Chloroplast DNA and Phylogenetic Relationships
S. K. Dutta in DNA Systematics, 2019
Figure 2A presents a phylogenetic tree, in this case based entirely on chloroplast DNA restriction site mutations,19,20 which depicts the molecular relationships among 21 species and cultivars in the genus Brassica, plus radish, Raphanus sativa.19 The level of restriction fragment variation encountered in this study necessitated a two-tiered approach for analyzing mutations. Certain enzymes, operationally designated Type I enzymes, cut the DNAs relatively infrequently and revealed sufficiently little variation to allow mutational analysis of all restriction fragment changes found in the 22 DNAs. Mutations found with these enzymes divided the DNAs into four major lineages, consisting of DNAs 1, 2, 15 to 17; 13, 14; 18; and 3 to 12, 19 to 22 (Figure 2A). The more complex patterns produced by enzymes operationally defined as belonging to the Type II class allowed further discrimination within each of these four lineages. The evolutionary direction (polarity) of Type II mutations was determined by a hybridization protocol using members of the other three lineages as outgroups.19
Molecular Approaches Towards the Isolation of Pediatric Cancer Predisposition Genes
John T. Kemshead in Pediatric Tumors: Immunological and Molecular Markers, 2020
When total genomic DNA is “cut” with a particular enzyme, a heterogeneous population of characteristic restriction fragments is produced. Each fragment has a fixed length, the restriction fragment length (RFL). Agarose gels can be used to separate these fragments according to size. Using standard techniques, developed by Southern5 and Rigby et. al.,6 radioactive DNA probes can be used to hybridize with, i.e., “recognize”, homologous sequences within the heterogenous population. This homology is manifested as a band (or set of bands)on an autoradiograph at one or more specific locations determined by the size of the fragment being recognized (Figure 1). If there is no sequence variation between individuals in the region of the chromosome recognized by the probe, a consistent band pattern is produced at the same position on the gel. However, any variation in DNA sequence that affects a given restriction site, such that an existing one is removed or a new one is created, will generate variations in the band pattern observed. Since the bands produced are polymorphic, the variation is referred to as a restriction fragment length polymorphism (RFLP). This principle is illustrated in Figure 2. If an individual is heterozygous for the polymorphic variants, then both of the individual homologous chromosomes can be identified (see Figure 2). Differences in DNA sequence at the same locus are allelic and the bands produced on the autoradiograph are conveniently referred to as alleles.
Identifying and Localizing Gene Expression Important for the Actions of Abused Drugs
Edythe D. London in Imaging Drug Action in the Brain, 2017
If a single gene defect caused drug abuse in humans, chromosomal gene mapping techniques could be readily applied to families expressing the defective gene. These techniques utilize genetic markers that include the DNA sites comprising restriction fragment length polymorphisms (RFLPs) (Blum et al., 1990). The degree to which inheritance plays a role in drug abusing behavior in humans is not clear-cut. One recent study of concordance rates for drug abuse among fraternal and identical twins estimates that ca. 20% of the etiology of substance abuse may be genetic (Pickens et al., 1991). Since susceptibility to drug abuse might also be influenced by combinations of several genes, direct genetic mapping using chromosomal markers presents a difficult prospect. Using specific gene markers, however, individual candidate genes may be identified much more rapidly. Interesting recent work has suggested that an A1 allele of the gene encoding a major neurotransmitter receptor of reward pathways, the dopamine D2 receptor, may be present at higher frequency in alcoholic than in nonalcoholic individuals; but this finding has been questioned (Bolos et al., 1990). Our recent examination of this question in drug abusing individuals reveals an allelic association in Caucasian polysubstance abusers. Confidence in this finding increases as this allele is examined in larger populations of individuals with varying degrees of drug use.
The relationship between KRAS LCS6 polymorphism and endometrium cancer
Published in Journal of Obstetrics and Gynaecology, 2020
Feyza Nur İncesu Çintesun, Özlem Seçilmiş Kerimoğlu, Ersin Çintesun, Süleyman Nergiz, Hasan Acar, Çetin Çelik
The Restriction Fragment Length Polymorphism (RFLP) method is a method which allows the examination of DNA profiles formed by separation in the agarose gel electrophoresis system of DNA segments and fragmentation with a restriction endonuclease enzyme specific to genomic DNA. By identifying specific sequences in the DNA of these enzymes, which are found in bacteria, the fragmentation process occurs in the identified region or in another specific sequence outside this region. Genotyping of single nucleotide polymorphisms occurs in two steps with this method. The first step is based on PCR product sequencing or enzyme fragmentation after gel electrophoresis for the genotyping of the polymorphic region by hundreds of thousands of proliferations with PCR of approximately 200 base pairs around the single nucleotide polymorphism (SNP) in the DNA region (Linn and Arber 1968).
Association of Glutathione S-Transferase Polymorphisms with Dietary Composition but Not Anthropometry in Obese as Well as Nonobese Individuals
Published in Journal of the American College of Nutrition, 2018
Barbara Klánová, Filip Zlámal, Aneta Pohořalá, Ondřej Slabý, Hynek Pikhart, Julie Bienertová-Vašků
DNA isolation and genotyping was carried out as described previously by Hezova et al. (14). Genomic DNA was isolated from whole peripheral blood and DNA concentration was measured using a Nanodrop ND-1000, Thermo Fisher Scientific, Waltham, USA. Real-time polymerase chain reaction (PCR) allelic discrimination was carried out using a StepOne Real-Time PCR, Thermo Fisher Scientific, Waltham, USA. PCR-restriction fragment length polymorphism was used for analysis (15). PCR was performed with a total volume of 12.5 μl; thermocycler parameters included an initial 5-minute denaturation step at 94°C, followed by 30 cycles of denaturation, annealing, and extension and a final 7-minute extension and cooling reaction to 4°C. Digestion was performed for 12 hours at 37°C and stopped at 65°C for 20 minutes. Digested products were isolated on 2% agarose gel stained with ethidium bromide. GSTM1 and GSTT1 deletions were genotyped using a duplex PCR with the albumin gene serving as an internal positive control. The total reaction volume was 12.5 μl. PCR conditions were the same as for the amplification of GSTA1. PCR products were isolated on 2% agarose gel stained with ethidium bromide. The wild-type genotype for GSTM1 corresponded to 215 bp, GSTT1 to 480 bp, and the internal control to 380 bp (16,17).
Cytogenetic and molecular genetic methods for chromosomal translocations detection with reference to the KMT2A/MLL gene
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Nikolai Lomov, Elena Zerkalenkova, Svetlana Lebedeva, Vladimir Viushkov, Mikhail A. Rubtsov
The Southern blot [43,44] was the first method to detect changes in genomic DNA sequences detected by restriction fragment length polymorphisms (RFLPs). In this method, total genomic DNA is cleaved with restriction endonucleases. The obtained fragments are then separated in an agarose gel, transferred to a nylon membrane, hybridized with labeled probes, and finally, the bound probes are detected. This method has been used to determine the rearranged genes in many AL-associated recurrent chromosomal translocations (see the analysis of the chimeric KMT2A-AFF1 gene in t(4;11)(q21;q23) [45]). Southern blot has also been utilized for the detection of rearrangements in diagnostic samples as the length of restricted DNA fragments differ in rearranged and wild-type alleles [46].
Related Knowledge Centers
- Agarose Gel Electrophoresis
- Allele
- DNA
- Gel Electrophoresis
- Nucleotide
- Recombinant DNA
- Restriction Enzyme
- Palindromic Sequence
- Sticky & Blunt Ends