Cids. A dNTP analog can be used to incorporate a fluorophore by PCR, nick translation or random priming, either directly into DNA1 or indirectly via a hapten such as biotin.2 Though high incorporation efficiencies have been reported, 3 all of these approaches require the separation of unincorporated label prior to downstream applications. Lawler Scientific, LLC has developed a series of reagents called Internally Quenched Nucleotides or IQNs. These reagents consist of a nucleoside triphosphate with a fluorescent reporter attached to the nucleobase and a quencher moiety attached to the gamma-phosphate. The nucleotides remain non-fluorescent until the quencher is enzymatically separated from the parent nucleotide. Since the IQNs are non-fluorescent until incorporated into a nucleic acid, they should not give rise to the background fluorescence signals commonly observed when DNA labeled by standard means is inadequately purified. Nucleic Acid Labeling The first generation IQN consists of a fluorescein-dUTP with a dabsyl quencher linked to the gamma phosphate (see Figure1). The first generation molecules were developed to address the cDNA labeling application described above. Fluorescein and dabsyl were selected because of their superior optical properties and because the photophysics governing their interaction is well described in the literature.4 In addition, this IQN is soluble and stable in aqueous solution. Molecular modeling of the fluorescein and dabsyl moieties of the IQN predicts that these moieties will be well within effective quenching distances.284028-89-3 Biological Activity Consistent with this prediction is the observation that fluorescence emission of this first generation molecule is 98% quenched (see Figure 2). Chemical fragmentation of the triphosphate backbone restores fluorescence emission as does hydrolysis by snake venom phosphodiesterase (SVP).58-05-9 Description As shown in the accompanying Figure 3, SVP rapidly hydrolyzes the IQN with a concomitant increase in fluorescence intensity at 520nm. Reverse transcriptases are commonly used in cDNA labeling protocols. An oligonucleotide primer extension assay was performed using Avian Myeloblastosis Virus (AMV) reverse transcriptase (RT). AMV RT correctly incorporated the IQN opposite dA
Figure 2: The fluorescence spectrum of equimolar amounts of FluoresceindUTP (black) and Fluorescein-dUTP-dabsyl (red) were recorded at an excitation wavelength of 440nm.
Figure 3: The hydrolysis of fluorescein-dUTP-dabsyl by snake venom phosphodiesterase. Note: The background fluorescence was essentially zero prior to the addition of the SVP.
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Figure 4: The fluorescence spectrum of equimolar amounts of PyrrolodCTP (black) and Pyrrolo-dCTP-dabcyl (red) were recorded at an excitation wavelength of 340nm.PMID:31421662
Figure 5: The hydrolysis of pyrrolo-dCTP-dabcyl by snake venom phosphodiesterase.
residues in the template strand. In a second set of experiments, this IQN was added to an AMV RT mediated cDNA synthesis reaction. The addition of the IQN resulted in label incorporation in the resulting 351nt cDNA. cDNA synthesis reactions can therefore be “doped” with IQNs to achieve the desired amount of label incorporation. The IQN technology may extend microarray and real-time PCR techniques but it would do so in different ways. Microarray hybridization experiments may be made more time- and cost-efficient by eliminating the fluid handling steps associated with the cDNA labeling and purification processes. Real-time PCR based experiments may also be made mo.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
