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A novel endonuclease IV post-PCR genotyping system.

Kutyavin IV, Milesi D, Belousov Y, Podyminogin M, Vorobiev A, Gorn V, Lukhtanov EA, Vermeulen NM, Mahoney W - Nucleic Acids Res. (2006)

Bottom Line: Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA.In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated.High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

View Article: PubMed Central - PubMed

Affiliation: Nanogen, 21720 23rd Drive SE, Suite 150, Bothell, WA 98021, USA.

ABSTRACT
Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5' end and fluorophore attached to the 3' end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3' end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

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Related in: MedlinePlus

Synthesis of methyl 3-(3-chloro-2,4-dihydroxyphenyl)propanoate (4a) required for the preparation of PFP esters 11(a and c).
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Related In: Results  -  Collection


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sch1: Synthesis of methyl 3-(3-chloro-2,4-dihydroxyphenyl)propanoate (4a) required for the preparation of PFP esters 11(a and c).

Mentions: Fluorogenic probes were synthesized on fluorophore-linker-based CPG supports (Scheme 1) using standard 3′-DNA-phosphoramidites. An Eclipse Dark Quencher was introduced at the 5′ end using the corresponding phosphoramidite (). Probes were purified by reverse-phase high-performance liquid chromatography (HPLC), dried and re-dissolved in 1× TE buffer. A nearest-neighbor model was applied to calculate extinction coefficient (ɛ260) of oligonucleotides (17). A260 measurements were made in PBS (pH 7.2) at ambient temperature and assumed to be a random coil DNA structure in solution. For each Eclipse Quencher, fluorophore 1 (FL1), fluorophore 2 (FL2) or fluorophore 3 (FL3) substitution an ɛ260 correction of +6600, +18 500, or +25 100 or 28 600 M−1 cm−1 was used, respectively.


A novel endonuclease IV post-PCR genotyping system.

Kutyavin IV, Milesi D, Belousov Y, Podyminogin M, Vorobiev A, Gorn V, Lukhtanov EA, Vermeulen NM, Mahoney W - Nucleic Acids Res. (2006)

Synthesis of methyl 3-(3-chloro-2,4-dihydroxyphenyl)propanoate (4a) required for the preparation of PFP esters 11(a and c).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636472&req=5

sch1: Synthesis of methyl 3-(3-chloro-2,4-dihydroxyphenyl)propanoate (4a) required for the preparation of PFP esters 11(a and c).
Mentions: Fluorogenic probes were synthesized on fluorophore-linker-based CPG supports (Scheme 1) using standard 3′-DNA-phosphoramidites. An Eclipse Dark Quencher was introduced at the 5′ end using the corresponding phosphoramidite (). Probes were purified by reverse-phase high-performance liquid chromatography (HPLC), dried and re-dissolved in 1× TE buffer. A nearest-neighbor model was applied to calculate extinction coefficient (ɛ260) of oligonucleotides (17). A260 measurements were made in PBS (pH 7.2) at ambient temperature and assumed to be a random coil DNA structure in solution. For each Eclipse Quencher, fluorophore 1 (FL1), fluorophore 2 (FL2) or fluorophore 3 (FL3) substitution an ɛ260 correction of +6600, +18 500, or +25 100 or 28 600 M−1 cm−1 was used, respectively.

Bottom Line: Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA.In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated.High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

View Article: PubMed Central - PubMed

Affiliation: Nanogen, 21720 23rd Drive SE, Suite 150, Bothell, WA 98021, USA.

ABSTRACT
Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5' end and fluorophore attached to the 3' end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3' end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

Show MeSH
Related in: MedlinePlus