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Recombinant DNA Technology and Gene Therapy Using Viruses
Published in Patricia G. Melloy, Viruses and Society, 2023
When one first isolates the gene, it is called gene or DNA cloning (Figure 7.1). The ability to do a cut and paste, where one removes a gene from one source, the “cut” part, and then places it in a vector to be expressed in a new organism, the “paste” part, critically depends on enzymes known as restriction enzymes (Pray 2008). These are enzymes, identified first in bacteria, that reliably cut the DNA at a particular short nucleotide sequence. Restriction enzyme sites can be mapped out in any gene sequence and used to cut the DNA strategically (Kurreck and Stein 2016; Alberts et al. 2019; Minkoff and Baker 2004; Mukherjee 2016).
PCR-RFLP
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
Table 13 lists 16 groups defined by 9 restriction enzymes (as described above). These give 120 heterozygous combinations. Ten combinations of them (DRB1*110(1 or 3 = 4)/1201 and 1102/1202, DRB1*110 (1 or 3 = 4)/130(l or 2) and 1102/1305, DRB1*1201/1305 and 1202/130 (1 or 2), DRB1*110(1 or 3 = 4)/1405 and 1305/140(1 or 4), DRB1*110(1 or 3 = 4)/0803 and 1102/0801, DRB1*1201/0801 and 1202/0803, DRB1*130 (1 or 2)/0801,130(3 or 4)/080(2 or 4) and 1305/0803, DRB1*1305/1403 and 1402/080(2 or 4), DRB1*1102/1405 and 130(1 or 2)/140(l or 4), and DRB1*130(3 or 4)/01403 and 1402/0803) which cannot be distinguished from each other by the cleavage patterns with the nine restriction enzymes, can be discriminated by investigation of the RFLP band patterns obtained after treatment with C/rl3I + Fokl, Fokl + SfaNl, Avail + SfaNl, Haell + Fokl, Fokl + Apal, Sacll + Avail double digestion, or Rsal single digestion (Table 14).
Cancer: A Genetic Disease
Published in Jeremy R. Jass, Understanding Pathology, 2020
The variability between maternal and paternal junk DNA means that sites where DNA can be ‘cut’ with DNA cutting enzymes will vary. These enzymes are called restriction enzymes because they cut DNA only at restricted sites comprising specific base sequences. A single strand of DNA with a radioactive label will bind to a complementary sequence in both the maternal and paternal DNA. The DNA will have been extracted from millions of cells and so there will be millions of copies of the sequence of interest. The restriction enzyme then cuts the DNA into short lengths. However, the cutting sites or maternal and paternal DNA will differ and so the lengths of DNA bound to the probe will also differ. We can now imagine multiple bits of DNA, but of only two lengths only. The shorter lengths will travel faster than the longer ones when they are suspended in a gel through which a current is passed (electrophoresis). The difference is detected as the different movement of bands of DNA, still carrying the radioactive probe. In order to see the signal from the radioactive probe, the DNA must be ‘blotted’ from the gel onto paper. Since the technique was invented by Southern (1975) it is called a Southern blot. (There are also Northern blots for RNA and Western blots for protein—a sort of biochemical joke.)
The γH2AX DSB marker may not be a suitable biodosimeter to measure the biological MRT valley dose
Published in International Journal of Radiation Biology, 2021
Jessica A. Ventura, Jacqueline F. Donoghue, Cameron J. Nowell, Leonie M. Cann, Liam R. J. Day, Lloyd M. L. Smyth, Helen B. Forrester, Peter A. W. Rogers, Jeffrey C. Crosbie
qPCR-based methods can estimate the absolute DSB frequency at specific loci (Van Houten et al. 2000; Chailleux et al. 2014; Furda et al. 2014) and can provide information on the in vivo chromatin structure (Pfeifer et al. 1999; Liu et al. 2003). A promising qPCR-based technique called quantitative DSB sequencing (qDSB-Seq) was used to measure DSB frequencies per cell and their precise genomic coordinates, employing the use of site-specific endonuclease and a DSB labeling method to quantify DSBs (Zhu et al. 2019). Zhu et al. used qDSB-Seq in yeast where DSBs induced by radiomimetric drugs were quantified and the location of these DSBs was characterized. This technique can be used in both in vitro and in vivo settings provided the organism-specific restriction enzymes (Aymard et al. 2014) and a DNA labeling method are available.
Increased risk of polycystic ovary syndrome (PCOS) associated with CC genotype of miR-146a gene variation
Published in Gynecological Endocrinology, 2018
Seyed Omar Ebrahimi, Somayeh Reiisi, Shahrbanou Parchami Barjui
For genotyping SNP of rs2910164 in mir-146a used PCR restriction fragment length polymorphism (RFLP). The corresponding restriction enzymes and appropriate primers used to amplify the target DNA by polymerase chain reaction (PCR) are showed in Table 1. The forward primer for genotyping rs2910164 was changed to construct a restriction site for SacI (Fermentase) enzyme. Polymerase chain reaction (PCR) containing: 0.3 μL each of the primers (10 pmol), 100 ng of genomic DNA (1 μL), 2.5 μL PCR buffer (10X), 1.5 μL MgCl2 (50 mM), 0.5 μL of dNTP mix, and 0.5 U of Taq DNA polymerase 5 U/μL (0.1 μL) was performed in a final reaction volume of 25 μL. Amplification was carried out for 35 cycles comprising denaturation at 94 °C and annealing at 61 °C temperature and synthesis of the fragments (extension) at 72 °C, all three for 40 s, finally terminal extension at 72 °C for 5 min. The product was electrophoresed in polyacryamid gel (PAGE) 8% at 200 voltage for 1 h and bands were visualized by silver staining. Then, digestion of PCR products was performed with 1 U of SacI at 37 °C overnight and digested product was separated by 10% polyacrylamide gel electrophoresis. The lengths of fragments resulting from digestion were 147 bp for CC genotype 120 bp and 27 bp for GG genotype and 147 bp, 120 bp and 27 bp for GC genotype.
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.