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Roles of Nucleotide Sequence Analysis in Human Genetics and Genomics
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
S. S. D. Mohammed, I. Abraham, D. Enoma, L. E. Okoror
Restriction analysis is the application of specific restriction enzymes to analyze the DNA molecules (Kessler and Vicentiu, 1990). Fragments of the DNA molecules that have been generated as a result of the cleaving by the restriction enzyme are generally separated by using the technique of gel electrophoresis (Churchill, 2012). The pattern of restriction fragments produced can be used for various purposes: to detect fragments of a specific gene using a nucleic acid probe, to detect the presence of mutations, to compare DNA from different individual, and to construct physical maps, called restriction maps, of DNA molecules that indicate the position of restriction sites in the molecules. Restriction enzymes can scan an entire length of DNA in search for particular sequence of bases which they can recognize (Kessler and Vicentiu, 1990; Churchill, 2012). The site for the recognition of the sequence is normally between 4 and 6 base pairs in size. Immediately after the identification has been done, the restriction enzyme attaches to precise location of the sequence and excise it. The repeated occurrence of this process throughout the entire length of the double-stranded DNA molecule eventually leads to the production of DNA fragments (Churchill, 2012).
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.
L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression
Published in Preparative Biochemistry and Biotechnology, 2019
Brian Effer, Guilherme Meira Lima, Sindy Cabarca, Adalberto Pessoa, Jorge G. Farías, Gisele Monteiro
The Glycoswitch® SuperMan5 (his−) strain from P. pastoris and expression vector pJAG-s1 containing α mating factor (αMF) from S. cerevisiae (used for extracellular secretion of recombinant proteins) was acquired from Biogrammatics Inc. The synthetic gene asnB (UniprotKB – P06608) (sequence in Supplemental data 1) was purchased from GenScript, and further used as a template to be amplified in two different ways: (1) adding the His-tag in the C-terminus (forward 5’ggcGGTCTCgGGCTGAACGCTGGTTTAAGTCC3’ and reverse5’ggcGGTCTCcATTAATGATGGTGGTGATGGTGTGGACCCTGGAACAAAACTTCAAGGTAGGTGTGGAAGTATTCTTGG3’), and (2) excluding the His-tag in the reverse primer (5’ggcGGTCTCcATTAGTAGGTGTGGAAGTATTCTTGG3’). The underlined sequences in the primers are the restriction sites of the BsaI enzyme (Biolabs Inc.). PCR was performed using the following conditions: denaturation at 95 °C/30 s, annealing at 69 and 65 °C/30 s for the insert including His-tag and excluding His-tag, respectively; and extension at 72 °C/7 min.