Genetics and deoxyribonucleic acid-based technology in clinical biochemistry
Martin Andrew Crook in Clinical Biochemistry & Metabolic Medicine, 2013
DNA can be cut into smaller fragments by restriction endonucleases derived from bacteria. These are named by abbreviating the names of the originating bacteria. These endonucleases often work in a palindromic way, that is, the sequence of bases on one DNA strand is repeated in reverse on the other. A restriction enzyme/restriction endonuclease is an enzyme that recognizes a specific nucleotide sequence (restriction site) and cuts (restricts) the nucleic acid at that particular site. An example of a restriction endonuclease is EcoRI made by Escherichia coli, which is specific for the following sequence: 5′ … GAATTC … 3′3′ … CTTAAG … 5′.
Methods in Molecular Biology
Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman in Molecular Imaging in Oncology, 2008
This technique was developed by the British biologist Edwin Southern (6) in 1975. The method utilizes a radioactive or chemiluminescent probe to visualize genes and others DNA fragments that have been previously separated by electrophoresis (Fig. 4). First, restriction endonucleases are used to cleave high–molecular weight DNA into smaller fragments. After the digestion, these fragments are separated by agarose gel electrophoresis and then transferred onto a nitrocellulose or nylon membrane. The DNA is denatured either prior to or during the transfer using alkaline solution to produce single-stranded DNA. After the complete transfer, the DNA is immobilized on the membrane by high temperature drying or ultraviolet irradiation. This process creates an exact replicate of the gel on the nylon or nitrocellulose membrane. The membrane is now ready to be hybridized or annealed with a DNA or RNA probe containing the sequence of interest. This hybridization is based on the complementary nucleotide sequences between the probe and the DNA molecule on the membrane. Radioactive or chemiluminescent probes are visualized with autoradiography or color development of the membrane, respectively.
Repeated DNA Sequences and Polyploidy in Cereal Crops
S. K. Dutta in DNA Systematics, 2019
When repeated sequences do not contain the sites for a restriction endonuclease, they are not cut and remain in high molecular form. After digestion of the total DNA, such undigested “spared” sequences are found at the top of the gel and have been referred to as “relic” DNA. Repeated digestion of DNA with restriction enzymes followed by steep, linear sucrose gradient centrifugation provides a method for enrichment of the spared, repeated sequences. Various restriction endonuclease spared fractions of rye were purified by velocity sedimentation. Each of the Hind III, Eco RI, Bam HI, and Bgl II digests of Secale cereale and S. silvestre, DNA showed a higher proportion of “relic” DNA in S. cereale. “Hind III relic” in the two species were 5 and 2.3% of the total DNA of S. cereale and S. silvestre respectively.
A Rapid, Affordable and Feasible Method for Detection of the HBG1: g.-225_-222delAGCA Polymorphism
Published in Hemoglobin, 2018
Restriction endonuclease digestion represents a simple and inexpensive tool to detect single nucleotide polymorphisms, but unfortunately, most of them do not span DNA sequences recognized by restriction enzymes. The 4 bp deletion from –225 to –222 in the Aγ-globin gene promoter destroys a Tru1I (MseI) recognition site (TTAA). The undigested PCR product can be easily separated by agarose gel electrophoresis from other shorter cleaved products, making a distinction between homozygous, heterozygous and wild type-state for this deletion. In conclusion, this is a simple, rapid and inexpensive method, based upon the principles of DNA fingerprinting, as a practical alternative to more advanced and expensive molecular techniques to diagnose and identify the 4 bp deletion in the Aγ-globin gene promoter.
DPYD and TYMS polymorphisms as predictors of 5 fluorouracil toxicity in colorectal cancer patients
Published in Journal of Chemotherapy, 2023
Yassine Khalij, Imtinen Belaid, Sana Chouchane, Dorra Amor, Asma Omezzine, Nabila Ben rejeb, Slim ben Ahmed, Ali Bouslama
The study of genetic polymorphisms focused on the DPYD and TYMS genes. We carried out DNA extraction from whole blood leukocytes taken from EDTA using the salting-out method and genotyping by direct PCR for TYMS 5′UTR VNTR and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method, using specific restriction enzymes (Table A.1). We used the following PCR protocols for all 8 SNPs: first denaturation, 5 min; at 95 °C, denaturation 45 s; at 95 °C, hybridization 45 s (see Table A.1 for temperature), elongation 1 min; at 72 °C, final elongation 7 min; and at 72 °C for 32 cycles. We used DreamTaq Green DNA Polymerase (5 U/µL, Thermofisher; EP0712) following the recommended PCR protocol. The digestion reactions with restriction endonuclease contained 1X buffer, 5 U of RE (restriction enzyme), and 3 µl of PCR-amplified DNA. We incubated the restriction endonuclease digestions at the temperature recommended by the manufacturer (Table A.1).
Biological challenges of phage therapy and proposed solutions: a literature review
Published in Expert Review of Anti-infective Therapy, 2019
Katherine M Caflisch, Gina A Suh, Robin Patel
The most ubiquitous mechanism of defense among bacteria and archaea, RM systems detect and cleave foreign DNA, including phage double-stranded DNA, based on nucleotide methylation of host DNA [109]. (RM systems are considered not to degrade single-stranded DNA or RNA phage.) Harbored by more than 90% of prokaryotes according to some estimates, RM systems play a role in prokaryotic homeostasis analogous to the innate immune system of higher-order organisms [109]. Restriction-modification systems are comprised of two functional subunits – a restriction endonuclease which cleaves (degrades) un-methylated DNA, and a related methyltransferase which methylates host DNA [109]. Such epigenetic modification forms the basis for recognizing and sequestering foreign genomic material [109,110] and is phylogenetically conserved, with several hypotheses attempting to explain why this might be the case [109]. Korona et al. predicts that RM systems stave off global infection of the bacterial community upon phage introduction to allow for the expansion of genetically diverse bacterial sub-populations [111].
Related Knowledge Centers
- Archaea
- Bacteria
- DNA
- Endonuclease
- Enzyme
- Restriction Site
- Virus
- Substrate
- Polymer Backbone
- Nucleic Acid Double Helix