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in soils. METHODS AND RESULTS: Oospores of P. myriotylum were separated from large soil particles by flotation in sucrose solution. The thick-walled oospores were disrupted by vortex with sea sand and its DNA was extracted by the Cetyl trimethyl Ammonium Bromide (CTAB) method. The recovered DNA was verified by PCR amplification of a 150-bp target sequence of P. myriotylum. Samples of 10 g of soil were assayed; thus, the detection limit by PCR-based method was 10 oospores per gram soil. The method was successfully applied for the detection of P. myriotylum in soils collected in March, prior to planting of ginger crops. CONCLUSIONS: A PCR-based method for detecting P. myriotylum from soil was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR method has allowed us to monitor the presence of P. myriotylum in soil prior planting season as a way of reducing or eliminating disease.     

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.     

Detection of Sclerotinia sclerotiorum in Planta by a Real-time PCR Assay

Stem rot caused by Sclerotinia sclerotiorum is a very serious disease on oilseed rape worldwide. In this study, a pair of polymerase chain reaction (PCR) primers was designed based on the nucleotide sequence of a DNA region amplified by a microsatellite primer M13. The primer pair amplified a 252-bp fragment from all S. sclerotiorum isolates collected from oilseed rapes at different locations in different years, but not from any other fungus tested. Using this pair of primers, a real-time PCR assay was developed to rapidly detect early infection of S. sclerotiorum on petals of oilseed rape. The real-time PCR assay developed in this study could help growers make a timely decision on fungicide application.     

Stress induces tRNA cleavage by angiogenin in mammalian cells

tRNAs play a central role in protein translation, acting as the carrier of amino acids. By cloning microRNAs, we unexpectedly obtained some tRNA fragments generated by tRNA cleavage in the anticodon loop. These tRNA fragments are present in many cell lines and different mouse tissues. In addition, various stress conditions can induce this tRNA cleavage event in mammalian cells. More importantly, angiogenin (ANG), a member of RNase A superfamily, appears to be the nuclease which cleaves tRNAs into tRNA halves in vitro and in vivo. These results imply that angiogenin plays an important physiological role in cell stress response, except for the known function of inducing angiogenesis.     

Following the course of human leptospirosis: evidence of a critical threshold for the vital prognosis using a quantitative PCR assay

In order to follow the course of acute human leptospirosis, an ELISA microtiter plate hybridization method was developed for the quantitative determination of Leptospira spp. in biological samples after PCR. The biotin-labelled amplified product (331 bp from the rrs gene) was hybridized with a complementary capture probe covalently linked onto aminated polystyrene wells, and detected using a colorimetric reaction. The mean detection limit was 50 copies per 10 microl. In a prospective study of human leptospirosis cases, we obtained evidence that a density of 10(4) leptospires per ml of blood is a critical threshold for the vital prognosis of the patients. The practicability of the method makes it suitable for use in tropical areas for multicentric studies. Such studies could lead to a better knowledge of the natural history of the human disease. The method is also suitable for experimental evaluation of improved antibiotic treatments for leptospirosis.     

Screening of transgenic plants by multiplex PCR

A protocol is described, for the rapid screening of a large number of putative transgenic shoots. Genomic DNA is isolated and screened by PCR. To validate the purity of the DNA, PCR amplification is done with primers homologous to an endogenous gene. Multiplex PCR is used to screen for the transgenic shoots with two sets of primers, one set against the endogenous gene (internal control) and the other set against the gene used in transformation. This protocol has been successfully used on maize, melon, oil-seed rape, pepper, petunia, potato, squash, sugar beet and tobacco.     

DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

Summary Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms. Fourteen human cell lines previously found by other methods to be either related or disparate were subjected to DNA fingerprinting by PCR amplification of selected fragment length polymorphism loci. Cell identification patterns by this method were concordant with those obtained by isoenzyme phenotyping and restriction fragment length polymorphism-DNA fingerprinting, and were reproducible within and between assays on different DNA extracts of the same cell line. High precision was achieved with electrophoretic separation of amplified DNA products on high resolution agarose or polyacrylamide gels, and with fragment length polymorphism (FLP) loci-specific “allelic ladders” to identify individual FLP alleles. Determination of the composite fingerprint of a cell line at six appropriately chosen fragment length polymorphism loci should achieve a minimum discrimination power of 0.999. The ability of PCR-based fragment length polymorphism DNA fingerprinting to precisely and accurately identify the alleles of different human cell lines at multiple polymorphic fragment length polymorphism loci demonstrates the feasibility of developing a cell line DNA fingerprint reference database as a powerful additional tool for future cell line identification and authentication.     

Simultaneous PCR amplification of two barley 5S rDNA sequence types in the same PCR reaction

Many researchers are currently using PCR technology to amplify individual members of multigene families, including 5S rDNA, for sequencing and related purposes. When members of the family differ in length, analyses would be facilitated if different units could be simultaneously and efficiently amplified. In the present paper we describe conditions that can be used to amplify simultaneously both the “long” and “short” 5S rDNA repeats found in barley (Hordeum vulgare L.).     

Detection of mitochondrial DNA deletions by a screening procedure using the Polymerase Chain Reaction

We describe an accurate procedure for a rapid diagnosis of heteroplasmic mtDNA deletions based on the polymerase chain reaction (PCR). For a selective amplification of deleted mtDNA across the breakpoints of the deletion, we used seven combinations of primers surrounding the most common deleted region between the two origins of mtDNA replication. This procedure was performed on muscle biopsies of twenty patients harboring a single mtDNA deletion and one patient with multiple mtDNA deletions. The results were compared with Southern-blotting analysis. We conclude that this PCR procedure is a sensitive and convenient screening method for the detection of mtDNA deletions. (Mol Cell Biochem 174: 221–225, 1997)     

Cloning and partial sequence of transferrin-binding protein 2 of Neisseria meningitidis using a novel method: Twin N-terminal PCR

Abstract The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae .     

A型肉毒神经毒素基因的PCR检测

目的:建立快速筛查A型肉毒毒素的PCR方法。方法:根据GenBank中报道的肉毒毒素基因序列,综合应用多种生物软件分析设计特异的检测引物,从提取的基因组DNA、热裂解产物和菌液等不同形式的模板中扩增大小为457bp的A型肉毒毒素特异基因片段,以肉毒梭菌其他血清型及破伤风梭菌为对照。结果:检测方法无交叉反应,灵敏度可达10pgDNA,3×103个菌。结论:建立的检测方法特异性强、灵敏度高,可以用于A型肉毒毒素基因的快速筛查。     

Analysis of phylogenetic relationships in the Triticeae tribe using RFLPs

The use of restriction fragment length polymorphisms in combination with other approaches is very useful for the reconstruction of evolutionary events revealing phylogenetic relationships. A set of 21 cDNA probes hybridizing to different chromosome arms in hexaploid wheat was used in a series of experiments designed to estimate the phylogenetic relationships among and within 16 species of the Triticeae tribe. A high degree of polymorphism was found both between and within the species examined. The RFLP data were used to generate a cladogram and a phenogram in order to compare the two different methods of constructing phylogenetic trees. The results of both methods were consistent with each other and with the general taxonomic information provided by earlier morphological studies, meiotic pairing analysis, isozyme tests, and sequence alignment in theTer,NOR, and5s DNA loci. In addition, several correlations were found between the geographical origin of accessions from the same species and their phylogenetic relationships as shown by the cladogram and phenogram.This paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or the University of Missouri.This paper is a contribution of the U.S. Department of Agriculture, Agricultural Research Service, and Missouri Agricultural Experimental Station, Journal Series No. 11706.     

PRaTo: a web-tool to select optimal primer pairs for qPCR

An essential pre-requisite to perform sound quantitative real-time polymerase chain reaction (qPCR) assays is to design outstanding primer pairs. This means they must have a good efficiency and be not prone to produce multiple amplicons or primer dimer products. To circumvent these issues, several softwares are available to help primer design. Although satisfactory computer-aided primer design tools are available for standard PCR, less efforts were done to provide specific methods for selection of optimal primer pairs for qPCR. We have developed PRaTo a web-based tool that enables checking and ranking of primers pairs for their attitude to perform optimally and reliably when used in qPCR experiments. PRaTo is available at http://prato.daapv.unipd.it.     

Identification of Vibrio harveyi using PCR amplification of the toxR gene

AIMS: The aim of this study was to develop an effective method for the identification of Vibrio harveyi based on using the toxR gene as a taxonomic marker. METHODS AND RESULTS: Primers for the toxR gene were designed for specificity to V. harveyi, and incorporated in a polymerase chain reaction (PCR). The results of the PCR, which took <5 h from DNA extraction to amplification, revealed positive amplification of the toxR gene fragment in 20 V. harveyi isolates including type strains, whereas DNA from 23 other Vibrionaceae type strains and 13 Vibrio parahaemolyticus strains were negative. The detection limit of the PCR was 4.0 x 10(3) cells ml(-1). In addition, the technique enabled the recognition of V. harveyi from diseased fish. CONCLUSIONS: The PCR was specific and sensitive, enabling the identification of V. harveyi within 5 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR allowed the rapid and sensitive detection of V. harveyi.