A computer program for selection of oligonucleotide primers for polymerase chain reactions.

We have designed a computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification by the polymerase chain reaction. This program is based on a set of rules which define in generic terms both the sequence composition of the primers and the amplified region of DNA. These rules (1) enhance primer-to-target sequence hybridization avidity at critical 3'-end extension initiation sites, (2) facilitate attainment of full length extension during the 72 degrees C phase, by minimizing generation of incomplete or nonspecific product and (3) limit primer losses occurring from primer-self or primer-primer homologies. Three examples of primer sets chosen by the program that correctly amplified the target regions starting from RNA are shown. This program should facilitate the rapid selection of effective and specific primers from long gene sequences while providing a flexible choice of various primers to focus study on particular regions of interest.     

Mutation-replication statistics of polymerase chain reactions.

The variability of the products of polymerase chain reactions, due to mutations and to incomplete replications, can have important clinical consequences. Sun (1995) and Weiss and von Haeseler (1995) modeled these errors by a branching process and introduced estimators of the mutation rate and of the efficiency of the reaction based, for example, on the empirical distribution of the mutations of a random sequence. This distribution involves a noncanonical branching Markov chain which, although easy to describe, is not analytically tractable except in the infinite-population limit. These authors for the infinite-target limit, and Wang et al. (2000) for finite targets, solved the infinite-population limit. In this paper, we provide bounds of the difference between the finite-target finite-population case and its finite-target infinite-population approximation. The bounds are explicit functions of the efficiency of the reaction, the mutation rate per site and per cycle, the size of the target, the number of cycles, and the size of the initial population. They concern every moment and, what might be more surprising, the histogram itself of the distributions. The bounds for the moments exhibit a phase transition at the value 1 - 1/N = 3/4 of the mutation rate per site and per cycle, where N = 4 is the number of letters in the encoding alphabet of DNA and RNA. Of course, in biological contexts, the mutation rates are much smaller than 3/4.     

Williopsis saturnus and Williopsis beijerinckii-different taxons from polymerase chain reaction data with nonspecific primers]

Fifteen strains of the yeast Williopsis sensu stricto were analyzed by means of UP-PCR. With the N21 universal primer, this approach showed that the strains could be clearly divided into two groups corresponding to the species W. saturnus (Kl?cker) Zender and W. beijerinckii (van der Walt) Naumov et Vustin. The results obtained are in good agreement with data of genetic and isoenzyme analyses and provide no support for the conspecificity of W. saturnus and W. beijerinckii commonly accepted in modern determination manuals.     

Chemically modified primers for improved multiplex polymerase chain reaction

Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance.     

Differentiation of Trichinella genotypes by polymerase chain reaction using sequence-specific primers.

A method was developed to identify species and genotypes within the genus Trichinella using polymerase chain reaction (PCR) and specific primers. Enzymatic amplification of 2 partially conserved and repetitive genomic DNA sequences that have been shown to be variable in length within the different Trichinella genotypes form the basis of this test. Within these regions of the genome, 4 sets of primers were evaluated from which 2 were chosen for their ability to differentiate among the genotypes under stringent primer annealing conditions while maintaining high yields of amplification product. Differences in the size of PCR products from multiple isolates of each genotype indicate sufficient variation to identify 7 of the 8 parasite groups within this genus. One primer set can differentiate among some genotypes working from a single larva. Identification of Trichinella genotypes will assist in distinguishing between sylvatic and synanthropic life cycles. Such information will be critical in tracing sources of trichinellosis by easily and unambiguously identifying likely host reservoirs and will provide valuable information for instituting methods of control.     

Escherichia coli mutants deficient in exonuclease VII.

Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains.     

Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.

Clinical isolates of the fungal respiratory and systemic pathogen Histoplasma capsulatum have been placed in several different classes by using genomic restriction fragment length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RFLP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA fragments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not distinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsulatum are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.     

Cross-species amplification of glutamine synthetase cDNA by polymerase chain reaction with degenerate primers

We have developed and optimized a consistent polymerase chain reaction (PCR)-based strategy to quickly obtain specific sequence information on novel plant glutamine synthetase (GS, EC 6.3.1.2) cDNAs. Two sets of degenerate primer pairs were designed to discriminate regions conserved in either any kind of GS messenger or exclusively in those for the chloroplastic GS. Novel GS cDNA sequences were successfully amplified from total RNA obtained from 14 different monocotyledonous and dicotyledonous plants. The procedure, coupled with a further restriction analysis, allowed us to uncover the presence of GS cDNA polymorphism, which most likely stems from the different GS gene family members within a single species. Contrary to previously reported strategies in other systems, GS cDNA oligonucleotide primers were designed keeping the degeneracy level to a minimum, together with a high melting temperature. This approach proved to be particularly effective, generating high yields of the expected products without requiring extra nested amplification steps or time-consuming optimization steps for each species GS cDNA amplification. Different clones containing sequence information from either the coding or the 3'-untranslated regions were further sequenced and characterized, confirming the high sequence identity and size uniformity the of GS cDNAs across higher plant species. Therefore, this approach is proposed as a stand-alone procedure to quickly determine the sequence of unknown GS cDNAs, as well as to speed up and complement classical molecular cloning methodologies.     

Monomorphic species specific DNA, detected by polymerase chain reaction with random primers

Intra- and interspecific variability of total DNA isolated from haploid megagametophytes of coniferous species was examined using polymerase chain reaction with random primers. Based on this technique, one can with certainty detect heterozygosity at gene loci carrying null alleles and thus reveal cryptic intraspecific genetic variation. Large population samples were used. Along with random amplified polymorphic DNA, i.e., widely known fragments (amplicons) polymorphic within a species, we found invariant loci lacking individual or geographic variability but differentiating species within genera and other taxa. This DNA was termed RAMD (random amplified monomorphic DNA) to distinguish it from polymorphic DNA. Our findings suggest that genetic monomorphism of species and the dual structure of the eukaryotic genome can be detected at the DNA level as was previously shown for protein gene markers.     

Detection of somatic mosaicism in humans using polymerase chain reaction with random primers

Using the method PCR amplification with random primers, DNA samples from human embryonic organs and tissues were examined. Among 27 oligonucleotide primers tested, 10 primers, producing stable, well-reproducible profiles of amplification products, were chosen for further analysis. With the help of two primers (447 and R45), the differences in RAPD PCR profiles obtained from the tissues of one embryo, were revealed. These differences were associated with the change of mobility, or with the fragment gain/loss in the RAPD profile, and could be caused either by genomic rearrangements, or mutations involving the regions of the DNA-primer pairing. Different epigenetic factors, like methylation, can also play the role in this process.     

Improved single-strand conformation polymorphism analysis by asymmetric polymerase chain reaction with end-labeled primers

An improved form of single-strand conformation polymorphism (SSCP) assay has been developed for the analysis of bovine major histocompatibility complex class II alleles. The method uses asymmetric polymerase chain reaction (PCR) amplification from each of two end-labeled primers to generate individual single-stranded products that are analyzed by electrophoresis in nondenaturing polyacrylamide gels. This technique gives good resolution of labeled single strands derived from 392-bp bovine DRB exon-2 PCR products, without interference from double-stranded products, and enables assignment of SSCP bands to the individual strands of the template DNA. The allelic groupings defined by this method in a panel of test animals were confirmed by independent typing by restriction fragment-length polymorphism.     

Development of microsatellite primers and two multiplex polymerase chain reactions for the common elder (Sambucus nigra)

Primer pairs were developed for eight microsatellite loci isolated from a genomic DNA library of Sambucus nigra‘Black Beauty’. In a survey of 21 accessions of S. nigra, all revealed polymorphism and the number of alleles per locus ranged from three to 18. Two multiplex polymerase chain reactions (PCRs) were optimized and these discriminated all the accessions tested providing a cost‐effective genotyping solution for the species. Six primer pairs (EMSn002, 003, 010, 016, 017, and 023) were informative in two Sambucus canadensis accessions.     

Detection of somatic mosaicism in humans using polymerase chain reaction with random primers

Using the method PCR amplification with random primers, DNA samples from human embryonic organs and tissues were examined. Among 27 oligonucleotide primers tested, 10 primers, producing stable, well-reproducible profiles of amplification products, were chosen for further analysis. With the help of two primers (447 and R45), the differences in RAPD PCR profiles obtained from the tissues of one embryo, were revealed. These differences were associated with the change of mobility, or with the fragment gain/loss in the RAPD profile, and could be caused either by genomic rearrangements, or mutations involving the regions of the DNA-primer pairing. Different epigenetic factors, like methylation, can also play the role in this process.     

Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.     

Flow-through polymerase chain reactions in chip thermocyclers

The miniaturization of analytical devices by micromachining technology is destined to have a major impact on medical and bioanalytical fields. To meet the current demands for rapid DNA amplification, various instruments and innovative technologies have been introduced by several groups in recent years. The development of the devices was extended in different directions and adapted to corresponding applications. In this review the development of a variety of devices and components for performing DNA amplification as well as the comparison of batch-process thermocyclers with reaction chambers and flow-through devices for different purposes are discussed. The main attention is turned to a flow device concept for thermocycling using microfabricated elements for local heat flow management, for which simulations and considerations for further improvement regarding design, material choice and applied technology were performed. The present review article mainly discusses and compares thermocycling devices for rapid thermocycling made of silicon or of silicon and glass with a short excursion to the possibility of plastic chip devices. In order to perform polymerase chain reactions (PCRs) in the microreactors, special attention must be paid to the conditions of the internal surfaces. For microchips, surface effects are generally pronounced because the surface to volume ratio increases upon miniaturization. Solutions for solving this problem are presented. We propose an overview of layouts for batch-process thermocyclers with different parallelization of reaction chambers and also of different designs of continuous flow thermocycling chips, paying particular attention to the parameters which influence the efficiency of such chip devices. Finally we point out some recent issues for applications in the field of clinical diagnostics.     

Alumorphs--human DNA polymorphisms detected by polymerase chain reaction using Alu-specific primers

The simultaneous analysis of multiple loci could substantially increase the efficiency of mapping studies. Toward this goal, we used the polymerase chain reaction to amplify multiple DNA fragments originating from dispersed genomic segments that are flanked by Alu repeats. Analysis of different human DNA samples revealed numerous amplification products distinguishable by size, some of which vary between individuals. A family study demonstrated that these polymorphic fragments are inherited in a Mendelian fashion. Because of the ubiquitous distribution of Alu repeats, these markers, called "alumorphs," could be useful for linkage mapping of the human genome. A major advantage of alumorphs is that no prior knowledge of DNA sequence of marker loci is required. This approach may find general application for any genome where interspersed repetitive sequences are found.