Fluorescence polarization

When a fluorophore is excited by plane-polarized light, the fluorescence emitted by the dye is also polarized. This phenomenon is termed fluorescence polarization (FP). Complete FP occurs only when the dye molecule is stationary. Therefore the degree of observed FP is dependent on how fast a molecule tumbles in solution, and this is in turn dependent on the volume of the molecule, which is related to its molecular mass. Therefore changes in molecular mass (e.g., caused by primer extension, probe hydrolysis, or invasive cleavage) can be detected by changes in FP as long as all other conditions (temperature, viscosity, etc.) remain constant. FP is used as the detection method in the SnaPshot (Applied Biosciences) and Acycloprime (Perkin Elmer) commercial genotyping systems.

Mass Spectrometry

Genotyping assays involving mass spectrometry are distinct from those discussed above in that signal detection is direct, based on differing molecular weights of small DNA fragments rather than the behavior of a label.[14] The analysis of DNA by mass spectrometry requires soft ionization (i.e., without fragmentation) and is usually achieved by matrix-assisted laser desorption/ ionization time of flight (MALDI-TOF) analysis. The MALDI procedure involves mixing the allele-specific products of the discrimination assay with a matrix compound on a metal plate. The mixture is then heated with a short laser pulse, causing it to expand into the gas phase where ionization is achieved by applying a strong potential difference. Ions are accelerated toward the detector and the time of flight (the time taken to reach the detector) is measured, allowing the mass/charge ratio to be calculated.

High-resolution mass spectrophotometers can accurately discriminate between alternative alleles in DNA fragments of 3-20 nucleotides in length. The smallest difference in mass between alternative allelic products is about 9 Da, representing an adenosine/thymidine polymorphism. Advantages of MS-based methods include its accuracy (as long as the sample is very pure) and the fact that each genotyping reaction takes a fraction of a second, allowing thousands of reactions to be carried out serially in a single day.

The majority of current MS genotyping platforms involve the use of DNA chips because the chip can be used directly as a MALDI plate. Thus far, the technique has been used to determine the masses of allele-specific hybridization probes, allele-specific primer extension products, and the products of invasive cleavage reactions. Commercial systems utilizing mass spectrometry include MassArray (Sequenom, Inc.) and PinPoint (PerSeptive Biosystems, Inc.).

Another promising MS genotyping technology involve the use of cleavable mass tags (chemical moieties of known molecular mass) as labels to replace fluoro-phores.[15] The advantage of mass tags is that, due to the accuracy of MS-based mass determination, the range of labels that can be used is virtually limitless, so ultra-high throughput genotyping is possible. The MassCode system (Qiagen Genomics) uses this technology.


Pyrosequencing is a novel method for sequencing short stretches of DNA based on the detection of pyrophosphate, a normal by-product of DNA synthesis.[16] Although similar in principle to primer extension allele discrimination methods, pyrosequencing is suitable not only for typing SNPs but also for scoring entire haplotypes (groups of linked SNPs). Pyrosequencing works on the basis that pyrophosphate can be used to generate adenosine triphosphate (ATP), which then stimulates luciferase activity, causing the emission of a chemilumi-nescent signal. To achieve this, the primer extension reaction must include adenosine 5' phosphosulfate (APS) and the enzyme ATP sulfurylase, which converts APS into ATP in the presence of pyrophosphate. Also present is luciferin (the substrate of luciferase) and the enzyme apyrase, which continuously degrades unincorporated dNTPs and excess ATP. The dNTPs are added to the reaction one by one. Only if the incoming dNTP is complementary to the template will it extend the primer and release pyrophosphate, resulting in the production of an equimolar amount of ATP. Visible light is generated in proportion to the amount of ATP and is detected as a peak on a pyrogram. When the degradation of the present dNTP is completed by apyrase, the next is added. A thioderivative of dATP must be used to avoid constant stimulation of luciferase activity.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment