PCR Southern Hybridization Research Articles

Sensitivity of polymerase chain reaction (PCR)-southern hybridization and conventional PCR analysis for Halal authentication of gelatin capsules

Halal authentication of gelatin capsules was conducted using polymerase chain reaction (PCR)-southern hybridization on chip and conventional PCR analysis. The primers used in PCR-southern hybridization were targeted on mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene and its amplicon was 276 bp. In conventional PCR, three pairs of mtDNA primers targeted cyt b, cytochrome oxidase II (COII) and ATP6 gene were tested, resulting of 398, 212 and 83 bp amplicons, respectively. Of 20 brands examined using PCR-southern hybridization, 6 capsules (C1–C6) were found to be porcine DNA positive but none were positive using conventional PCR method. The sensitivity of each primer in the detection of porcine DNA was 0.25 ng (cyt b), 0.1 ng (COII), 0.001 ng (Olipro™ Chip) and 0.0001 ng (ATP6). Results demonstrated that the PCR-southern hybridization on chip was useful and reliable for verifying porcine DNA in gelatin capsules compared to conventional PCR.

Transformation of Antisense Wheat Mlo (Ta-Mlo) Gene and Wheat Powdery Mildew Resistance Analysis of Transgenic Plants

The antisense wheat Mlo gene, Ta-Mlo, was transformed into wheat (Tritivum aestivum L.) varieties Yangmai 158 and Jimai 20 via biolistic transformation using immature embryo calli as explants. After two rounds of bialaphos selection and regeneration, herbicide-resistant plants were obtained, which were subsequently confirmed by PCR, PCR-Southern hybridization, genomic dot hybridization, and BASTA resistance analysis. The results showed that the Ta-Mlo antisense transgenic Yangmai 158 and Jimai 20 plants were obtained. The real time fluorescence quantitative PCR analysis proved that the transcript of Ta-Mlo was knocked down in these transgenic plants. The disease resistance test showed that the six transgenic lines appeared highly resistance to powdery mildew pathogen Blumeria graminis f. sp. tritici (Bgt). The transgenic lines showed distinct acceleration of the production and stabilization of papillae, and effective suppression to further development of haustoria of Bgt. Therefore, the transgenic lines showed high resistance to Bgt.

Molecular cloning and mapping of casein kinase 2 alpha and beta subunit genes in barley

Casein kinase 2 (CK2) is a ubiquitous, highly pleiotropic, constitutively active, and messenger-independent Ser/Thr protein kinase. It is found in two different forms: the heterotetrameric CK2, composed of two alpha catalytic subunits and two beta regulatory subunits, and the monomeric CK2 alpha, consisting of the alpha catalytic subunit. In the present study, we isolated barley cDNA clones of the CK2 alpha and beta subunit genes, designated HvCK2A and HvCK2B, respectively. Chromosome assignment, using a set of wheat-barley disomic chromosome addition lines, and RFLP mapping, using two doubled haploid populations, showed that HvCK2A was duplicated on the short arm of chromosome 2H and the long arm of chromosome 5H (designated HvCK2a-2H and HvCK2a-5H, respectively), and a single copy of HvCK2B was located on the long arm of chromosome 1H (designated HvCK2b). A PCR-Southern hybridization experiment demonstrated that the HvCK2A sequence originated from the HvCK2a-5H locus, showing that at least HvCK2a-5H was expressed. The present cDNA sequences and genomic organization of the two subunits will facilitate further functional analysis of CK2 in barley.

Steroid hormones (17β-estradiol and hydrocortisone) upregulate hepatocyte nuclear factor (HNF)-3β and insulin-like growth factors I and II expression in the gonads of tilapia ( Oreochromis mossambicus) in vitro

Hepatocyte nuclear factors (HNF-1α, -1β and -3β) and insulin-like growth factors (IGF-I and -II), which are involved in liver-specific gene expression, metabolism, development and cell growth, have been found in the gonads of tilapia ( Oreochromis mossambicus). However, the functions of these factors and how they interact within the gonads of bony fish are not understood. In the present study, we provided experimental evidence that the expression of HNF-3β in the gonads of tilapia, but not HNF-1α and -1β, was affected in vitro by 17β-estradiol and hydrocortisone. Immunohistochemical staining confirmed that tilapia HNF-3β was mainly found in the nuclei of hepatocytes, the follicular granulosa cells of the ovaries, and the interstitial cells of the testes of adult tilapia. Further data were gathered at various steroid concentrations (0.1, 1, 10, 100, and 1000 nM) over various culture intervals (6, 12, 18, 24, 30, and 36 h) and subjected to semi-quantitative RT-PCR analysis. The expression of downstream genes (IGF-I and -II) followed the same temporal patterns as HNF-3β, albeit at decreased levels for 30 and 36 h culture intervals. Both hormones upregulated HNF-3β mRNA expression at concentrations of 0.1–10 nM, and reached optimal physiological concentrations for induction of IGFs at 1–10 nM. The identity of the PCR fragments was concurrently verified by sequencing and PCR-Southern hybridization. We inferred that HNF-3β and IGFs may play a regulatory role in tilapia gonads during oocyte maturation and spermatogenesis.

Hairy roots induced by Agrobacterium rhizogenes and production of regenerative plants in hairy root cultures in maize

Hairy roots of maize were induced by infecting 15-d calli with Agrobacterium rhizogenes. The hairy roots cultured in hormone-free media showed the vigorous growth and typical hairy root features. The regenerated plants were produced from hairy roots in MS media supplemented with 1.6 mg/L ZT and 0.4 mg/L NAA. The PCR-Southern hybridization demonstrated that T-DNA had been integrated into the chromosome of regenerated plants.

Genetic Transformation of ‘Kyoho’ Grape with a GFP Gene

‘Kyoho’ grapevine (Vitis × labruscana Bailey) was successfully transformed by Agrobacterium-mediated transformation. Embryogenic calli (EC) were co-cultured with A. tumefaciens strain, EHA105 that harbors a green fluorescent protein (GFP) gene with the CaMV35S promoter. Kanamycin at 15 mg·L−1 was suitable for isolating transformed EC from which three plants that express GFP fluorescence were regenerated. DNA analyses, including polymerase chain reaction (PCR) and PCR-Southern hybridization, confirm that these three plants contain sequences comparable to the GFP gene to indicate that it was successfully incorporated into the ‘Kyoho’ grapevine genome.

Transformation of tomato cultivar ‘Pusa Ruby’ with bspA gene from Populus tremula for drought tolerance

Boiling stable proteins are known to play a role in desiccation tolerance against water stress by protecting proteins in membranes and cytosol. A novel 66 kDa boiling stable protein was highly expressed in Populus tremula in response to gradual water stress. The plasmid carrying cDNA encoding this protein was transferred to tomato (Lycopersicon esculentum cultivar ‘Pusa Ruby’) through Agrobacteriumtumefaciens. Initially, hygromycin resistance was used for selection of transient gene expression. Stable integration of the T-DNA into nuclear genome was confirmed by PCR and PCR-Southern hybridization. Northern blot analysis detected the different levels of transcripts of bspA gene in individual transgenic lines. Drought tolerance of putative transgenic lines was assayed by PEG test, biomass analysis, proline estimation and electrolyte leakage measurement. All of them showed slightly increased tolerance of transgenic plants to water stress compared to non-transgenics.

Comparison of PCR-southern hybridization and quantitative real-time PCR for the detection of JC and BK viral nucleotide sequences in urine and cerebrospinal fluid

The presence of the human polyomaviruses JCV and BKV in immunocompromised patients can lead to lethal diseases and conditions including progressive multifocal leukoencephalopathy (PML), interstitial nephritis, hemorrhagic cystitis, and kidney allograft rejection. Typically, detection of JCV and BKV in clinical samples has employed standard PCR amplification for viral nucleotide sequences, with subsequent confirmation for viral genome specificity of PCR products by southern blot hybridization. Here, we directly tested a validated PCR-southern protocol with a TaqMan real-time PCR protocol (Applied Biosystems) to assay clinical samples of urine and cerebrospinal fluid. We found equal specificity and sensitivity with both methods. However, real-time allowed for absolute viral-genome quantitation without the use of radionucleotides and was performed more rapidly, in as little as 24 h. Such advantages are important to consider in the effort to establish international standardization of controls for the detection of JCV and BKV, which would aid in screening confidence and the reliable assessment of anti-viral therapies.

Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens.

An efficient procedure is described for the transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens via callus regeneration. Calli derived from ovules were co-cultivated with A. tumefaciens strains EHA101 and LBA4404, which harbored the binary vector plasmids pIG121Hm and pTOK233, respectively. These plasmids contain the beta-glucuronidase gene ( gusA) as a reporter gene and the hygromycin phosphotransferase and neomycin phosphotransferase II ( nptII) genes as selective markers. Inoculated calli were first plated for 4 weeks on medium containing cefotaxime to eliminate bacteria, following which time transformed cells were selected on medium that contained 20 mg/l hygromycin. A histochemical assay for GUS activity revealed that hygromycin-based selection was completed after 8 weeks. The integration of the T-DNA of pIG121Hm and pTOK233 into the genome of the cells was confirmed by PCR analysis. Efficient shoot regeneration from the transformed calli was observed on half-strength MS medium supplemented with 0.5 mg/l naphthaleneacetic acid and 0.5 mg/l benzyladenine after about 5 months of culture. The presence of the gusA and nptII genes in the genomic DNA of regenerated plants was detected by means of PCR and PCR-Southern hybridization, and the expression of these transgenes was verified by reverse transcription-PCR.

Isolation and identification of a novel satellite DNA family highly conserved in several Cervidae species.

In an attempt to amplify cervid satellite II DNA from the genomes of Indian muntjac and Chinese muntjac, a pair of primers derived from the white tailed deer satellite II DNA clone (OvDII) yielded a prominent approximately 1 kb polymerase chain reaction (PCR) product (in addition to the expected 0.7 kb satellite II DNA fragments) in both species. The approximately 1 kb products were cloned, sequenced, and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). This revealed that the approximately 1 kb cloned sequences indeed represent a previously unknown cervid satellite DNA family, which is now designated as cervid satellite IV DNA. Approximately 1 kb PCR clones were also obtained from the genomes of the black tailed deer and Canadian woodland caribou with similar primer pairs. Extremely high sequence conservation (over 90% homology) was observed among the clones generated from all four deer species and PCR-Southern hybridization experiments further verified the co-amplification of two kinds of satellite DNA sequences with the same pair of primers. This satellite DNA was found to co-localize with centromeric proteins at the kinetochore by a simultaneous FISH and immunofluorescence study. Due to its high sequence conservation and close association with kinetochores, the newly identified satellite DNA may have a functional centromeric role.

Trypanosoma cruzi:Polymerase Chain Reaction-Based Detection in Dried Feces ofTriatoma infestans

PCR was employed to detectTrypanosoma cruziDNA inTriatoma infestansdry fecal spots collected on filter papers. Both insects fed on experimentally infected monkeys and insects collected in a Paraguayan endemic area for Chagas' disease were examined. When the insects fed on a chronically infected monkey with low parasitemia as revealed by direct microscopic observation (DMO),T. cruziwas detected in the insect feces by PCR as soon as 2 days postfeeding. When the same experiment was performed on monkeys with parasitemia levels below the limit of detection by DMO, the degree of positivity found through PCR–Southern hybridization, applied on Day 8 postfeeding, was superior to that obtained through xenoculture. These results suggest that PCR can be used to speed the xenodiagnosis results with great sensitivity. On the other hand, when applied to the feces of triatomines collected in the field, 84% were positive by PCR–Southern hybridization, whereas only 26% were positive by DMO. Therefore, PCR could also be applied to the monitoring of the infection status of triatomines which infest rural dwellings by examining only the feces left on paper sensors hung on the walls of the houses.