Point mutations detection

Zhu DB, Xing D, Shen XY, Liu JF, Chen Q. High sensitive approach for point mutation detection based on electrochemiluminescence. Biosens Bioelectron 2004 20 448-53. 

The various types of point mutations detected via mtDNA sequencing can also be targeted in nuclear DNA. SNPs are particularly useful in ethnicity testing and in the analysis of highly degraded or compromised samples [57,75,76]. In the human genome SNPs occur every 1000-2000 bp, thereby... 

Piezoelectric Based on nucleic acids To point mutation detection in... 

A. Okumura,Y. Sato, M. Kyo, H. Kawaguchi, Point mutation detection with the sandwich method employing hydrogel nanosptieres by the surface plasmon resonance imaging technique, Anai. Biochem. 339 (2005) 328-337. 

GaUuzzi, J. R. Ordovas, J. M. Genotyping method for point mutation detection in the intestinal fatty... 

Myal Y., Blanchard A., Watson P., CoRRiN M., Shiu R., Iwasiow B. Detection of genetic point mutations by peptide nucleic acid-mediated polymerase chain reaction clamping using paraffin-embedded specimens. Anal. Biochem. 2000 285 169-172. 

In 1995, Nagata et al. [16] identified a point mutation consisting of a substitution of valine for aspartic acid in the catalytic domain of c-kit (D816V) in the peripheral blood of patients with mastocytosis and predominately myelodysplastic features. Subsequently, the same mutation was identified in adult patients with different forms of mastocytosis in tissues where mast cells are abundant, such as bone marrow, skin and spleen [17]. It is now believed that more than 90% of adults with mastocytosis have the D816V mutation, if bone marrow mononuclear cells are examined [17]. In a subset of patients, primarily those with more severe disease, the clone expands sufficiently to be detected in peripheral blood [16]. 

Detection of a codon 816 c-kit point mutation in blood, bone marrow, or lesional tissue Mast cells in the bone marrow, blood, or other lesional tissue expressing CD25 or CD2... 

Sotlar K, Escribano L, Landt O, et al One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes. Am J Pathol 2003 162 737-746. 

A similar analysis could be made for a number of other diseases. Point mutations are usually defined by sequencing the gene in question, though occasionally, if the mutation destroys or creates a restriction enzyme site, the technique of restriction fragment analysis can be used to pinpoint the lesion. Deletions or insertions of DNA larger than 50 bp can often be detected by the Southern blotting procedure. 

Figure 49-10. Simplified scheme of the causation of malignant hyperthermia (MIM 145600). At least 17 different point mutations have been detected in the RYRl gene, some of which are associated with central core disease (MIM 117000). It is estimated that at least 50% of families with members who have malignant hyperthermia are linked to the RYRl gene. Some individuals with mutations in the gene encoding DHPR have also been detected it is possible that mutations in other genes for proteins involved in certain aspects of muscle metabolism will also be found.

Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874-879. 

Protein-protein interactions between heterodimeric protein pairs that form only transient interactions can be detected, y-secre-tase is presenilin-1 (PS1) dependent [51-53]. PS1 is a 467-amino acid, 9-transmembrane domain protein. Over 100 documented single point mutations are known to cause autosomal-dominant familial AD (FAD) [54], in which the ratio of the more fibrilogenic variety of A ft (A/142) to the less fibrilogenic variety (A/140) is increased. Chinese hamster ovary (CHO) cells were stably transfected with human APP and either wild type or mutant PS1 [4, 55]. 

V. Chan, Y. Chong, L. Cheung, J. Vielmetter, and D.H. Farkas, Bioelectronic detection of point mutations using discrimination of the H63D polymorphism of the Hfe gene as a model. Mol. Diagn. 5, 321-328 (2000). 

Point mutations can occur when one base is substituted for another (base substitution). Substitution of another purine for a purine base or of another pyrimidine for pyrimidine is called a transition, while substitutions of purine for pyrimidine or pyrimidine for purine are called transversions. Both types of base substitution have been identified within mutated genes. These changes lead to a codon change which can cause the wrong amino acid to be inserted into the relevant polypeptide and are known as mis-sense mutations. Such polypeptides may have dramatically altered properties if the new amino acid is close to the active center of an enzyme or affects the three-dimensional makeup of an enzyme or a structural protein. These changes, in turn, can lead to change or reduction in function, which can be detected as a change in phenotype of the affected cells. 

Automated programmable instruments that can carry out the repeated thermal cycles necessary for PCR and that can accommodate multiple samples simultaneously are now widely available. The procedure is usually performed with thermostable DNA polymerases. PCR is widely used to facilitate detection of minute amounts of viral DNA. The technique can also be used to detect specific point mutations, provided the approximate site of mutation is known. One limiting feature of this approach arises from the fact that the bacterial polymerases frequently make errors when synthesizing new strands and so can introduce mutations that are not present in the original sample. 

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