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A Survey of Newer Gene Probing Techniques
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
The electrophoretic mobility of single-stranded nucleic acids in nondenaturing polyacrylamide gels is affected not only by size but also by the sequence characteristics, being sensitive to even single-base substitutions. Under nondenaturing conditions the single-stranded DNA conformation is stabilized by interstrand interactions that influence DNA mobility in the gel. Therefore, the conformation characteristics reflected in altered mobility are determined, in turn, by changes in the sequence, justifying the term single-strand conformation polymorphism (SSCP).
Burkholderia
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
Danielle L. Peters, Jaclyn G. McCutcheon, Karlene H. Lynch, Jonathan J. Dennis
Capillary electrophoresis–single-strand conformation polymorphism (CE-SSCP) analysis is a second technique that uses 16S rRNA sequences to identify B. gladioli pv. Cocovenenans.110 In this method, 16S rRNA sequences are amplified using polymerase chain reaction (PCR) primers with fluorescent labels. Capillary gel electrophoresis is performed using an automated DNA sequencer following denaturation of the PCR product. The resulting electropherograms differ based on the conformation of the two labeled single-strand products. Using this protocol, B. gladioli pvs. cocovenenans, gladioli, and alliicola, B. glumae, and B. plantarii all produce similar electropherograms as expected based on their homologous 16S rRNA sequences, and can be differentiated from other gram-negative nonfermenters but not from one another.
Common Inherited Genetic Disorders
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
In the remaining 30% of DMD and BMD cases that exhibit no detectable deletion or duplication, the mutations are assumed to be either microdeletions or point mutations. Several methods have been suggested for the detection of point mutations in the dystrophin gene.71,72 They include single strand conformational polymorphism (SSCP), heteroduplex analysis (HA), protein truncation testing (PTT), chemical mismatch cleavage analysis (CCM), denaturing gradient gel electrophoresis (DGGE), and denaturing high pressure liquid chromatography (DHPLC).
Relationship between genetic polymorphism of MTHFR C677T and lower extremities deep venous thrombosis
Published in Hematology, 2019
Jiasheng Xu, Kexin Li, Weimin Zhou
5, 10- methylenetetrahydrofolate reductase (MTHFR) is a key enzyme with homocysteine metabolism pathway. It catalyzes the reduction of N5, N10- methylenetetrahydrofolate into N5 methylenetetrahydrofolate which is the most important methyl donor in the body with significant physiological functions. Goyette et al. successfully cloned MT HFR gene for the first time in 1994 and located it on chromosome 1p36.3. Then, they used DNA single-strand conformation polymorphism (SSCP) and DNA sequencing technology to find more than ten polymorphisms in the M TH FR gene, among of which, the most common one is C677 T polymorphism. Mutations of MTHFR gene may lead to the imbalance between vascular relaxing factor and vascular contraction factor, resulting in deep vein thrombosis (DVT). The purpose of this study was to investigate the relationship between the C677 T polymorphisms of MTHFR gene and DVT to provide a theoretical reference for the etiology, prevention and treatment of DVT.
The relationship between gene polymorphism of MTRR A66G and lower extremity deep venous thrombosis
Published in Hematology, 2018
Polymorphism refers to there may be more than two genotypes in the same genetic locus in random mating population. For this population, there are differences in the nucleotide sequence of individual genes called genetic polymorphism [21]. This kind of polymorphism can be divided into two categories, the polymorphism of DNA site and DNA length polymorphism [22,23]. In the past, genetic polymorphism analysis mostly used restriction fragment length polymorphism, single strand conformation polymorphism, polymerase chain reaction (PCR), DNA sequencing, PCR oligonucleotide probe hybridization (PCR-SSO) method/sequence-specific oligonucleotide method and other methods [24]. The above-mentioned methods have the disadvantages of high technical requirements, complicated operation, expensive instruments and reagents required, long term typing detection, which is not conducive to popularization in the laboratory [25]. PCR-SSP utilizes the lack of 3'-5' exonuclease activity of Taq enzymes; when the SSPs are not complementary to the 3-terminal base of the template DNA, it will cause the extension of the taq enzyme to be blocked. The genotypes of HLA were analyzed by gel electrophoresis amplification. The specificity of amplification product can be accurate to the difference in one base, and the amplified product can be analyzed by agarose gel electrophoresis, which has the characteristics of high resolution, strong specificity, simple technique, rapidness, easy application and promotion. Therefore, this experiment chose PCR-SSP method for genetic polymorphism detection.
Adaptive immunity in the joint of rheumatoid arthritis
Published in Immunological Medicine, 2022
Despite the longstanding belief that RA is a T cell-mediated autoimmune disease, demonstrating autoreactivity of joint-infiltrating T cells is still a challenging issue. An expansion of TCR clonotype in RA joint has long been observed and is thought to reflect their autoreactivity. Earlier studies examined TCRVβ usage but showed somewhat conflicting results on the clonality of synovial T cells [93,94]. Among those studies, similarly biased TCR Vβ usage by synovial T cells in different joints was reported [95]. Yamamoto has developed an improved method for evaluating TCR clonality, single-strand conformation polymorphism (SSCP) analysis, and demonstrated clonal expansion of T cells, which is shared among different areas of the same joint [96]. Analysis of complementary determining region 3 (CDR3) sequence has later been used to demonstrate the presence of dominant clones in the joint [97]. Interestingly, a study showed higher extent of clonal expansion in ACPA-positive synovium [5]. This might be related with the presence of Tph cells in seropositive RA, as we observed a biased TCR usage by Tph cells (Sakuragi et al., in press). Recent studies utilized non-biased, next-generation sequencing of TCR to investigate T cell expansion in RA joint. Klarenbeek et al. showed highly expanded clones, which are shared between different joints of the same patients [98]. They later showed that the overlap between synovial tissue and SF was limited, indicating these are two separate compartments [99]. However, the phenotype, functions, as well as antigen specificity of the expanded T cell clones identified by these studies are unclear.