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Recombinant DNA technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
In the Maxam and Gilbert procedure, the DNA fragment to be sequenced is end-labeled by the addition of 32p-dATP, either at the 5′-ends (by the enzyme polynucleotide kinase) or at the 3′-ends (by the enzyme deoxynucleotidyl transferase) of its two strands. The end-labeled fragment is now digested with a restriction endonuclease, which cleaves it into only two fragments of unequal lengths. As a result, only one end of each of the two fragments thus produced will be labeled. The two unequal fragments are separated through gel electrophoresis, and they are sequenced separately. Alternatively, the end-labeled fragment is denatured and its two complementary strands are separated through gel electrophoresis. For some reasons, the two complementary strands of a DNA molecule generally show different mobilities during gel electrophoresis. The samples of complementary strands thus separated are sequenced separately. It may be noted that each strand will be labeled only at one end (either the 5′-end or the 3′-end).
Production of Life-Saving Drugs from Marine Sources
Published in Prasenjit Mondal, Ajay K. Dalai, Sustainable Utilization of Natural Resources, 2017
Vidarabine (4) or 9-β-D-arabinofuranosyladenine (ara-A) is a glycoside analog of adenosine where D-ribose is replaced with D-arabinose which is inspired by the knowledge gained from the chemical structure of spongouridine (2). The synthesis of vidarabine was first achieved in the laboratory of Bernard Randall Baker at the Stanford Research Institute (now known as SRI International) and finally natural vidarabine was obtained from the fermentation broth of Streptomyces antibioticus. It is the first systemic nucleoside analog antiviral agent that has been licensed for the management of herpes virus infection in human. Vidarabine interferes with the synthesis of viral DNA. Diphosphorylated and triphosphorylated vidarabine are active drugs produced in vivo that interfere with the synthesis of viral DNA. Diphosphorylated vidarabine prevents the reduction of nucleotide diphosphates by inhibiting ribonucleotide reductase enzyme, thereby inducing a decline of viral replication. Triphosphorylated vidarabine competitively inhibits dATP leading to the formation of “faulty” DNA. Vidarabine has some significant limitations such as rapid metabolism by adenosine deaminase, low intramuscular and intestinal absorption, and requirement of large fluid volumes for intravenous administration over prolonged periods (Cohen 1979; Whitley et al. 2012).
Molecular Machines
Published in Thomas M. Nordlund, Peter M. Hoffmann, Quantitative Understanding of Biosystems, 2019
Thomas M. Nordlund, Peter M. Hoffmann
EcoRI requires no obvious fuel source for its cleavage activity. When DNA is synthesized, the polymerases work with the high-energy form of the nucleotides, dATP, dCTP, dTTP, and dGTP, so the building blocks also provide free energy for assembly. Because double-stranded DNA is a higher free-energy form than the cleaved strands, one could claim that no energy is required for cleavage. However, before cleavage the two phosphate bonds between two sets of ribose rings were intact; after cleavage, they are not. This will not spontaneously happen, at least not on a time scale of tenths of a second. (There is some truth to the Jurassic Park assertion that DNA can be stable for millions of years, under the proper circumstances.)
Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application
Published in Preparative Biochemistry & Biotechnology, 2023
Kashif Maseh, Syed Farhat Ali, Shazeel Ahmad, Naeem Rashid
Assay for DNA polymerase activity was done as describe previously[14] by measuring the incorporation of TTP [methyl-3H] by using activated calf thymus DNA. The reaction mixture, in 20 µL, contained: 25 mM Tris–Cl pH 8.5, 4 mM MgCl2, 100 µM each of dATP, dGTP, dCTP, dTTP, 0.5 µCi TTP [methyl-3H] (78 Ci/mmol), 5 µg activated calf thymus DNA, 0.2 mg/ml BSA and 0.1% Tween 20. The reaction mixture was incubated at 75 °C for 5 min before enzyme addition. After adding the enzyme, aliquots were removed from the reaction mixture at various time intervals and spotted onto DE-81 filter paper disks (23 mm diameter, Whatman). The disks were air-dried, washed three times in sodium phosphate buffer pH 7.0 and finally washed with 70% ethanol. All washing steps were done for 2 min each. The filter paper disks were then dried and incorporated radioactivity on the filter disks was measured in counts per second (cps) by using Raytest Malisa scintillation counter (Berlin, Germany). One unit of DNA polymerase activity was defined as the amount of the enzyme required to incorporate 10 nmol [methyl 3H] TTP into a polynucleotide fraction (adsorbed on DE-81 filter disc) at 75 °C in 30 min.
A new hydrophobin candidate from Cladosporium macrocarpum with super-hydrophobic surface
Published in Preparative Biochemistry & Biotechnology, 2023
Büşra Albayrak Turgut, Serkan Örtücü
PCR was performed with ITS and D1-D2 regions. For ITS region, ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-GCTGCGTTCTTCATCGATGC-3′) primers, and for D1-D2 region NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) primers were used. ITS primers amplify the DNA region containing a partial sequence of small subunit ribosomal RNA gene, the complete sequence of the internal transcribed spacer 1, 5.8 ribosomal RNA gene, and internal transcribed spacer 2, and partial sequence of large subunit ribosomal RNA gene, and D1-D2 region includes a partial sequence of large subunit ribosomal RNA gene. For PCR reactions, 50 µl PCR reaction including 50 ng of genomic DNA, 37.5 µl of dH2O, 5 µl of 10X reaction buffer, 0.8 µl of 200 µM primers (synthesized by OLIGOMER), dNTP mix (QIAGEN, Germany) containing 10 mM of each dATP, dCTP, dGTP, and dTTP and 2.5 U of Taq DNA polymerase (QIAGEN, Germany) was prepared. As a negative control, dH2O instead of gDNA was used in the same composition. The following PCR conditions were used: one cycle at 95 °C for 2 min, 35 cycles at 95 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR reaction was occurred in a thermal cycler (Sensoquest-Labcycler, Germany). After the PCR reactions were completed, a EcoSpin PCR purification kit (Ecotech Biotechnology, Türkiye) was used to purify the PCR products by following the manufacturer’s instructions.
Augmentation of metal-tolerant bacteria elevates growth and reduces metal toxicity in spinach
Published in Bioremediation Journal, 2021
K. M. Sarim, U. Sahu, M. S. Bhoyar, D. P. Singh, U. B. Singh, A. Sahu, A. Gupta, A. Mandal, J. K. Thakur, M. C. Manna
Total genomic DNA was extracted and purified from bacterial pellets, and collected after growth on nutrient agar, using the CTAB-method (Wilson 2001) with slight modifications. DNA concentration was measured in a UV-Visible spectrophotometer (Shimadazu) and stored at −80 °C until further use. Primers 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492 (5′-AAGGAGGTGATCCAGCCGCA-3′) were used for the amplification of 16S rDNA (Edwards et al. 1989). The DNA amplification was performed in 50 µl reaction volume by incorporating 50 ng of template DNA in the 10X polymerase reaction buffer; 100 µM of dATP, dCTP, dTTP and dGTP (each); forward and reverse gene primers (100 ng each) along with 1.0 U of Taq polymerase. The PCR amplification conditions were as follows: initial denaturation of DNA strand for 5 min at 94 °C, followed by 40 cycles of 40 s at 94 °C, 40 s at 52 °C and 50 s at 72 °C and a last extension for 7 min at 72 °C. The PCRs were carried out in a BioRad MyCyler thermal cycler. Amplified PCR product was visualized by electrophoresis in 1.2% agarose gel containing ethidium bromide (10 mg ml−1) and further documented in Biorad gel documentation system. The amplified 16S rDNA PCR product was further purified with a PCR purification kit (Nucleopore). The nucleotide sequences of amplified product were determined by Applied Biosystems ABI prism (3130xl) sequencer.