一种热激PCR法高效鉴定重组克隆

利用热激PCR法和菌落直接PCR法进行大肠杆菌和农杆菌的鉴定,筛选含有GFP-linker-C-TAPa或wtGPA1-C-TAPa的pCAMBIA2300EC的阳性克隆,比较热激PCR法和菌落直接PCR法的优劣。热激PCR法阳性检出率是100%,菌落直接PCR法阳性检出率是60%左右。热激PCR法是一种快速可靠的鉴定重组阳性克隆的方法。     

A highly specific PCR assay for detecting the fish ectoparasite Amyloodinium ocellatum

Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.     

A highly reliable sex diagnostic PCR assay for mass screening of papaya seedlings

Female plants of several dioecious angiosperms are commercially valued for production of fruits or seeds, viz. papaya, nutmeg, pistachio, kiwi fruit and jojoba. To make the cultivation profitable it is necessary to grow more female than male plants. To discriminate between male and female plants, sex-specific molecular markers have been identified in a few dioecious species such as Silene and pistachio. However, accurate and convenient sex diagnostic methods for early sexing of seedlings are not available to date. For the first time, we report here a PCR-based Seedling Sex Diagnostic Assay (SSDA) specially designed for early sexing of papaya seedlings. We have developed a male-specific SCAR marker in papaya by cloning a male-specific RAPD (831 bp) fragment and designing longer primers. The potential of this SCAR marker is further exploited to develop a simplified and highly accurate sex diagnostic assay by (1) including an internal PCR control, (2) following a single-step DNA extraction procedure and (3) optimising the PCR conditions to simultaneously amplify male-specific and control bands from the crude leaf extract. This diagnostic approach would be of great commercial significance to papaya growers as well as to seed companies and plant nurseries for early identification of female seedlings of dioecious species. In principle, this experimental design could be easily applied to molecular analysis of any agriculturally important trait for which specific DNA probes could be identified and hence opens new avenues of research in the field of genetic diagnostics of plants.     

Design of primers for PCR ampiification of highly variable genomes

A program to aid in the search of primers for specific polymerasechain reaction (PCR) amplification of highly variable genomesis presented. It involves the derivation of variability profilesto identify optimal regions for PCR amplification, taking intoaccount stability of DNA-primer hybrids. An application of theprogram to foot-and-mouth disease virus diagnosis is presented.     

In-vitro selection of highly stabilized protein variants with optimized surface

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.     

A novel Real Time micro PCR based Point-of-Care device for Salmonella detection in human clinical samples

Our POC (Point of Care) device is intended to be a diagnostic tool for routine use in the clinical sector. The validation of the whole procedure, including bacterial genomic DNA isolation and the Real Time detection of Salmonella spp., was conducted on 29 clinical stool samples that had been diagnosed with Salmonella spp. by a routine culture technique. The entire process was achieved in a single microfluidic chip within 35 min. In comparison to the culture reference method that is used in the clinical laboratories, this new device performed well in regards to the analytical parameters of sensitivity, specificity and accuracy. Therefore, the POC device reported in this study proved to be very appropriate for the fully integrated analysis system. To the best of our knowledge, this is the first work to report the sample preparation and followed by Real Time PCR (Polymerase Chain Reaction) on a single 2.5 μl chamber chip for the detection of Salmonella spp. bacteria in stool samples.     

Effect of highly fragmented DNA on PCR.

We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.     

PCR use of highly conserved DNA regions for identification of Sinorhizobium meliloti

A PCR identification method in which four primers that recognize homologous conserved regions in the Sinorhizobium meliloti genome are used was developed and tested. The regions used for identification were the nodbox 4 locus, which is located in one of the symbiotic megaplasmids, and the mucR gene, which is located in the chromosome. The new method was used to establish a collection of S. meliloti strains from polluted soils.     

A highly sensitive quantitative real-time PCR assay for determination of mutant JAK2 exon 12 allele burden

Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.     

A stabilized moment estimator for the beta-binomial distribution

The beta-binomial distribution has been proposed as a model for the incorporation of historical control data in the analysis of rodent carcinogenesis bioassays. Low spontaneous tumor incidences along with the small number and sizes of historical control groups combine to make the moment and maximum likelihood estimates of the beta-binomial parameters deficient. We therefore propose a stabilized moment estimator for one of the parameters. The stabilized moment estimator is similar to the ridge regression estimator and introduces a shrinkage parameter. Computer simulations were run to examine the behavior of the stabilized moment estimator. The effect of the stabilized moment estimator on the score test for dose-related trend is considered both on simulated data and on an example from the literature.     

A new approach to apple proliferation detection: a highly sensitive real-time PCR assay

The present paper describes a new approach for diagnosis of apple proliferation (AP) phytoplasma in plant material using a multiplex real-time PCR assay simultaneously amplifying a fragment of the pathogen 16S rRNA gene and the host, Malus domestica, chloroplast gene coding for tRNA leucine. For the first time, such an approach, with an internal analytical control, is described in a diagnostic procedure for plant pathogenic phytoplasmas enabling distinction between uninfected plant material and false-negative results caused by PCR inhibition. Pathogen detection is based on the highly conserved 16S rRNA gene to ensure amplification of different AP phytoplasma strains. The newly designed primer/probe set allows specific detection of all examined AP strains, without amplifying other fruit tree phytoplasmas or more distantly related phytoplasma strains. Apart from its specificity, real-time PCR with serial dilutions of initial template DNA ranging over almost five orders of magnitude (undiluted to 80,000-fold diluted) demonstrated linear amplification over the whole range, while conventional PCR showed a reliable detection only up to 500-fold or 10,000-fold dilutions, respectively. Compared to existing analytical diagnostic procedures for phytoplasmas, a rapid, highly specific and highly sensitive diagnostic method becomes now available.     

A thermally controlled microinjector.

An economical device for 0.1-2 microliter microinjections in animals is described. The expansions of electrically heated mercury is used to provide precise and repeatable injections. The rate of injection can be controlled either manually or by a motor drive.     

A fast and highly sensitive method for detecting freemartinism in bovine twins using immunomagnetic beads and Y-specific PCR primers

A method for detecting male cells in the blood of the female calf in bovine heterosexual twin pregnancies has been established. Nucleated cells were isolated from full blood by immunomagnetic separation, lysed by boiling and then subjected to polymerase chain reaction (PCR) amplification with Y chromosome specific primers. Diagnosis was achieved within one day. The method was successfully used on blood samples that had been stored at +4°C for more than one month. Dilution of male blood in female blood showed that XY cells were detectable down to a concentration of 0.1%. This method should be amenable to automatization and can be adapted to any PCR-based genetic test.     

A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry

In this work we present a rapid and fully automated method to purify and desalt PCR products prior to analysis by electrospray ionization mass spectrometry. The protocol employs a commercial pipette tip packed with an anion-exchange resin and comprises four primary steps: tip pretreatment, sample loading, rinsing, and sample elution. This tip-based purification/desalting protocol has two distinct advantages over previously published methods. First, the protocol can be performed either manually (1-12 samples at a time), using a standard p10 manual pipette, or in a fully automated microtiter plate format (96 samples at a time) employing standard laboratory robotics. Additionally, the entire protocol from crude PCR product to an "electrosprayable" analyte solution requires only 10 microl of crude product and takes less than 20 min. Using capillary gel electrophoresis, we demonstrate an overall recovery efficiency of approximately 80% and demonstrate the exquisite desalting efficiency with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Using an internal mass standard we demonstrate sub-ppm mass measurement error which provides an unambiguous base composition for a 120-mer PCR product.     

A stabilized molten globule protein

A predominant conformational isomer of non-native alpha-lactalbumin (alpha-LA) has been purified by thermal denaturation of the native alpha-LA using the technique of disulfide scrambling. This unique isomer retains a substantial content of alpha-helical structure. It is stabilized by two native disulfide bonds within the alpha-helical domain and two scrambled non-native disulfide bonds at the beta-sheet domain. This denatured isomer of alpha-LA exhibits structural characteristics that are consistent with the well-documented molten globule state. The ability to prepare a stabilized and structurally defined molten globule provides a useful model for studying the folding and unfolding pathways of proteins.