Agarose Gel Electrophoresis of DNA

Boffey, S.

doi:10.1385/0-89603-064-4:43

Cited by 16 publications

(7 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1 minute at 94ºC, Annealing: 30seconds at 60 ºC, and Extension: 1 minute at 72 ºC) and Final extension: 10 minute at 72ºC. Agarose Gel Electrophoresis of the MSP Products: (20).…”

Section: Methylation-specific Pcr (Msp)mentioning

confidence: 99%

Evaluation of Hypermethylation of Ras Association Domain Family-1 a and Glutathione S-Transferase Protein-1 Genes as Diagnostic Marker for Hepatocellular Carcinoma

El-Sabakawy

Assaf

Arafa

et al. 2015

Zagazig University Medical Journal

View full text Add to dashboard Cite

Background and study aims: The molecular pathogenesis of HCC involves well-defined genetic and epigenetic alterations. The Ras association domain family 1A (RASSF1A) and the Glutathione S-transferase P 1 (GSTP1) genes are two tumour suppressor genes that are reported to be silenced by CpG island promoter hypermethylation which is a key to the tumourigenic process in HCC. The aim of this study was to analyze the methylation frequency of RASSF1A and GSTP1 genes in early stages of HCC , chronic hepatitis C and healthy subjects to evaluate its value as a diagnostic marker for early HCC. Patients and methods: Methylation-specific polymerase chain reaction (MSP) was used to detect RASSF1A and GSTP1 promotor methylation in DNA extracted from plasma samples of 25 patients with HCC, 25 patients with chronic hepatitis C and 25 healthy controls. Assessment of alpha fetoprotein (AFP) was performed in all groups by ELISA using commercially available kits. Results: Methylated RASSF1A was detected in 76 % of the HCC group (19/25), in 20% of the chronic hepatitis C patients (5/25) and in 16% of the healthy controls(4/25). The methylation frequencies were significantly higher in patients with HCC compared to the controls (P ≤ 0.001) and chronic hepatitis C patients ( P ≤ 0.001). While methylated GSTP1 was detected in 44% of the HCC group(11/25) ,in 12% of the chronic hepatitis C group (3/25) and in 8% of the controls (2/25). Although the sensitivity and specificity, for each gene as an epigenetic biomarker was moderate (76% and 44% for RASSF1A and GSTP1 respectively), the combination analysis of both genes resulted in an increased sensitivity and specificity to 88%,and 76% respectively) in discriminating HCC from normal control and chronic hepatitis C pateints. As regard AFP, Receiver operating characteristic curves were plotted and showed an optimal cutoff value of 9.5 ng/ml with sensitivity of 88 % and specificity of 58% when the area under the receiver operator characteristic (AUROC) curve was 0.87 with 95% Confidence Interval .Conclusion: The epigenetic changes observed in this study indicate that examination of methylation status of RASSF1A and GSTP1 could be of value for early diagnosis of HCC especially when using a combination of more than one epigenetic marker .

show abstract

“…1 minute at 94ºC, Annealing: 30seconds at 60 ºC, and Extension: 1 minute at 72 ºC) and Final extension: 10 minute at 72ºC. Agarose Gel Electrophoresis of the MSP Products: (20).…”

Section: Methylation-specific Pcr (Msp)mentioning

confidence: 99%

Evaluation of Hypermethylation of Ras Association Domain Family-1 a and Glutathione S-Transferase Protein-1 Genes as Diagnostic Marker for Hepatocellular Carcinoma

El-Sabakawy

Assaf

Arafa

et al. 2015

Zagazig University Medical Journal

View full text Add to dashboard Cite

show abstract

“…Genomic DNA was extracted from promastigotes in stationary phase, then LACK and TSA genes were amplified by PCR .…”

Section: Methodsmentioning

confidence: 99%

Enhancement of immune response induced by DNA vaccine cocktail expressing complete LACK and TSA genes against Leishmania major

Ghaffarifar

Jorjani

Sharifi

et al. 2012

APMIS

View full text Add to dashboard Cite

Leishmaniasis is an important disease in humans. Leishmania homologue of receptor for Activated C Kinase (LACK) and thiol specific antioxidant (TSA) as immuno-dominant antigens of Leishmania major are considered the most promising molecules for a DNA vaccine. We constructed a DNA cocktail, containing plasmids encoding LACK and TSA genes of Leishmania major and evaluated the immune response and survival rate in BALB/c mice. IgG and Interferon gamma values were noticeably increased in the immunized group with DNA cocktail vaccine, which were significantly higher than those in the single-gene vaccinated and control groups (p < 0.05) following the immunization and after challenging with Leishmania major. Interleukin 4 values were decreased in all immunized groups, but only in DNA vaccine cocktail and single-gene vaccination with pc-LACK there were statistical differences with control groups (p > 0.05). The immunized mice with the cocktail DNA vaccine presented a considerable reduction in diameter of lesion compared to other groups and a significant difference was observed (p < 0.05) in this regard. The survival time of the immunized mice with the cocktail DNA vaccine was significantly higher than that in the other groups (p < 0.05) after their being challenged with Leishmania major. The findings of this study indicated that the cocktail DNA vaccine increased the cellular response and survival rate and induced protection against infection with Leishmania in the mice.

show abstract

“…White colonies were selected as containing recombinant plasmids (Bothwell et al, 1990), mass-cultured and plasmid extraction was performed using miniprep alkaline method (Feliciello and Chinali, 1993). Electrophoresis on 0.8% agarose gel was done (Boffey, 1984) and recombination was confirmed using restriction analysis using SacI and BamHI restriction enzymes.…”

Section: Cloning and Transformationmentioning

confidence: 99%

Cloning and expression of Toxoplasma gondii tachyzoite P22 protein

Maryam¹,

Mojgan²,

ehpour³

et al. 2011

Afr. J. Biotechnol.

View full text Add to dashboard Cite

Delay in diagnosis ofToxoplasma gondii infection in pregnant women who have been infected during the first trimester of gestation can lead to death of her fetus. Serological tests based on recombinant proteins are the main diagnosis methods for the detection of anti Toxoplasma antibody in serum samples. The aim of this study was to clone and express a gene encoding a P22 protein of T. gondii tachyzoite as using antigen for ELISA serology method. DNA was extracted from T. gondii (RH-strain) tachyzoites and PCR reaction was done using corresponding primers. The PCR product was purified, ligated to PTZ57R plasmid via T/A cloning method and subcloned into SacI and BamHI digested pET32a expression vector. Recombinant plasmid was transformed in E. coli (Bl21 DE3) and induced by 1 mM IPTG and analyzed by 12% SDS-PAGE. Expressd protein was purified by affinity chromatography and confirmed by western blot analysis. We successfully cloned and expressed T. gondii P22 protein.

show abstract

Agarose Gel Electrophoresis of DNA

Cited by 16 publications

References 0 publications

Evaluation of Hypermethylation of Ras Association Domain Family-1 a and Glutathione S-Transferase Protein-1 Genes as Diagnostic Marker for Hepatocellular Carcinoma

Evaluation of Hypermethylation of Ras Association Domain Family-1 a and Glutathione S-Transferase Protein-1 Genes as Diagnostic Marker for Hepatocellular Carcinoma

Enhancement of immune response induced by DNA vaccine cocktail expressing complete LACK and TSA genes against Leishmania major

Cloning and expression of Toxoplasma gondii tachyzoite P22 protein

Contact Info

Product

Resources

About