restriction enzyme
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Gene Reports ◽  
2022 ◽  
pp. 101487
Author(s):  
S.N.J. Pathirana ◽  
D.A.S. Elvitigala ◽  
C.M. Nanayakkara ◽  
Prashanth Suravajhala ◽  
Sanath Rajapakse ◽  
...  

2021 ◽  
Author(s):  
Magal Saphier ◽  
Lea Moshkovich ◽  
Stanislav Popov ◽  
Yoram Shotland ◽  
Eldad Silberstein ◽  
...  

Abstract The effect of monovalent copper ions on enzymatic systems has hardly been studied to date; this is due to the low stability of monovalent copper ions in aqueous solutions, which led to the assumption that their concentration is negligible in biological systems. However, in an anaerobic atmosphere, and in the presence of a ligand that stabilizes the monovalent copper ions over the divalent copper ions, high and stable concentrations of monovalent copper ions can be reached. Moreover, the cell cytoplasm has a substantial concentration of potential stabilizers that can explain significant concentrations of monovalent copper ions in the cytoplasm. This study demonstrates the effect of monovalent and divalent copper ions on DNA polymerase, ligaseT4 DNA, the restriction enzymes EcoP15I and EcoR I, acid phosphatase, and α and βamylase enzymes. These systems were chosen because they can be monitored under conditions necessary for maintaining a stable concentration of monovalent copper ions, and since they exhibit a wide range of dependency on ATP. Previous studies indicated that ATP interacts with monovalent and divalent copper ions and stabilizing monovalent copper ions over divalent copper ions. The results showed that monovalent copper ions dramatically inhibit DNA polymerase and acid phosphatase, inhibit ligaseT4 DNA and the restriction enzyme EcoP15I, moderately inhibit α and β amylase, and have no effect on the restriction enzyme EcoR I. From the results presented in this work, it can be concluded that the mechanism is not one of oxidative stress, even though monovalent copper ions generate reactive oxygen species (ROS). Molecular oxygen in the medium, which is supposed to increase the oxidative stress, impairs the inhibitory effect of monovalent and divalent copper ions, and the kinetics of the inhibition is not suitable for the ROS mechanism.ATP forms a complex with copper ions (di and monovalent ions, where the latter is more stable) in which the metal ion is bound both to the nitrogen base and to the oxygen charged on the phosphate groups, forming an unusually distorted complex. The results of this study indicate that these complexes have the ability to inhibit enzymatic systems that are dependent on ATP.This finding can provide an explanation for the strong antimicrobial activity of monovalent copper ions, suggesting that rapid and lethal metabolic damage is the main mechanism of monovalent copper ions’ antimicrobial effect.


2021 ◽  
Vol 62 (4) ◽  
pp. 371-377
Author(s):  
Rossana C. Jaspe ◽  
Yoneira Sulbaran ◽  
Mariana Hidalgo ◽  
Mariana Hidalgo ◽  
Carmen L. Loureiro ◽  
...  

Variants of Concern or Interest of SARS-CoV-2 (VOC or VOI), the coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 452 of the Spike protein are particularly important and associated with some of these variants: L452R, present in Delta VOC, and L452Q, present in Lambda VOI. These mutations have been associated with both increased infectivity and evasion of protective immune response. A search on GISAID to detect the number of sequences harboring the L452R mutation and the frequency of Delta VOC among them, showed that since August 2021, most of these sequences belong to the Delta VOC. Restriction enzyme analysis is proposed as a rapid method to detect L452R. A small amplicon from the Spike gene was digested with MspI. A 100% concordance was observed between digestion and sequencing results. The mutation L452Q can also be detected by restriction analysis, allowing the identification of putative Lambda VOIs. The proposed methodology, which allows screening of a great number of samples, could provide a faster information on the prevalence of Delta VOC cases.


2021 ◽  
Author(s):  
Merve-Zeynep Kesici ◽  
Philip Tinnefeld ◽  
Andres M Vera

DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays, and showed comparable performance to commercial hot-start enzymes.


2021 ◽  
Vol 45 (7) ◽  
pp. S37
Author(s):  
Elite Possik ◽  
Clémence Schmitt ◽  
Anfal Al-Mass ◽  
Johanne Morin ◽  
Heidi Erb ◽  
...  

2021 ◽  
Vol 888 (1) ◽  
pp. 012024
Author(s):  
P W Prihandini ◽  
A Primasari ◽  
M Luthfi ◽  
D Pamungkas ◽  
A P Z N L Sari ◽  
...  

Abstract The restriction enzyme is important for genotyping using the PCR-RFLP technique. Therefore, this study aims to identify the restriction enzyme mapping in the partial sequence of the follicle-stimulating hormone receptor (FSHR) gene in Indonesian local cattle. A total of 29 samples sized 306 bp, were aligned with Genbank sequence acc no. NC_032660, resulting three polymorphic sites, namely g.193G>C, g.227T>C, and g.275A>C. Furthermore, the restriction mapping analysis using the NEBcutter program V2.0 showed that no enzyme recognized the SNP g.275A>C, while the SNP g.193G>C and g.227T>C were identified by the AluI and MscI enzymes, respectively. The AluI enzyme cuts at two positions (193 bp and 243 bp) in the G allele sample producing three fragments namely 50 bp, 63 bp, and 193 bp, meanwhile, in the C allele, the AluI cuts only in position 243 bp, hence, the fragment products are 63 bp and 243 bp. In contrast, the MscI enzyme was only recognized in the T allele, producing fragments sized 77 bp and 229 bp but failed to identify the restriction site along with the PCR products in the C allele. Based on the results, the SNPs (g.193G>C and g.227T>C) and restriction enzymes (AluI and MscI) are applicable for genotyping local Indonesian cattle using the PCR-RFLP technique in future studies.


Author(s):  
S. Nihar ◽  
S. Naveen Kumar ◽  
Wilfred Ruban ◽  
H.M. Yathish ◽  
R. Nagaraja ◽  
...  

Background: Recent developments in molecular genetics lead to addressing certain poultry diseases via breeding for disease resistance. The present study was carried to identify and compare the genetic polymorphism in Chicken Mx1 and TVB genes among the indigenous and Giriraja chicken using PCR-RFLP technique. Methods: Blood samples were collected from 50 indigenous and 50 Giriraja birds and DNA isolation was done by Phenol: Chloroform: Isoamyl alcohol method. PCR amplification of Chicken Mx1 (exon 14) and Chicken TVB (exon 3) genes was carried out followed by RFLP analysis. Result: PCR product sizes of 301 bp and 303 bp of Mx1 and TVB genes, respectively were successfully amplified. RFLP analysis of Mx1 gene with Hyp8I restriction enzyme revealed three genotypes AA, AB and BB. In indigenous birds genotypic frequencies of AA, AB and BB were 0.314, 0.493 and 0.194, respectively and gene frequencies were 0.56 and 0.44 for alleles A and B, respectively. In Giriraja birds, genotypic frequencies for AA, AB and BB were 0.27, 0.499 and 0.23, respectively and gene frequencies were 0.52 and 0.48 for alleles A and B, respectively. RFLP analysis of TVB gene with NlaIII restriction enzyme revealed two genotypes viz., AA and AB. In indigenous birds genotypic frequencies of AA and AB were 0.81 and 0.18, respectively and gene frequencies were 0.9 and 0.1 for alleles A and B, respectively. In Giriraja birds genotypic frequencies for AA and AB were 0.774 and 0.211, respectively and gene frequencies were 0.88 and 0.12 for alleles A and B, respectively.


2021 ◽  
Author(s):  
Zhenfeng Fu

Enzyme digestion is to cut the DNA molecule and the carrier molecule at the sticky end to obtain the corresponding sticky end connection.


2021 ◽  
pp. 196-203
Author(s):  
Fitrine Ekawasti ◽  
Umi Cahyaningsih ◽  
N. L. P. Indi Dharmayanti ◽  
Siti Sa'diah ◽  
Didik Tulus Subekti ◽  
...  

Background and Aim: Toxoplasma gondii is a unicellular coccidian parasite distributed globally and is an important zoonotic pathogen. Approximately 30% of the human population worldwide is chronically infected with T. gondii. The pathogenicity of this species depends on the type originating from the clonal population. Techniques for more accurately determining the type of T. gondii have recently been developed using genetic markers. Specifically, T. gondii has been typed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This study aimed to identify sets of PCR-RFLP markers that have high power to discriminate genotyping of T. gondii and are easy to use and are easy to use. The objective of this study was to characterize virulent strain isolates of T. gondii by PCR-RFLP using 10 markers with DdeI. Materials and Methods: T. gondii tachyzoites (RH virulent strain) were derived from culture cells at the Indonesian Research Center for Veterinary Sciences. Genotyping was performed on T. gondii DNA extracted from cell cultured tachyzoites using 10 genetic markers of PCR-RFLP, namely, B1#1, B1#2, B1#3, SAG1#1, SAG1#2, P30, BAG1, ROP1, GRA1, and GRA7, with digestion using the restriction enzyme DdeI. Results: The 10 genes were amplified by PCR. Among them, three genetic markers, B1#3, ROP1, and GRA1, were genotyped by the PCR-RFLP using restriction enzyme DdeI. Overall, the findings showed that the specific RFLP profile of digestion of gene regions by DdeI could be used as a specific marker for the virulent biotype causative of toxoplasmosis. In addition, virulent strains of T. gondii can be easily detected by these markers. Conclusion: Three pairs of primers (B1#3, ROP1, and GRA1) with DdeI have proven useful for the diagnosis of acute toxoplasmosis (virulent strain biotype I). This proposed method is relatively simple, rapid, cheap, and can be performed in most laboratories, providing a practical approach for the routine analysis of T. gondii strains.


2021 ◽  
Vol 19 (3) ◽  
pp. 539-545
Author(s):  
Vo Thi Thuong Lan ◽  
Le Thi Thanh

DNA marker is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare recombinant plasmids pSY-60 which was partially digested by one restriction enzyme for generating DNA markers of 7 fragments from 60 to 420 bp. The procedure included a synthesis of 60 bp DNA fragment with EcoRI sites at both ends using PCR extension, self-ligation of the 60 bp fragments and subcloning the ligated product into plasmid, generating recombinant pSY-60. Once being cloned, 500 ng of 420 bp fragment purified from 100 µL PCR product was incompletely digested by EcoRI, sufficiently producing to 50 acrylamide gels. Our procedure for production of DNA markers could be simple, time saving and inexpensive in comparison with current ones widely used in most laboratories.


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