-Iga SNP vajab spetsiaalpraimereid -Kõige efektiivsemad väheste SNP-de ja suure hulga proovide puhul Introduction to TaqMan® probes TaqMan® probes are dual labeled hydrolysis probes and are a registered trademark of the Roche Molecular Systems, Inc. TaqMan® probes utilize the 5' exonuclease activity of the enzyme Taq Polymerase for measuring the amount of target sequences in the samples. TaqMan® probes consist of a 18- 22 bp oligonucleotide probe which is labeled with a reporter fluorophore at the 5' end and a quencher fluorophore at the 3' end. TaqMan® probe functioning While carrying out a TaqMan® experiment, a fluorogenic probe, complementary to the target sequence is added to the PCR reaction mixture. This probe is an oligonucleotide with a reporter dye attached to the 5' end and a quencher dye attached to the 3' end. Till the time the probe is not hydrolized, the quencher and the fluorophore remain in proximity to each other, separated only by the length of the probe
trast, hybridization probes are not hydrolyzed the scientific literature (Garcia-Canas et al. during PCR. The fluorescence is generated 2002; Burns et al. 2003; Feriotto et al. 2003; by a change in its secondary structure during Rudi et al. 2003; Glynou et al. 2004; Obeid the hybridization phase, which results in an et al. 2004; Fantozzi et al. 2008; Morisset et increase of the distance that separates the al. 2008). Recently, a novel DNA-based reporter fluorophore from the quencher technology has been developed, the NASBA moiety. The most relevant hybridization Implemented Microarray Analysis (NAIMA) probes are those containing hairpins such as (Morisset et al. 2008). In this technology, a Molecular Beacons, Scorpion primers, and cRNA product is obtained from a consecu- 508 Chapter 29 tive first primer extension reaction, using junction regions of DNA from different
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