Sequence And Positioning Of Taq Man PROBE

Many of the rules that apply to PCR primer design also apply to TaqMan probe design (see Primer/Probe Design chapter in EDGP). The sequence of the TaqMan probe should have GC contents within 40-60% preferably. Long sequences of identical nucleotides should be avoided in the probe. The sequence should be devoid of self-complementarity, or complementarity with the forward and reverse primers. The TaqMan probe should anneal as close as possible to the forward primer without overlapping. The size of the amplicon (dictated by the forward and reverse primers) should be quite short, never exceeding 300 bp.

The TaqMan probe should be 20-30 nucleotides in length, with a high melting temperature (65 °C to 67°C). This is ~ 10°C higher than the forward and reverse primers of the PCR reaction, and ensures that the probe is bound more stably than the primers when there is a perfect match with the target DNA sequence (Fig. 1A). This is

Fig. 2 Comparison of the classical TaqMan probe and the minor groove binder (MGB) TaqMan probe. Classical TaqMan probes (top) are longer than the MGB counterparts. Allelic discrimination is best achieved by positioning the polymorphic site (indicated by the asterisk) toward the center of the probe. The MGB probes (bottom) are shorter because of the additional duplex stabilization afforded by the MGB group over the span of approximately 5 base pairs. Allelic discrimination is best achieved by positioning the polymorphic site in the MGB portion of the TaqMan probe.

Fig. 2 Comparison of the classical TaqMan probe and the minor groove binder (MGB) TaqMan probe. Classical TaqMan probes (top) are longer than the MGB counterparts. Allelic discrimination is best achieved by positioning the polymorphic site (indicated by the asterisk) toward the center of the probe. The MGB probes (bottom) are shorter because of the additional duplex stabilization afforded by the MGB group over the span of approximately 5 base pairs. Allelic discrimination is best achieved by positioning the polymorphic site in the MGB portion of the TaqMan probe.

important, because hybridization of the probe with the template DNA activates the Taq 5' nuclease activity, which in turn generates the fluorescence increase because of cleavage. In the case of a TaqMan probe designed to distinguish between two alleles of a specific marker gene, the polymorphic site should be positioned near the middle of the probe to cause as much destabilization as possible of the mismatched duplex (Fig. 2).

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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