Polymerase Chain Reaction Reaction Research Articles

Comparison of molecular tests for the diagnosis of malaria in Honduras

BackgroundHonduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras.MethodsA total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrolment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species.ResultsOf the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite.ConclusionsMolecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions.

Abstract 4153: Usefulness of sensitive digital PCR assay to quantify microRNA-34b/c methylation in the circulating serum DNA of malignant mesothelioma patients

Abstract . Background: Malignant mesothelioma (MM) is an aggressive tumor but it can sometimes be cured if its clinical stage is not advanced. It is very expected to establish a useful screening test for early detection of MM. Recently we have found that DNA methylation of microRNA-34bc (miR34b/c) plays a dismal role on pathogenesis of MM and is frequently observed in MM. Circulating DNA from malignant tumors is known to be found in the serum of the patient. Digital polymerase chain reaction (PCR) is a high sensitive quantitative assay using real time PCR assay. In order to establish a new early detection system for MM, we quantify the extent of DNA methylation of miR-34b/c using a digital PCR assay in the circulating serum DNA from MM. Material and methods: We collected serum samples from 30 MM patients, 22 patients with benign asbestos pleurisy (BAP), and 10 healthy volunteers (HV) at Okayama Rosai Hospital, NHO Yamaguchi Ube Medical Center and our institute during January 2006 to August 2009. The circulating serum DNA was extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen) followed by bisulfite conversion using EpiTect Bisulfite Kits (Qiagen) according to manufactural protocols. The real time PCR assay of methylation-specific PCR (MSP) for miR-34b/c was performed using SYBR Green method. In addition, the digital PCR of real time MSP assay was explored for 40 wells per each sample with a suitable dilution to aim the increase of sensitivity of PCR reaction, and we quantified the extent of methylation in each sample by counting the number of positive well of PCR reaction per sample. As positive and negative controls, the supernatant cultured medium of NCI-H290 harboring heavy methylation of miR-34b/c and water blank was used, respectively. Results: By melting curve analysis, we distinguished the miR-34b/c methylated wells from unmethylated wells based on the temperatures of PCR products of them (78.2 degree on methylated samples vs. 76.2 degree on un-methylated samples). Using the criteria above, each 40 wells per sample was defined as positive or negative for miR-34b/c methylation. The miR-34b/c methylated samples were defined as having more than three positive wells by ROC curve analyses. Finally, we found that the number of positive wells was significantly higher in MMs than that in other groups, and the miR-34b/c methylated samples was observed in 87% of MM patients, 45% of the patients with BAP, and 10% of HV, respectively, indicating that the miR-34b/c methylation in MM patients were significantly frequent than that in others (P <0.0001). Conclusion: The digital PCR assay can detect miR-34b/c methylation in the circulation serum DNA of MM patients to find that miR-34b/c methylation is more frequent in MM than in BAP, suggesting that it may become a useful high-sensitive screening test to distinguish MM patients from the patients with BAP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4153. doi:1538-7445.AM2012-4153

Raman Spectroscopic Detection of Anthrax Endospores in Powder Samples

Polymerase chain reaction (PCR) is becoming the method of choice for detecting microorganisms concealed in complex matrices or “hoax materials” like household dry powders, food, and soil.[1] However, adding samples directly to a PCR reaction is in most cases not possible because of the presence of PCR inhibitors in the sample.[2] Thus, in order to reliably use PCR, one must either enrich the culture prior to the analysis, which is time-consuming for fastidious organisms, or extract the total DNA directly from the sample, which requires an extraction technique capable of processing different sample types. However, since most DNA extraction methods are often not generic in that sense, they lead to unreliable DNA recovery yields.[2, 3]

Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids

QuickChange mutagenesis is the method of choice for site-directed mutagenesis (SDM) of target sequences in a plasmid. It can be applied successfully to small plasmids (up to 10 kb). However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography (HPLC) purified primers, we were able to achieve SDM in big plasmids (up to 16 kb) involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction (PCR) cycles and 0.5 µl of polymerase (instead of 18 PCR cycles and 1.0 µl of enzyme in the standard protocol) were sufficient for the reaction.

Use of the polymerase chain reaction for detection of Fusarium graminearum in bulgur wheat

The detection of mycotoxigenic fungi in foodstuff is important because their presence may indicate the possible associated mycotoxin contamination. Fusarium graminearum is a wheat pathogen and a producer of micotoxins. The polymerase chain reaction (PCR) has been employed for the specific identification of F. graminearum. However, this methodology has not been commonly used for detection of F. graminearum in food. Thus, the objective of the present study was to develop a molecular methodology to detect F. graminearum in commercial samples of bulgur wheat. Two methods were tested. In the first method, a sample of this cereal was contaminated with F. graminearum mycelia. The genomic DNA was extracted from this mixture and used in a F. graminearum specific PCR reaction. The F. graminearum species was detected only in samples that were heavily contaminated. In the second method, samples of bulgur wheat were inoculated on a solid medium, and isolates having F. graminearum culture characteristics were obtained. The DNA extracted from these isolates was tested in F. graminearum specific PCR reactions. An isolate obtained had its trichothecene genotype identified by PCR. The established methodology could be used in surveys of food contamination with F. graminearum.

New Molecular Bridge between RelA/p65 and NF-κB Target Genes via Histone Acetyltransferase TIP60 Cofactor

The nuclear factor-κB (NF-κB) family is involved in the expressions of numerous genes, in development, apoptosis, inflammatory responses, and oncogenesis. In this study we identified four NF-κB target genes that are modulated by TIP60. We also found that TIP60 interacts with the NF-κB RelA/p65 subunit and increases its transcriptional activity through protein-protein interaction. Although TIP60 binds with RelA/p65 using its histone acetyltransferase domain, TIP60 does not directly acetylate RelA/p65. However, TIP60 maintained acetylated Lys-310 RelA/p65 levels in the TNF-α-dependent NF-κB signaling pathway. In chromatin immunoprecipitation assay, TIP60 was primarily recruited to the IL-6, IL-8, C-IAP1, and XIAP promoters in TNF-α stimulation followed by acetylation of histones H3 and H4. Chromatin remodeling by TIP60 involved the sequential recruitment of acetyl-Lys-310 RelA/p65 to its target gene promoters. Furthermore, we showed that up-regulated TIP60 expression was correlated with acetyl-Lys-310 RelA/p65 expressions in hepatocarcinoma tissues. Taken together these results suggest that TIP60 is involved in the NF-κB pathway through protein interaction with RelA/p65 and that it modulates the transcriptional activity of RelA/p65 in NF-κB-dependent gene expression.

Real-Time Droplet DNA Amplification with a New Tablet Platform

We present a novel droplet-based tablet platform for temporal polymerase chain reaction (PCR) in microliter droplets. The simple design of the device does not require extensive processing or external equipment, which allows for greater ease of use and integration as a point-of-care diagnostic. We demonstrate its functionality to perform both PCR and reverse-transcription PCR for λ phage DNA and H3 influenza RNA with ramp rates and cycle times consistent with traditional PCR thermal cyclers. We additionally investigate the effect of performing PCR in small volumes on the reaction performance by specifically examining adsorption of reagents at the oil/water interface. We determined that adsorption of Taq polymerase at the biphasic interface reduces yield and impairs reaction performance at standard concentrations. Thus, microdroplet PCR reactions require additional polymerase to achieve sufficient amplification and we project for applications utilizing nanodroplets or picodroplets like digital applications, even greater concentrations of polymerase are required to achieve desired results. Following the adsorption investigation, we evaluated the sensitivity of λ phage PCR on our platform to be less than 2.0 copies/μL with an efficiency of 104.4% and similar sensitivity for reverse-transcription PCR for influenza H3 RNA.

Data Clustering for the DNA Computing Readout Method Implemented on LightCycler and Based on Particle Swarm Optimization

In this work, particle swarm optimization (PSO) is applied to automate the DNA computing readout method based on a real-time polymerase chain reaction (PCR). Moreover, real-time amplification was performed and the TaqMan detection approach was used for the plan and the readout approach development. The most important part of the readout method is identifying two different reactions in the real-time PCR, which involve in vitro and in silico processes in order to inspect the placement of pairs of nodes in the Hamiltonian path problem. In addition, the real-time PCR experiment is implemented on the LightCycler System. Previously, manual method was exploited to classify two different output reactions of real-time PCR that was a time consuming process. In this study, by exploiting MATLAB the PSO has been implemented for clustering output reactions of real-time PCR and experimental results depict that the amplification response for “YES” and “NO” reactions can be clustered correctly.

No evidence of XMRV infection in immunocompromised patients and HIV-positive individuals from Germany

Xenotropic murine leukaemia virus-related virus (XMRV) has been detected in patients with prostate cancer and chronic fatigue syndrome (CFS). The detection of XMRV in healthy individuals has raised concern about a possible virus transmission by blood products. However, recent studies challenge the association between XMRV and human disease. This study investigated whether or not XMRV is present in patients with altered immune function and individuals at increased risk of blood-borne viral infections in Germany. We investigated 503 peripheral blood mononuclear cell (PBMC) samples from 240 patients with iatrogenic immune suppression (71 haematopoietic stem cell recipients, 132 solid organ transplant recipients, 37 others) and 311 PBMC samples from 302 patients with HIV-1 infection for the presence of proviral XMRV by real-time polymerase chain reaction (PCR). All 814 PBMC samples from 542 patients tested negative for XMRV DNA and positive for an internal herpesvirus saimiri (HVS) control. Human genomic DNA was detected in all samples, and 90% of the samples contained >10,000 cell equivalents per XMRV PCR reaction. Our failure to detect proviral XMRV provides evidence against the presence of XMRV in patients at increased risk of viral infections in Germany.

Evaluation of nested PCR method for diagnosis of meningitis due to Neisseria meningitidis and Haemophilus influenzae

Meningitis due to Neisseria meningitidis and Haemophilus influenzae is a common infectious disease among infants and children that can be fatal in the absence of rapid and proper diagnosis and treatment. Therefore, a rapid and sensitive method for diagnosis is a high priority. The aim of this study was to compare the performance of culture, conventional polymerase chain reaction (PCR), and nested PCR methods for diagnosis of meningitis caused by N. meningitidis and H. influenzae. A total of 106 cerebrospinal fluid samples were collected from infants and children suspected of having meningitis. A portion of these samples were used for culture and DNA extraction. The extracted DNA was placed in a PCR reaction that contained specific primers for N. meningitidis and H. influenzae. PCR products from negative samples were reamplified with internal primers by nested PCR reaction. The results indicated that among the 106 samples evaluated for N. meningitides, 1 (0.9%) sample was positive by culture, whereas 3 (2.8%) samples were positive by PCR and 7 (6.6%) were positive by nested PCR. For H. influenzae, 2 (1.9%) samples were positive by culture, whereas 3 (2.8%) were positive by PCR and 9 (8.5%) were positive by nested PCR methods. In conclusion, among the culture, conventional PCR, and nested PCR methods evaluated, nested PCR showed higher sensitivity than culture or PCR methods.

The analysis of the rDNA sequence in populations of western flower thrips (Frankliniella occidentalis Pergande) from WielkopolskaAnaliza sekwencji rDNA wielkopolskich populacji wciornastka zachodniego (Frankliniella occidentalis Pergande)

Summary Western flower thrips (Frankliniella occidentalis Pergande) is an economically important pest in greenhouse crops. Successful control of this pest is very difficult. Larvae of this pest are vectors of Tomato spotted wilt virus (TSWV) and Tobacco streak virus (TSV). The aim of this work was to analyze ribosomal DNA sequences of western flower thrips populations originating from different regions of the Wielkopolska province and to make phylogenetic studies based on those sequences. In this work new Polymerase Chain Reaction (PCR) primers were used which enable rDNA fragment amplification. PCR reaction was carried out and its products were sequenced, followed by bioinformatic analysis. The identity level of analyzed populations from Wielkopolska was 98.2–100% and identity between analyzed populations and reference population from the GeneBank database was about 98.7–99.7%. Phylogenetic studies were also made. The results of this work will be useful to develop new diagnostic methods to distinguish western flower thrips and other thrips species presenting a threat in Poland.

An integrated PCR microfluidic chip incorporating aseptic electrochemical cell lysis and capillary electrophoresis amperometric DNA detection for rapid and quantitative genetic analysis

A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 μl min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.

Quadruplex-Based Technology for Nucleic Acid Amplification and Detection

Polymerase chain reaction (PCR) has impacted nearly every field in molecular biology, genetics and forensic science. Numerous applications of PCR have been described for basic scientific purposes, as well as for diagnosis of hereditary and infectious diseases. This is particularly true for real-time PCR (RT-PCR), which detects and quantifies product molecules within PCR reaction vessels. However, PCR is still considered a daunting task due to many variables in the reaction, temperature cycling and complicated quantification methods. Current RT-PCR specific probes (Molecular Beacons, TaqMan, Scorpions) require costly synthesis and considerable effort to achieve optimal sensitivity. Typically, a fluorophore-quencher pair is attached to the ends of a probe, which doesn’t fluoresce when free in solution. Upon probe hybridization to an amplicon, the fluorophore is separated from the quencher and a signal is released.Recently we have discovered that the free energy of DNA quadruplexes can be used to drive unfavorable (endergonic) reactions of nucleic acids (e.g., isothermal PCR). The key point of quadruplex-driven reactions is that some G-rich sequences are capable of forming quadruplexes with significantly more favorable thermodynamics than the corresponding DNA duplexes. The energy of quadruplex formation can be used to drive PCR at constant temperature or DNA signal amplification. In addition, fluorescent nucleotide analogs incorporated and fully quenched within the primers regain maximum emission upon quadruplex formation, allowing very simple and accurate detection of product DNA. Thermodynamic and fluorescent bases of quadruplex priming amplification will be discussed.Supported by Bill & Melinda Gates Foundation.

One-stop polymerase chain reaction (PCR): An improved PCR method with speedy operation and comparable efficiency

The widely used polymerase chain reaction (PCR) protocol requires several post-cycling steps to visualize amplicons, decelerating PCR sample processing and result calling. “One-stop PCR” was developed by including both the loading buffer and nontoxic staining dye within a single PCR tube, allowing direct loading and simultaneous staining of amplicons for visualization. The efficiency and sensitivity of one-stop PCR were comparable to commercially available PCR kits and it is compatible with downstream uses and is not mutagenic. One-stop PCR robustly minimizes the time and labor required for PCR reaction setup and product visualization. Key words : Polymerase chain reaction (PCR), post-cycling, compatible dye, Gelred.

Effects of Recent Leptospira Vaccination on Whole Blood Real‐Time PCR Testing in Healthy Client‐Owned Dogs

Bacterin-based canine Leptospira vaccines could present a challenge for the use of whole blood real-time polymerase chain reaction (PCR) as a diagnostic tool. Recent vaccination could induce positive results if the targeted DNA fragment is present within the vaccine and in the blood of the recently vaccinated dog. The objective of this study was to assess whether 2 available 4-serovar vaccines induce a positive real-time PCR reaction in the blood of healthy recently vaccinated dogs. Twenty healthy dogs. This was a prospective study. Dogs were assigned to 1 of 2 vaccine groups. Both vaccines were culture-based and include Leptospira interrogans serovars Pomona, Canicola, and Icterohaemorrhagiae and Leptospira kirschneri serovar Grippotyphosa. Whole blood for real-time PCR and serum for the microscopic agglutination test (MAT) were collected prior to and 3 and 7 days after vaccination and weekly thereafter for 8 weeks. Two real-time PCR tests targeting 2 different genes were performed independently in a blinded fashion. Both Leptospira vaccines produced positive real-time PCR reactions when assayed undiluted or diluted 1 : 100 in canine blood. However, blood samples drawn from all dogs at all time points after vaccination were negative on PCR. All dogs developed MAT titers. Recent vaccination with 2 commercially available vaccines does not interfere with the use of real-time PCR for the identification of acute Leptospira infection in dogs.

Analytical performance of a PCR assay for the detection of KRAS mutations (codons 12/13 and 61) in formalin-fixed paraffin-embedded tissue samples of colorectal carcinoma

KRAS mutation testing is mandatory before prescribing anti-epidermal growth factor monoclonal antibodies in the treatment of advanced colorectal cancer. We describe the performance of a TaqMelt polymerase chain reaction (PCR) assay—the cobas® KRAS Mutation Test—designed to detect 19 mutations in codons 12, 13, and 61. The limit of detection was determined using DNA blends from cell lines, plasmids, and formalin-fixed paraffin-embedded tissue specimens. Assay performance was compared to Sanger sequencing using a panel of 188 specimens. Discordant specimens were subjected to next generation pyrosequencing (454). Assay repeatability was assessed using a panel of six specimens. A >95% correct mutation call rate was obtained in all specimen types with ~5% mutant alleles at DNA inputs of 0.8–6.3 ng per PCR reaction; 100% detection rate was observed at the recommended DNA input of 50 ng. The positive percent agreement with Sanger was 97.5% (79/81) for codons 12/13 and 85.7% (6/7) for codon 61. Negative percent agreement was 94.4% (101/107) for codon 12/13 and 99.4% (180/181) for codon 61. Nine of 10 discordant specimens yielded 454 results consistent with the cobas® results. With repeated testing, the assay showed a correct call rate of 100% (192/192) for all operators, instruments, reagent lots, and days tested. The cobas® test detects KRAS mutations in codons 12, 13, and 61 at a limit of detection of <5%. The PCR assay was more sensitive and specific than Sanger sequencing, and performance was highly reproducible. Test performance was not influenced by various endogenous interfering substances or common gut microbes.

An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in Eucalyptus

Abstract Integration of fluorescent-dUTP in polymerase chain reaction (PCR) appears to be a sound method for fluorescence labelling of amplicons in genotyping with simple sequence repeats (SSRs) using an automated sequence analyser. However, the method has not been explored in terms of performance optimisation and cost control. In this paper, we optimised the protocol for fluorescent-dUTP based SSR genotyping in a case study with Eucalyptus. A combination of low dNTP concentration (25 μM each) in PCR reaction and a touchdown PCR programme contributed to increase dramatically the fluorescent intensity of SSR amplicons, thereby facilitating accurate and multiplexed scoring of SSR alleles. The usefulness of the optimised protocol was demonstrated in its application to genetic mapping of SSR loci onto E. urophylla and E. tereticornis linkage maps constructed previously. The protocol optimised here would provide a reliable and economical assay for sequencer-based SSR genotyping in a wide range of biological applications.

Screening of banana bunchy top virus through multiplex PCR approach

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. An attempt was made to develop a sensitive polymerase chain reaction (PCR) and multiplex PCR-based method for detection of BBTV in hill banana. DNA was isolated from the experimental plants at third and sixth months after planting. Multiplex PCR was done with Coat Protein (CP) and Replicase (Rep) gene-specific primer, and banana ethylene insensitive like protein (EISL) primer as internal control to identify failure in PCR reaction. This study revealed that multiplex PCR is effective for BBTV screening in hill banana with the advantage of overcoming the false positive in PCR amplification.

Two Isoforms of Flk-1 Transcripts in Early Diabetic Rat Retinas

Background: We studied the expression levels of genes encoding the two isoforms of fetal liver kinase-1 (Flk-1) and the effect of intravitreal injection of triamcinolone acetonide (IVTA) in diabetic rat retinas on the isoforms.Methods: The right eyes of both streptozotocin-induced diabetic and non-diabetic rats received triamcinolone treatment, while the left eyes were sham-treated, thereby providing four treatment groups. Three pairs of polymerase chain reaction (PCR) primers were designed to specifically amplify the total, long and truncated isoforms of rat Flk-1 mRNA. Gene transcriptional levels of the two isoforms were evaluated using quantitative PCR reaction (real time RT-PCR). To detect the gene activities, standard efficiency standard curves were set up for each of the candidate isoforms.Results: The transcripts level of the long form Flk-1 is about 4.3 times higher than the level of the short form in the sham-treated normal rat retinas. The expression of the total, long and short form of Flk-1 was up-regulated by 1.5, 1.8 and 0.7 fold, respectively in sham-treated diabetic retinas compared with the sham-treated non-diabetic retinas. IVTA inhibited the expression of the total, long and short forms of Flk-1 by 1.2, 2.0 and 0.3 fold, respectively in the IVTA-treated diabetic compared with sham-treated retinas. There was no statistically significant difference in the expression of the total and short form of Flk-1 in IVTA-treated diabetic/non-diabetic retinas compared with the sham-treated diabetic/non diabetic retinas.Conclusion: The long form of Flk-1 is the predominant mediator of VEGF-A in the pathogenesis of diabetic retinopathy (DR) and can be significantly inhibited by the IVTA treatment. The short form, which cannot be phosphorylated, does not appear to contribute to the pathogenesis of DR. Further research is warranted to establish whether the truncated form of Flk-1 can be used clinically as a dominant negative inhibitor of the effects of Vascular Endothelial Growth Factor (VEGF).

Discrimination of almond self compatible genotypes by different methods in a breeding program in Iran

Self-incompatibility is one of the most important difficulties in almond production, which reduces fruit set dramatically and makes orchard management difficult. Therefore, producing breeding self-compatible almond genotypes is of great importance. In this research work, the growth of pollen tube was studied in 38 genotypes obtained after crossing “Touno” (male parent) with “Shahrood 12” (female parent). To study fruit set during years 2009 and 2010, four branches of each genotype were bagged in stage D. Results showed that some genotypes were self- compatible. On the other hand, cross-pollination increased the fruit set of self-compatible genotypes. For microscopic study, flowers were harvested at 24, 72 and 120 h after hand pollination. Results of two years (2009 and 2010) study indicated that 24 and 72 h were not enough for the pollen tube to reach the base of style, whereas 120 h were an appropriate time. To double check the status of self-compatible those genotypes and to determinate the situation of genotypes recognized as doubtful by the use of fluorescent microscopy, PCR reaction was carried out with specific primers of SfF and SfR. PCR results confirmed the situation of self-compatible genotypes. In addition, it indicated that five doubtful genotypes were self-compatible. However, another four genotypes were recognized as self-incompatible. In general, 50% of genotypes were realized as self-compatible and another 50% were self-incompatible. Key words: Self compatibility, self-incompatibility, cross compatibility, self pollination, fruit set, fluorescent microscopy, polymerase chain reaction (PCR).