Detection of Point Mutations

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. 

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). 

Goldstein DB (2001) Islands of linkage disequilibrium. Nat Genet 29 217-222 Haliassos A, Chomel JC, Tesson L, Baudis M, Kruh J, Kaplan JC, Kitzis A (1989) Modification of enzymatically amplified DNA for the detection of point mutations. Nucleic Acids Res 17 3606... 

Kumar R, Dunn LL (1989) Designed diagnostic restriction fragment length polymorphisms for the detection of point mutations in ras oncogenes. Oncogene Res 1 235-241... 

G. Wang, and W. Knoll, Detection of Point Mutations and Insertion Mutations in DNA Using a Quartz Crystal Microbalance and MutS, a Mismatch Binding Protein, Anal. Clwm. 2004, 76. 489. 

The MLA is used for the detection of point mutations, structural aberrations and aneugenicity. The principle of the assay is that cells deficient in thymidine kinase (TK) due to the tk+/" or tk/ mutation are resistant to the cytotoxic effects of the pyrimidine analogue trifluoro-thymidine (TFT). TK proficient cells are sensitive to TFT. This influences the cellular metabolism and leads finally to an inhibition of further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain TK, are not. The major advantage of the assay is its ability to detect a broad range of mutagenic events represented by optimal detection of both large and small colonies. 

Kuypers AWHM, Willems PMW, van der Schans MJ, Linssen PCM, Wessels HMC, de Bruijn CHMM, Everaerts FM, Mensink EJBM (1993) Detection of point mutations in DNA using capillary electrophoresis in a polymer network. J Chromatogr 621 149-156. 

Condie, A., Ecles, R., Bprresen, A.-L., Coles, C., Cooper, C., and Prosser, J. (1993) Detection of point mutations in the p53 gene comparison of single-strand conformation polymorphism, constant denaturant gel electrophoresis, and hydroxylamine and osmium tetroxide techniques. Human Mutat. 2, 58-66. 

Fig. 5. Strategy for color complementation assay detection of point mutations. A cytidine-to-thymidine mutation is illustrated in the example. Two allele-specific primers corresponding to this region are labeled with red dye (corresponding to the wild-type allele) or green dye (mutant allele). The primer amplifying the opposite strand is unlabeled. After PCR and removal of unincorporated primers, the amplified products for normal, heterozygous, and homozygous DNA are ted, yellow, and green, respectively. Reprinted with the permission of Chehab and Kan (C2) and the Proc. Natl. Acad. ScL (USA.).

Once the PCR procedure is complete, the products of amplification should be visualized for analysis and interpretation. Agarose gel electrophoresis and ethidium bromide staining can achieve this however, they cannot separate amplification products that differ in size by only a few nucleotides. Polyacrylamide gel electrophoresis or capillary gel electrophoresis can achieve a finer separation (Fig 2.3A,B). PCR amplification followed by gel electrophoresis is frequently used for detection of small deletions or insertions, microsatellite instability, and LOH. For detection of point mutations, the PCR products should be interrogated by other molecular techniques. 

A variety of molecular techniques are available to detect point mutations at a specific hot spot (e.g., the KRAS mutation at codons 12 and 13 or the BRAF mutation at codon 600). These methods include realtime PCR amplification and post-PCR melting curve analysis, allele-specific PCR, direct DNA sequencing, pyrosequencing, and PCR-RFLP. o,31-37 q methods demonstrate reliable detection of point mutations, such as the KRAS mutation in colorectal cancer or the BRAF mutation in thyroid cancer. 

Occhialini, A., Urdaci, M Doucet-Populaire, F., Bdbdar, C. M., Lamouliatte, H., and Megraud, F. (1997). Macrolide resistance in Helicobacter pylori. Rapid detection of point mutations and assays of macrolide binding to ribosomes. Antimicrob. Agents Chemother 41, 2724-2728. 

Prediction of carcinogenic potential of pesticides was taken a step further by Dr. S. Nesnow, who described EPA s "phased approach" for the application of short-term tests to these compounds. Phase 1 involved detection of point mutations, DNA damage and chromosomal effects in appropriate microorganisms. 

General method for multiplex detection of point mutations (combination of ASD and DNA-sequencing)... 

Kerman, K., Ozkan, D., Kara, P., Erdem, A., Meric, B., Nielsen, P. E., Ozsoz, M. (2003). Label-free bioelectronic detection of point mutation by using peptide nucleic acid probes. Electroanalysis 15, 667-670. 

Palecek, E., Masarik, M., Kizek, R., Kuhlmeier, D., Hassmann, I., Schulein, I. (2004). Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutations in DNA. Anal Chem 76, 5930-5936. 

Single-strand conformation polymorphism was originally described by Orita et al. [94] for the detection of point mutations. The idea of SSCP is to perform electrophoresis on a non-denaturing polyacrylamide gel using small PCR products after denaturation of the DNA. As the PCR product moves through the gel, it will regain a secondary structure that is sequence dependent (similar to RNA secondary structure). The mobility of the single-stranded PCR products depends on their secondary structure. Therefore, PCR products that possess sequence differences (mutations, insertions or deletions) will have different mobilities. 

Figure 5 Typical record of an SSCP analysis. Detection of point mutation (substitution of thymine to cytosine) causing phenylketonuria in a heterozygote. Four completely dissociated DNA strands with different sequences (two couples of complementary strands with and without the point mutation) are resolved as four conformers in a native separation environment. The large peak represents a portion of undissociated double-stranded DNA.

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