Polymerase Chain Reaction

The gut bacteria exert phenotypic traits to the host but the factors which determine the gut bacterial profile (GBP) is poorly understood. This study aimed to understand the effect of ethnicity and geography on GBP of Mongoloid and Proto-Australoid tribes of India. Fecal bacterial diversity was studied in fifteen tribal populations representing four geographic regions (Assam, Telangana, Manipur and Sikkim) by DGGE followed by NGS analysis on Illumina MiSeq platform. Geography and diet had significant effect on GBP of the Indian tribes which was dominated by Prevotella. The effects were more prominent with lower taxonomic levels, indicating probable functional redundancy of the core GBP. A comparison with the worldwide data revealed that GBP of the Indian population was similar to the Mongolian population (Mongolia). The bacterial genera Faecalibacterium, Eubacterium, Clostridium, Blautia, Ruminococcus and Roseburia were found to be core genera in the representative populations of th...

Transforming growth factor alpha (TGFα) belongs to the epidermal growth factor (EGF) family and is known to play an important role during eyelid morphogenesis. In this study, we showed that ectopic expression of TGFα in the stroma of Kera-rtTA/tet-O-TGFα bitransgenic mice results in precocious eye opening, abnormal morphogenesis of the meibomian gland, tendon and tarsal plate malformation and epithelium hyperplasia. TGFα did not change proliferation and differentiation of meibocytes, but promoted proliferation and inhibited differentiation of the tarsal plate tenocytes. These results suggest that proper formation of the tendon and tarsal plate in the mouse eyelid is required for normal morphogenesis of the meibomian gland.

To determine the prevalence, molecular characteristics and hematological study of thalassemia in Tha Kradarn Subdistrict Chachoengsao Province. The present study population consisted of266 participants from Moo 19 Baan Na-Ngam, Chachoengsao Province, Thailand. After blood collection, all samples were screened for thalassemia by initial screening with the OF and DCIP tests and additional testing by CBC, RBC indices, hemoglobin typing and determination of Hb A2 and Hb E. All common alpha-thalassemia mutations were determined using the PCR with allele specific primers and Gap PCR for common deletions. The prevalence of alpha-thal 1, alpha-thal 2 and beta-thal were found as 2.72%, 11.26% and 0.97%, respectively. Regarding the abnormal hemoglobins, the prevalence of Hb E, Hb Constant Spring and Hb Pakse was 38.45%, 3.69% and 0.78%, respectively. MCV and MCH were significantly different between P-thalassemia as well as a-thal 1 carriers and normal subjects. In all alpha-thal 1 traits, it ...

Mutations in the DJ-1 gene have recently been shown to cause autosomal recessive Parkinson's disease. To estimate the prevalence of this mutation, an analysis was undertaken of 39 index cases of Parkinson's disease in whom a family history suggested autosomal recessive inheritance. No DJ-1 mutations were found in these patients, indicating that this gene is unlikely to be of numerical significance in clinical practice. The hypothesis was also tested that young onset Parkinson's disease patients in whom, despite extensive analysis, only a single heterozygous parkin mutation was found, might harbour a second mutation in the DJ-1 gene--that is, digenic inheritance. No patient was found with a single mutation in both DJ-1 and parkin genes, making this mode of inheritance unlikely. Finally it was confirmed that PARK6 and PARK7 (DJ-1), despite being phenotypically similar and mapping to the same small chromosomal region of 1p36, are caused by mutations in separate genes.

Identification of Candida cultured from various clinical specimens to the species level is increasingly necessary for clinical laboratories. Although sn PCR identifies the species within hours but its cost-effectiveness is to be considered. So there is always a need for media which help in the isolation and identification at the species level. The study aimed to evaluate the performance of different chromogenic media and to compare the effectiveness of the traditional phenotypic methods vs. seminested polymerase chain reaction (sn PCR) for identification of Candida species. One hundred and twenty seven Candida strains isolated from various clinical specimens were identified by conventional methods, four different chromogenic media and sn PCR. HiCrome Candida Differential and CHROMagar Candida media showed comparably high sensitivities and specificities in the identification of C. albicans, C. tropicalis, C. glabrata and C. krusei. CHROMagar Candida had an extra advantage of identify...

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecula...

With the widespread use of protein electrophoresis in verte- brate systematics in the 1960s and 70s, several museums and universities established extensive collections of frozen tissues. Use of these special collections expanded exponentially with the development of the polymerase chain reaction (PCR) and improvements in obtaining nucle- otide sequences. Moreover, using PCR, DNA sequences can now be routinely obtained directly

The cacti family is a morphologically heterogeneous group comprising 100 genera and about 1500 species (Hernandez and Barcenas, 1996). With the exception of one genus, all members of this family are native to America (Hernandez and Barcenas, 1996). There are three subfamilies, Opuntioideae, Cactoideae, and Pereskioideae (Gibson and Nobel, 1986). DNA isolation from cacti is notoriously difficult because they contain high amounts of polysaccharides and secondary metabolites which form insoluble complexes with nucleic acids during extraction (Guillemaut and Marechal-Drouard, 1992). Like in other groups of plants, the secondary metabolites and polysaccharides in cacti inhibit enzyme action (Porebski et al., 1997). The polysaccharides are visually evident by their viscous, glue-like texture and they make the DNA unmanageable when pipeting and hard to amplify by the polymerase chain reaction (PCR) (Poresbski et al., 1997). We report an easy and inexpensive protocol to isolate DNA from cacti. We used this method to isolate DNA from 85 species (170 individuals) of 39 genera of the subfamilies Pereskioideae, Opuntioidea, and Cactoideae. This procedure is a modification of a protocol described by De la Cruz et al. (1995) for the Cacti family. It requires only a few grams of tissue and does not require destruction of the whole plant to produce high molecular weight genomic DNA. The DNA from this procedure can be amplified consistently by PCR and used for RAPD analysis.

Genetic manipulation of the oil-yielding crop plants for better oil quality through biotechnological methods is an important aspect of crop improvement. Due to the inherent absence of the Δ6-desaturase (d6D) function, Brassica juncea, an oil-yielding crop plant, is unable to synthesize γ-linolenic acid (GLA), a nutritionally important fatty acid although the crop plant synthesizes the precursor fatty acids required for GLA production. Cyanobacterial d6D introduces carbon–carbon double bond onto linoleic acid (C18:2) and α-linolenic acid (C18:3) by desaturation processes for production of GLA and octadecatetraenoic acid (OTA) respectively. In the present investigation, d6D coding sequence from Synechocystis sp. PCC6803 was cloned by polymerase chain reaction and introduced into B. juncea through Agrobacterium-mediated transformation technique. Both cytosolic as well as seed-specific expression of d6D were attempted. The transformed plants show production of GLA and OTA in contrast to their absence in the untransformed control plants adducing evidence for introgression and functional expression of the cyanobacterial d6D gene in B. juncea.

We compared PCR to conventional culture for the detection of vancomycin-resistant enterococci (VRE) in 30,835 rectal samples over a 3-year period. The positive and negative predictive values of vanB PCR were 1.42% and 99.9%, respectively. A positive vanB result by PCR is poorly predictive and necessitates culture for differentiation of VRE-positive and -negative individuals.