SNPs, detection

A scanner with two lasers for Cy3 and Cy5 labeling is fairly good enough for most of the microarray experiments. However, multiple lasers are necessary for simultaneous detection of all four nucleotide polymorphisms in chip-based SNPs detection. Besides, an extra third flurophore attached to a sequence that specifically binds to a linker region of the DNA spots could be used for spotting quality control. 

Gaylord BS, Massie MR, Feinstein SC, Bazan GC (2005) SNP detection using peptide nucleic acid probes and conjugated polymers applications in neurodegenerative disease identification. Proc Natl Acad Sci USA 102 34—39... 

Duan X, Li Z, He F, Wang S (2007) A sensitive and homogeneous SNP detection using cationic conjugated polymers. J Am Chem Soc 129 4154-4155... 

Figure 2.9 SNP detection using Orchid s SNPstream. (From Bell, J. et al.. Biotechniques, 32, S70-S77, 2002. With permission.)...

Schmalzing, D., Belenky, A., Novotny, M.A., Koutny, L., Salas-Solano, O., El-Difrawy, S., Adourian, A., Matsudaira, P., Ehrlich, D., Microchip electrophoresis a method for high-speed SNP detection. Nucleic Acids Res. 2000, 28, e43. 

Rule-based approaches are predicated on the analysis of expressions that describe mutations in text the rule set developed is based on patterns that have been analyzed by human experts. Learning of patterns is the basis for machine learning approaches for SNP detection. Similar to the human expert, the machine program makes use of features (attributes) that are used with different frequency and in different combinations when information on mutations is communicated in natural language.21... 

Solid-phase hybridization Multiple probe DNA immobilization SNPs detection... 

The one-base extension method [73], rolling circle amplification (RCA) method [74]and Taqman PCR method [75], have been applied to SNP detection. Also a DNA chip which anchored oligonucleotide on the substrate has been developed as a simple hybridization detection tool [76-82], For SNP detection, it is not easy to determine the difference of one base mismatch with this hybridization detection method due to unstable hybridization between probe and SNP and nonspecific adsorption. 

Another method for nonfluorescent detection of DNA involves the noncovalent binding of an inexpensive cyanine dye to PNA-DNA duplexes (27). The dye assembles into a helical aggregate in the presence of PNA-DNA, which results in a vivid blue-purple color change. This phenomenon has been used in SNP detection assays. 

An important feature in the cost per assay is the capability of easy multiplexing. Multiplexed assays are conducted together in one tube (or one well of a micro plate) and combined for example, assays are combined for different SNPs to save time and reagents. With fluorescent detection, the level of multiplexing is somewhat coupled to the amount of available dyes - which is in most applications only four. For MALDI-based SNP detection, a multiplex level of up to 12 assays has already been described [60]. 

One specialty in dehybridization detection is that of dHPLC (denaturing high-performance liquid chromatography). Hybridization products differ in melting temperature and mobility on a specific support if a mismatch is introduced by a SNP. In the case of a SNP, a heteroduplex is formed in the analyzed sequence, whereas in the case of identical sequences (no SNP present), a perfect homoduplex is formed. These events can be discriminated by dHPLC at a low to medium throughput for SNP discovery or SNP detection. 

A different experimental approach to SNP detection combines mass spectrometric detection with enzymatic extension of primers hybridized to immobilized DNA taiget aiiays. Tlie advantage of this combination is high specificity and high accmacy of allele identification. 

SNP detection kit (AcycloPrime), including lOX reaction buffer, AcycloPol enzyme (PerkinElmer, Boston, MA). 

---