Combinations Of Specific Primers Research Articles

MethyMer: Design of combinations of specific primers for bisulfite sequencing of complete CpG islands.

We present MethyMer, a Python-based tool aimed at selecting primers for amplification of complete CpG islands. These regions are difficult in terms of selecting appropriate primers because of their low-complexity, high GC content. Moreover, bisulfite treatment, in fact, leads to the reduction of the 4-letter alphabet (ATGC) to 3-letter one (ATG, except for methylated cytosines), and this also reduces region complexity and increases mispriming potential. MethyMer has a flexible scoring system, which optimizes the balance between various characteristics such as nucleotide composition, thermodynamic features (melting temperature, dimers [Formula: see text]G, etc.), the presence of CpG sites and polyN tracts, and primer specificity, which is assessed with aligning primers to the bisulfite-treated genome using bowtie (up to three mismatches are allowed). Users are able to customize desired or limit ranges of various parameters as well as penalties for non-desired values. Moreover, MethyMer allows picking up the optimal combination of PCR primer pairs to perform the amplification of a large genomic locus, e.g. CpG island or other hard-to-study region, with minimal overlap of the individual amplicons. MethyMer incorporates ENCODE genome annotation records (promoter/enhancer/insulator), The Cancer Genome Atlas (TCGA) CpG methylation data derived with Illumina Infinium 450K microarrays, and records on correlations between TCGA RNA-Seq and CpG methylation data for 20 cancer types. These databases are included in the MethyMer release. Our tool is available at https://sourceforge.net/projects/methymer/ .

Molecular genetical characterization of vernalization genesVrn-A1, Vrn-B1andVrn-D1in spring wheat germplasm from Russia and adjacent regions

We characterized a representative set of 42 spring wheat cultivars from Russia and adjacent regions for 3 Vrn loci. The 42 genotypes were screened, along with 3 genotypes of known Vrn genes, using previously published genome-specific polymerase chain reaction (PCR) primers designed for detecting the presence or absence of dominant or recessive alleles of the major Vrn loci: Vrn-A1, Vrn-B1 and Vrn-D1. The dominant promoter duplication allele Vrn-A1a was present in 28 of 42 cultivars, whereas the promoter deletion allele Vrn-A1b was present in only 1 of the Russian cultivars (Triticum aestivum L. ‘Pyrothrix 28’). The intron deletion allele Vrn-A1c was not present in any tested cultivar. The dominant Vrn-D1 allele was found in 1 of the cultivars. Thirteen of the spring wheat cultivars tested here carry the recessive vrn-A1 allele. However, for 6 cultivars, there were inconsistencies between PCR data and genetic segregation analysis, showing the presence of the dominant Vrn-A1 gene. No inconsistencies were found in the case of Vrn-B1 locus. A new combination of specific primers allowed amplification of the common Vrn-B1a allele along with the novel Vrn-B1c allele, which was present in 17 of the studied cultivars (40%). Twenty-five cultivars (59%) had dominant alleles of Vrn-A1a and Vrn-B1 in combination. We showed the predominance of the Vrn-B1c allele among cultivars with monogenic control of vernalization in West Siberia and Kazakhstan. In the absence of epistatic effects of Vrn-A1, this allele causes an earlier heading time compared to Vrn-B1a, thereby avoiding early fall frosts. Suggestions are made concerning the origin and distribution of the Vrn-B1c allele among Russian spring wheats.

Studies on Paeonia cultivars and hybrids identification based on SRAP analysis

Plants of Paeonia are valuable for their ornamental and medicinal values. Genetic relations and hybrids identification among different sections of Paeonia were studied using sequence related amplified polymorphism (SRAP) markers. A total of 29 cultivars including 2 intersectional hybrids, 13 sect. Moutan and 14 from sect. Paeonia were used. A total of 197 bands were produced using 24 primer combinations, among which 187 bands showed polymorphism. From the bands amplified, we can identify the peony cultivars using unique SRAP markers and specific primer combinations. Fourteen peony cultivars were distinguished among each other by using totally 35 SRAP markers, which were generated by 16 primer pairs. Two specific primer pairs of Me8/Em8 and Me8/Em1 can be used to identify cultivars from different sections. The mean genetic similarity coefficient (GS), the gene diversity (GD), and the Shannon's information index of peony cultivars were 0.45, 0.19 and 0.32, respectively. Both UPGMA (unweighted pair-group method of arithmetic average) dendrogram and PCA (principle component analysis) analysis showed clear genetic relationships among the 29 peony cultivars, and within section and its intersectional hybrids. The above results are valuable for estimating and analyzing genetic background of Paeonia, parent selection in crossing breeding programs, molecular marker assisted selection (MAS) breeding for further germplasm innovation programs.

Detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer with nested RT-PCR

Objectives: The development and validation of a nested RT-PCR methodology for the detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer and evaluation of its prognostic significance. Design and methods: Different combinations of specific primers were in silico designed and selected, so that false positive results due to genomic DNA contamination were avoided. The specificity of the primers used was evaluated in 30 healthy individuals, 20 patients with colorectal cancer and 20 patients with non-small cell lung cancer. The method was applied in 101 patients with operable breast cancer before the administration of adjuvant chemotherapy and 39 patients with metastatic breast cancer. Results: Mammaglobin A-mRNA-positive cells were detected in 14/101 (13.9%) of early breast cancer patients but not in the control population studied (0%); 9 of them (64.3%) relapsed during the follow-up period. Mammaglobin A was detected in 7/39 (17.9%) of patients with verified metastasis. Multivariate analysis revealed the detection of Mammaglobin A-mRNA-positive cells, as an independent risk factor for reduced DFI. Conclusions: Mammaglobin A is a highly specific molecular marker for the detection of circulating tumor cells in operable breast cancer, with important prognostic applications.

Abschätzung der Eignung einer molekularbiologischen Methode zur Charakterisierung der Grundwasserbiozönose

A molecularbiological technique was used to characterise the bacterial community structure of groundwater habitats. This method consists of the isolation of bacterial DNA from the samples, amplification of 16S rDNA by PCR (polymerase chain reaction), and separation of the amplified DNA by DGGE (denaturing gradient gel electrophoresis). By using more specific primer combinations in the PCR instead of universal eubacterial primers, also groups of microorganisms (Proteobacteria, sulfate reducer, Archaea) were determined. The resulting DGGE patterns that reflect the microbial diversity are compared and differences or similarities evaluated. In the present studies, groundwater from different sites (bank filtrate, artificially recharged groundwater, and natural groundwater) and with changing redox milieus (aerobic, anaerobic) were investigated as well as the solid aquifer material. Besides, samples were taken from the different stages of artificial groundwater recharge, i.e., from surface water to the drain tile. Samples from groundwater derived from sites with different hydrogeochemical or hydrological conditions like bank filtrate and recharged groundwater revealed great differences in DGGE patterns indicating a characteristic species composition in these habitats, while samples taken at different times from the same groundwater showed only small seasonal variations. Clearly different patterns were also found for groundwater and the adjacent solid material as well as for anaerobic and aerobic groundwaters. Looking at artificial groundwater recharge, almost identical patterns were found in raw water and samples from gravel and sand filtration. DGGE patterns from the resulting groundwater indicated a total change in community structure during underground passage. By using group specific primers, Desulfovibrionaceae, Desulfobacteriaceae, and Archaea could be detected in anaerobic groundwaters. The molecularbiological approach described here gives an increasingly comprehensive and more precise picture of the microbial population of different environments. It is especially suitable to compare the community structure from different habitats or to analyse changes for example due to environmental stress at the same site.

Specific primers for the detection of vip3A insecticidal gene within aBacillus thuringiensiscollection

A PCR-based method was developed for the rapid detection of vip3A gene encoding a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Specific primer combinations (three primers for the normal strand and two primers for the complementary strand) were capable of generating diagnostic fragments that successfully predicted the presence of the gene encoding the Vip3A insecticidal toxin in various B. thuringiensis strains. Specificity of amplicons generated by primer pairs was confirmed by restriction endonuclease digestion and DNA sequence analysis. The evaluation of B. thuringiensis strains for biological activity against insect pests of rice is thus aided by the grouping of strains based on their potential insecticidal spectrum.