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2021 ◽  
Vol 4 (2) ◽  
pp. 14
Author(s):  
Abdul Halim Sadikin ◽  
Irene Dian ◽  
Mukharjon Mukharjon ◽  
Rini Puspitaningtum ◽  
Septelia Inawati Wanandi

Background: In some people, acetaldehyde, a toxic product from ethanol oxidation, cannot be oxidized to acetate. The excess of acetaldehyde could cause facial flushing, dizziness, and hypertension when they consume ethanol. This ethanol sensitivity is caused by a deficiency of ALDH2. Objective: This study aims to analyze and count the polymorphism frequency of the ALDH2 gene in Indonesia’s Minang ethnic. Methods: DNA samples were taken randomly from hair bulbous of 60 subjects (male and female, 3rd generation). A nested polymerase chain reaction was conducted to amplify the ALDH2 in the samples. Afterward, restriction fragment length polymorphism (RFLP) was conducted to the amplicons using the EcoRI restriction enzyme. The measured parameters were the distribution of the wildtype, atypical homozygote, and heterozygote. Results: Results showed that out of 60 subjects, 53.33% have an atypical homozygote gene (subjects prone to hypersensitive to alcohol), 28.33% have a heterozygote gene, and 18.33% have a wildtype gene. The frequency of the atypical alleles in Minang ethnic is 0.675. Conclusion: The atypical ALDH2 allele was much higher than the normal ALDH2 allele, in which most participants have atypical homozygote ALDH2, suggesting the samples are sensitive to alcohol.


2020 ◽  
Vol 6 (1) ◽  
pp. 10-19
Author(s):  
Popi Asri Kurniatin ◽  
Laksmi Ambarsari ◽  
Annisa Dhiya Athiyyah Khanza ◽  
Inda Setyawati ◽  
Djarot Sasongko Hami Seno ◽  
...  

Glucose oxidase is used in various industries for the development of enzymatic fuel cell. Based on prior studies, this compound is sourced from the local isolates of Aspergillus niger IPBCC 08.610, although investigations on the encoding gene have not been conducted. The purpose of this research, therefore, is to identify and characterized the gene responsible for encoding glucose oxidase, in the aspect of sequence, length, and restriction patterns. This experiment involved the amplification of genomic DNA using specific primers for gene recognition, which was followed by the restriction technique with EcoRI and PstI endonucleases. Furthermore, the gene is inserted into vector pGEM®T-Easy and transformed into competent E. coli DH5α cells, in an attempt to perform sequencing. The glucose oxidase gene from A. niger IPBCC 08.610 was confirmed to possess a size of 1848 bp, and a GC content of 57.8%, with a possibility of restriction into two fragments of size 908 bp and 980 bp, using the EcoRI restriction.


2020 ◽  
Vol 3 (1) ◽  
pp. 3
Author(s):  
Leonid V. Gening ◽  
Alexandr A. Volodin ◽  
Konstantin Y. Kazachenko ◽  
Irina V. Makarova ◽  
Vyacheslav Z. Tarantul

We propose an improved earlier described “mirror” method for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to the misincorporation of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). This method is based on the synthesis of a complementary chain (“mirror”) by nuclear extracts of different mice organs on a template containing 8-oxoG and dideoxycytidine residue (ddC) at the 3′‑end. The “mirror” was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the “framed mirror” products. The misincorporation of dAMP in “framed mirror” products forms an EcoRI restriction site. The restriction analysis of double-stranded “framed mirror” products allows a quantification of the mutation frequency in nuclear extracts. The data obtained show that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos.


2019 ◽  
Vol 24 (1) ◽  
pp. 8
Author(s):  
Achmad Rodiansyah ◽  
Riyona Desvy Pratiwi ◽  
Sabighoh Zanjabila ◽  
Asrul Muhamad Fuad

Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5a. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained.


2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Erma Sulistyaningsih ◽  
Sukarti Moeljopawiro ◽  
Jarot Subandono ◽  
Wayan T. Artama

Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA


2011 ◽  
Vol 11 (1) ◽  
pp. 15-21
Author(s):  
Elmy Mariana

Polymorphism analysis of lactoferrin gene on holstein-friesian cow with PCR-RFLP methodABSTRACT. The purposes of this study were to identify the polymorphism of the lactoferrin gene in Holstein-Friesian (HF) cows. The study was conducted on 281 heads of HF lactating cows coming from dairy farmers in Lembang district. Investigation on variant genotypes of the lactoferrin gene used PCR-RFLP method. Genotyping of the lactoferrin gene with EcoRI restriction enzyme produced two genotypes, i.e. AA (65.5%) and AB (34.5%) genotypes.


2008 ◽  
Vol 10 (5) ◽  
pp. 469-474 ◽  
Author(s):  
Shujian Liang ◽  
Harold N. Bass ◽  
Hanlin Gao ◽  
Caroline Astbury ◽  
Mehdi R. Jamehdor ◽  
...  

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