Cancer: A Genetic Disease
Jeremy R. Jass in 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.)
PCR-RFLP
M. Kam, Jeffrey L. Bidwell in 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).
rDNA: Evolution Over a Billion Years
S. K. Dutta in DNA Systematics, 2019
Several types of sequence differences in addition to fragment length variation such as that seen in the spacer also occur in the rDNA locus at the population level. The first such difference is restriction enzyme site polymorphisms. In man, two restriction enzymes (Hind III and EcoRI) vary in terms of the presence or absence of additional sites from one repeat to another. The Hind III polymorphism occurs as a result of the presence or absence of an additional enzyme site in the 28S gene,259,262 and this polymorphism is old since it is widespread in other primate species.262 Krystal et al.235 determined the frequency of this polymorphism to be 30% for one Hind III site and 70% for two Hind III sites. The EcoRI site polymorphism has been mapped to the 5′ end of the spacer259 with an extra EcoRI site being present 80% of the time.235,258 This polymorphism also occurs in chimpanzee, pygmy chimpanzee, and gorilla.262 The second type of sequence variation is within individual variation resulting from sequence changes between subrepeats in the spacer. Unlike Xenopus, which has no sequence divergence between spacer subrepeat units, the mouse has 13% sequence divergence among the 13 copies of the 135 bp repeating unit.209 Unfortunately, data of this kind for man and Drosophila are not available.
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.
Association of cytotoxic T-lymphocyte antigen 4 gene with immune thrombocytopenia in Chinese Han children
Published in Hematology, 2019
Liqiong Yao, Bei Liu, Li Jiang, Lanxia Zhou, Xiaoju Liu
A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) detection method was used to genotype rs11571315 and rs3087243. The primers for PCR and restriction enzymes are listed in Table 2. The 80 μl PCR mixture consisting of 50–150 ng of genomic DNA, 10 pM of each primer and Premix Taq (Takara, Dalian, China) was processed under 94°C for 5 min, 35 cycles including 94°C for 30 s, annealing temperature for each SNP for 30 s and 72°C for 1 min; and then 72°C for 10 min. Cleavage was performed with restriction enzymes (New England BioLabs, Ipswich, MA, U.S.A.) according to the corresponding protocols. Digested fragments were separated on 2% agarose gels. At the end of electrophoresis, the bands were visualized by ethidium bromide staining and photographed under an ultraviolet transilluminator. To confirm the genotyping results, PCR products with special band patterns were purified for the Sanger sequencing.
Telomeres as a molecular marker of male infertility
Published in Human Fertility, 2019
Ewa Boniewska-Bernacka, Anna Pańczyszyn, Natalia Cybulska
In the Southern blot method genomic DNA is subjected to restriction endonuclease digestion, most often HinfI/RsaI, at sites that do not contain telomere sequences. The digested DNA is electrophoretically separated in agarose gel (Aviv et al., 2011), transferred to a nylon membrane and then hybridized to specific probes (Law & Lau, 2001). This method is used to determine the average length of telomeres; however, it is not suitable for analysis of short telomere sequences. The Southern blot method does not require specialized equipment; however, it requires time and experience to analyze the results. The disadvantages of this method include possibility of erroneous determination, which may be due to the occurrence of sequence-changing polymorphisms at restriction enzyme sites. This method is considered to be a reference method for validating newly introduced techniques (Kimura et al., 2010; Zong, Wang, Chen, & Cui, 2014).
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