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Recombinant DNA Technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
We know that restriction enzyme is critical for rDNA technology, and it has been shown that restriction enzyme or restriction endonuclease cuts double-stranded or single-stranded DNA at specific recognition nucleotide sequences, known as restriction sites. In fact, without restriction enzymes, it is not possible to make rDNA products. Restriction enzymes, which are found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction. Host DNA is then methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Collectively, these two processes form the restriction modification system. To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e., each strand) of the DNA double helix.
Recombinant DNA technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single-stranded DNA at specific recognition nucleotide sequences, known as restriction sites. These enzymes are very critical components of rDNA technology. In fact, without restriction enzymes, it is not possible to make rDNA products. Restriction enzymes, which are found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction. Host DNA is then methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Collectively, these two processes form the restriction modification system. To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e., each strand) of the DNA double helix.
Partial Digest Problem
Published in Adwitiya Sinha, Megha Rathi, Smart Healthcare Systems, 2019
Urvi Agarwal, Sanchi Prakash, Harshit Agarwal, Prantik Biswas, Suma Dawn, Aparajita Nanda
A DNA molecule’s composition includes four nucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G). Extensive research on the genome through many years has given major contributions. For example, the complete DNA sequence of the human genome is available as a free resource (Pääbo, 2001). Even after such developments, studying individual DNA molecules is an impractical task, and thus there exist several biological techniques that supply the information about restriction sites. Partial Digest, Double Digest (Sur-Kolay et al., 2009; Ganjtabesh et al., 2012), and Shotgun Sequencing (Venter et al., 1998) are some unique examples of such technique. Partial Digest itself has many classes: Simplified Partial Digest (Blazewicz et al., 2007), Labeled Partial Digest (Pandurangan and Ramesh, 2002), and Probed Partial Digest (Karp and Newberg, 1995; Newberg and Naor, 1993) to name a few. Many more problems include various kinds of difference sets. A detailed survey was given by Hall (1956). Our focus is on the Partial Digest technique. A restriction enzyme, when added to a DNA solution, cuts the DNA at specific positions. This results in DNA fragments of all possible distances between restriction sites. Obtaining the original set X containing the restriction sites requires considerable brainstorming, which makes the PDP an engaging problem, and no doubt, it has gained worldwide attention. An analogy to PDP exists in the sphere of computer science, known as the turnpike problem (Dakic, 2000), which is also an extensively researched topic. Another similar problem is the beltway problem that considers these locations as placed in a loop (Fomin, 2018). The inverse problem of Partial Digest is often referred as Chord’s Problem (Daurat et al., 2005).
Efficient production of endotoxin depleted bioactive α-hemolysin of uropathogenic Escherichia coli
Published in Preparative Biochemistry and Biotechnology, 2019
Vivek Verma, Surbhi Gupta, Parveen Kumar, Ankita Rawat, Rakesh Singh Dhanda, Manisha Yadav
Vector pET-28 a (+) (Cat# 69864, Novagen, Merck, India) and pTrcHis B (Kind gift from Dr. LR Singh, ACBR, DU) were used for cloning. Restriction enzymes BamHI (Cat# R0136), XhoI (Cat# R0146), EcoRI (Cat# R3101), and HindIII (Cat# R0104) were used for restriction digestions (NEB, Massachusetts, USA). Recombinant shrimp alkaline phosphatase (rSAP) (Cat# M0371, NEB, Massachusetts, USA) was used for dephosphorylation of free ends of the plasmid. For PCR amplification, Phusion DNA polymerase (Cat# F530, NEB, ThermoFisherScientific, USA) and Taq DNA polymerase (Cat# M3005, Promega, Wisconsin, USA) were used. Clonal selection was done by using Kanamycin (Cat# K-120-5, Gold Biotechnology, Missouri, USA) and Ampicillin (Cat# A-301-5, Gold Biotechnology, Missouri, USA). EZ-10 Spin Column DNA Gel Extraction Kit (Cat# BS353, Biobasic, New York, USA) was used for agarose gel extraction of DNA. T4 DNA ligase (Cat# M0202, NEB, ThermoFisherScientific, USA) was used for ligation protocol. Amicon Ultra-0.5 ml centrifugal filters (Cat# UFC505008, Merck, India) were used for desalting and protein concentration. Plasmid DNA Purification Kit (Cat# 17093, Intron Biotechnology, South Korea) was used for plasmid isolation. Anti-His antibody (Cat# sc-803, Santa Cruz, California, USA) was used for immunoblotting to detect overexpression.