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Another modification of allele-specific hybridization is allele-specific ligation, in which two oligonucleotides are annealed at adjacent sites and DNA ligase is used to join them together. One of the oligonucleotides has two allele-specific variants, and the discriminatory base is at the most 3' position. If there is perfect complementarity between the ASO and the template, it will be successfully ligated to the common oligonucleotide. If not, ligation will fail and the two oligonucleotides will remain separate. The assay in itself is not very sensitive, but sensitivity can be increased using additional steps such as the ligase chain reaction (in which the ligation reaction is reiterated with further oligonucleotides complementary to the first ligation product) or rolling circle amplification technology (RCAT; in which two ASOs about 80 nucleotides in length are designed to form a circle on the template, generating a closed loop or padlock probe when ligated). Primers annealing to this circle can be extended with a strand-displacing DNA polymerase, so that when the nascent strand completes the circle and encounters itself, it is continually displaced, generating a long concatemer that is easy to detect using fluorescence methods.[8]

Allele-Specific Single-Base Primer Extension

In allele-specific single-base extension (also called mini-sequencing), primers that anneal one nucleotide upstream of the polymorphic site are designed, and allele discrimination depends on the ability of this perfectly annealed primer to be extended (Fig. 1c).[9] This is distinct to allele-specific PCR where the discriminatory position lies within one of the primers and extension depends on the ability of the primer to anneal to its template. Allele-specific primer extension methods are more adaptable than hybridization/ annealing assays because a much greater diversity of labeling strategies can be used. For example, the free nucleotides in solution can be labeled with four different fluorescent tags, mass tags, or haptens, allowing the same mix to be used in the detection of multiple SNPs in parallel (e.g., on a microarray). Primer extension methods for genotyping have been extensively patented as Genetic Bit Analysis™ (GBA) technology (Orchid Biosciences, Inc.) and form the basis of many popular commercial genotyping systems (e.g., SnaPshot; Applied Biosystems).

Allele-Specific Enzymatic Cleavage

One of the earliest and most widely used genotyping methods, restriction fragment length polymorphism (RFLP) analysis, works on the principle of allele-specific enzymatic cleavage. An RFLP is generated when an SNP occurs at a restriction endonuclease recognition sequence, and one allele preserves the sequence while the other destroys it. If we consider any DNA fragment with three adjacent restriction sites, with the middle one containing an SNP, then digestion of amplified genomic DNA with the appropriate restriction endonuclease will produce either a single large fragment (if the central restriction site is absent) or two smaller fragments (if the central restriction site is present and cleavage occurs).

RFLP analysis is difficult to adapt for high-throughput genotyping, but this has been achieved for another cleavage-based assay known as Invader.[10] The Invader assay employs a unique method of allelic discrimination involving overlapping probes and an enzyme that specifically recognizes the resulting ''flap'' (Fig. 1d). An advantage of the Invader assay is that it requires no PCR amplification. Two signal probes are used, one recognizing each allele, plus a third invader probe. The signal and invader probes hybridize in tandem, and the signal probe overlaps the invader probe, generating a flap that is recognized by an enzyme called Cleavase, a modified form of the thermostable FEN-1 enzyme (flap endonuclease). A flap with the appropriate structure is generated only if the signal probe completely matches the template and there is a one-base invasion. The cleaved flap can then be used in a second round invader assay to generate a fluorescent signal, or the cleaved flap can be detected by mass spectrometry.

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.

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